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high - speed turbines and diamond burs have been the primary choice for tooth preparation to receive direct and indirect dental restorations . their use requires professional skill , as improper use can cause injuries to dental tissues . pulpal injury during cavity preparation remains an important concern , since heat generation during operative procedures can be considered one of the main sources of trauma . new methods have been suggested for the preparation of cavities in an attempt to preserve healthy dental tissue and improve the quality of its interface with restorative materials . however , they are generally expensive and are not suitable for all types of preparations , especially for indirect restorations , such as crowns and laminate veneers . ultrasonic devices on the other hand can be considered a good alternative to conventional high - speed turbines , since they produce less noise and are less expensive than lasers . recently , chemical vapor deposition ( cvd ) technology was used to produce new diamond tips to be used with ultrasonic devices . this technology utilizes a process for obtaining coalescent diamond films in grinding layers and allows the growth of a single diamond crystal as the active part of the bur . the integrity of the diamond and the high adherence to the metallic shank are responsible for its high efficiency and durability as compared to the conventional diamond coated burs . another advantage of this technology is the decreased injury to gingival tissues when operating at subgingival margins of the cavity [ 4 , 5 ] . little is known about the influence of different surface textures resulting from cutting with rotary and ultrasonic instruments on enamel bond strength [ 6 , 7 ] . investigated the microtensile bond strength of enamel surfaces gritted with regular- and superfine - grit diamond burs using three different adhesive systems and showed statistically similar results for both surface finishing . regarding ultrasonic diamond burs and tips on enamel , the majority of the studies investigated other properties , such as microleakage and surface roughness , but not the bond strength . the literature reports that the use of cvd diamond tips in ultrasonic device , when compared to diamond rotary instruments , can produce different morphological characteristics in the dentin surface , more specifically in relation to the thickness of the smear layer and the surface roughness . moreover , alternative methods of cavity preparation ( cvd bur in a high - speed turbine , a cvd tip in an ultrasound device , and an er , cr : ysgg laser ) compared to conventional diamond burs negatively influenced the microtensile bond strength of different adhesives systems , irrespective of their acidity or approach . the instrument used in the cavity preparation has a significant influence on the cut surface of the dentin . moreover , the completion of preparation with finishing burs seems to be the method of choice for achieving a smooth and better wetting surface [ 11 , 12 ] . surface roughness of dental hard tissues plays an important role in operative dentistry , since it affects how well the restorative material will adapt to the dental structure . , the smoothness of the surface plays an important role in the impression and cementation procedures . while a smooth surface is important during the impression , rougher surfaces could provide better substrates for adhesion . while dental surface finish characteristics may have been in a way neglected , new reinforced ceramic materials for esthetic purposes have been developed . glass - ceramics are indicated for anterior and posterior esthetic restorations because of their optical and mechanical properties . recently , a zirconia - reinforced , higher strength lithium silicate - reinforced glass - ceramic that features a fine - grained and homogenous microstructure and that supports a wide range of applications , including anterior and posterior restorations , has become available . thus , it is necessary to study the adhesive property of this new lithium silicate glass - ceramic to tooth structure and the influence of textures on the ceramic material . ceramic laminate veneers have high survival rates when bonded to enamel and provide a safe and predictable treatment option that preserves tooth structure . in a retrospective study evaluating porcelain laminate veneers up to 12 years , survival rates of 99% for veneers with preparations confined to enamel and 94% for veneers with enamel - only margins were observed . despite these high success rates , the authors also reported that laminate veneers bonded to dentin and teeth with preparation margins in dentin were approximately 10 times more likely to fail than when the veneers were bonded to enamel . hence , it is important to evaluate the effects of surface roughness on enamel and dentin surfaces created with different types and granulations of diamond burs and tips on bond strength using new glass - ceramic systems used for indirect laminate veneers . the objectives of the present study were to evaluate the influence of enamel and dentin surface finishing with medium and fine - grit diamond burs and tips mounted on a high - speed turbine handpiece and ultrasonic device on the surface roughness and influence of surface roughness on the microshear bond strength of a lithium silicate glass - ceramic to enamel and dentin . two null hypotheses were tested : ( 1 ) there are no differences in the surface roughness parameters evaluated on enamel and dentin surfaces finished with medium and fine - grit diamond burs and tips mounted on a high - speed turbine handpiece and ultrasonic device , and ( 2 ) there are no differences in the microshear bond strength of a glass - ceramic luted to enamel and dentin surfaces finished with medium and fine - grit diamond burs and tips mounted on a high - speed turbine handpiece and ultrasonic device . thirty - five bovine incisors were selected and stored in chloramine t 0.5% at 4c until use . the crowns were separated from the roots at the enamel - cemental junction and each crown was sectioned at the incisal third using a slow - speed diamond saw ( isomet 1000 , buehler , lake bluff , il , usa ) so enamel and dentin specimens could be obtained . the incisal third was used for enamel specimens and the remaining two thirds were used for dentin specimens . the tooth fragments were embedded in pvc cylinders with acrylic resin ( jet , classico artigos odontolgicos clssico ltda , so paulo , brazil ) . a semiautomatic polishing machine ( buehler metaserv 250 , lake bluff , il , usa ) with 600-grit sandpaper under water - cooling was used to expose flat enamel and dentin surfaces . the specimens were then randomly divided into seven groups , according to the type of surface finishing of dentin and enamel : g1 : 1200-grit sandpaper , under refrigeration , in semiautomatic polishing machine ( control);g2 : medium - grit diamond burs ( # 4138 , kg sorensen , barueri , sp , brazil ) in a water - cooled high - speed turbine;g3 : medium - grit diamond burs ( # 4138 ) , followed by fine - grit diamond burs ( # 4138f , kg sorensen ) in a water - cooled high - speed turbine;g4 : medium - grit diamond burs ( tr-26 , mani , tochigi , japan ) in a water - cooled high - speed turbine;g5 : medium - grit diamond burs ( tr-26 ) , followed by fine - grit diamond burs ( rt-26f , mani ) in a water - cooled high - speed turbine;g6 : medium - grit cvd tip ( cr1 , cvdentus , so jos dos campos , sp , brazil ) in a piezoelectric ultrasonic device;g7 : medium - grit cvd tip ( cr1 ) , followed by fine - grit cvd tip ( tf1 , cvdentus ) in a piezoelectric ultrasonic device . g1 : 1200-grit sandpaper , under refrigeration , in semiautomatic polishing machine ( control ) ; g2 : medium - grit diamond burs ( # 4138 , kg sorensen , barueri , sp , brazil ) in a water - cooled high - speed turbine ; g3 : medium - grit diamond burs ( # 4138 ) , followed by fine - grit diamond burs ( # 4138f , kg sorensen ) in a water - cooled high - speed turbine ; g4 : medium - grit diamond burs ( tr-26 , mani , tochigi , japan ) in a water - cooled high - speed turbine ; g5 : medium - grit diamond burs ( tr-26 ) , followed by fine - grit diamond burs ( rt-26f , mani ) in a water - cooled high - speed turbine ; g6 : medium - grit cvd tip ( cr1 , cvdentus , so jos dos campos , sp , brazil ) in a piezoelectric ultrasonic device ; g7 : medium - grit cvd tip ( cr1 ) , followed by fine - grit cvd tip ( tf1 , cvdentus ) in a piezoelectric ultrasonic device . the diamond burs were mounted in a high - speed turbine ( extra torque 605 high - speed turbine , kavo , joinville , sc , brazil ) and the grinding was performed using low pressure and intermittent cutting , with a water flow rate of 45 ml / min . for ultrasonic - gritted specimens , cvd diamond tips were adapted to a piezoelectric ultrasonic device ( cvdent 1000 , cvdentus ) , operating according to the parameters recommended by manufacturer ( 20 ml / min of water flow rate ; 70% of its maximum power for cr1 and 50% of its maximum power for tf1 ) . for standardization purposes , diamond burs and tips were passed 10 times in the same direction on the enamel and dentin surfaces , as uniformly as possible by using light pressure and keeping the active part of the burs and tips always in contact with the tooth surface . for the roughness test , the specimens ( n = 5 ) of each group were analyzed with a roughness tester ( sj-210p surftest , mitutoyo , japan ) equipped with a diamond needle ( radius of 5 m ) at a constant speed of 0.5 mm / s . before the readings , the roughness tester was calibrated according to the manufacturer 's recommendations with a roughness standard ( precision reference specimen 178 - 602 , mitutoyo , japan ) . for each reading , a length of 2.5 mm was analyzed with a cutoff of 0.25 mm . three measurements were made for each specimen and the average of these three readings was used as the roughness value for each specimen . the ra , rz , and rq roughness parameters were evaluated : ( i)ra ( roughness average ) is the mathematical average height of roughness irregularities measured from a mean line within the sampling length.(ii)rz ( low - point height ) is the average distance between the 5 highest peaks and 5 deepest valleys within the sampling length.(iii)rq ( root mean square ) is the geometric average of roughness component irregularities measured from the mean line within the sampling length . ra ( roughness average ) is the mathematical average height of roughness irregularities measured from a mean line within the sampling length . rz ( low - point height ) is the average distance between the 5 highest peaks and 5 deepest valleys within the sampling length . rq ( root mean square ) is the geometric average of roughness component irregularities measured from the mean line within the sampling length . for the microshear bond strength test , zirconia reinforced lithium silicate glass - ceramic ( suprinity , vita zahnfabrik , bad sackingen , germany ) cylinders with 1 mm in diameter and 1 mm in height were bonded to the enamel and dentin specimens . the glass - ceramic surface was treated with 10% hydrofluoric acid ( condac porcelana , fgm , joinville , sc , brazil ) for 20 s and rinsed and air - dried . a thin layer of silane ( prosil , fgm , joinville , sc , brazil ) was applied with a microbrush for 1 min . the tooth surfaces were etched with 37% phosphoric acid , ( condac 37 , fgm , joinville , sc , brazil ) 30 s for enamel and 15 s for dentin , rinsed with water for 20 s , and blot - dried using absorbent paper . an etch - and - rinse adhesive system ( ambar , fgm , joinville , sc , brazil ) was applied according to the manufacturer 's recommendations and air - dried for 5 s at an approximate distance of 20 cm to allow solvent evaporation . glass - ceramic cylinders were luted to enamel and dentine with a light - cured resin cement ( allcem veneer , fgm , joinville , sc , brazil ) . the excess of cement was removed and each cylinder was light - cured for 40 s , with a led light curing device ( poly wireless , kavo , joinville , sc , brazil ) operating on standard mode and emitting 800 mw / cm irradiance . the output irradiance was measured with a radiometer ( demetron , kerr , middleton , wi , usa ) . the microshear bond strength tests were performed with a universal testing machine ( dl2000 , emic , so jos dos pinhais , pr , brazil ) at a crosshead speed of 0.5 mm / min until fracture . a stainless steel wire - loop ( 0.2 mm diameter ) was used and the specimens were carefully aligned to allow the load to be applied as close as possible to the bonded interface . the fractured interfaces were examined in a light microscope under 57x magnification ( szx9 , olympus , tokyo , japan ) to determine the failure mode ( adhesive , cohesive , or mixed ) . data were statistically analyzed using one - way anova and tukey 's hsd test with a significance level of 5% . pearson 's product moment coefficient was used to correlate ra with microshear bond strength for enamel and dentin . means and standard deviations of the roughness parameters for enamel and dentin are shown in tables 1 and 2 , respectively . for both enamel and dentin , the one - way anova of each roughness parameter revealed significant differences among groups ( p < 0.0001 ) . for the three parameters analyzed , g1 ( control ) showed the lowest mean roughness readings . g2 and groups in which the tooth surface was finished with fine - grit diamond burs on a high - speed turbine or with medium versus fine - grit cvd tips in ultrasonic device showed intermediate values of surface roughness and were statistically similar . means and standard deviations of enamel and dentin microshear bond strengths are presented in table 3 . for both enamel and dentin , one - way anova revealed significant differences among groups ( p = 0.0035 and p = 0.012 , resp . ) . in all groups , the microshear bond strength values for enamel were higher than for dentin . in enamel groups , g7 ( fine - grit cvd tip ) presented the highest microshear and was statistically different from g1 but statistically similar to g6 ( medium - grit cvd tip ) , g3 ( kg fine - grit diamond bur in high - speed turbine ) , g2 ( kg medium - grit diamond bur in high - speed turbine ) , and g5 ( mani fine - grit diamond bur in high - speed turbine ) . g1 ( 1200-grit sandpaper ) and g4 ( mani medium - grit diamond bur in high - speed turbine ) presented the lowest microshear bond strength values . for dentin , g7 ( fine - grit cvd tip in ultrasound ) also presented the highest microshear bond strength , being statistically similar to g1 ( 1200-grit sandpaper ) , g6 ( medium - grit cvd tip ) , g4 , and g2 ( mani and kg medium - grit diamond burs in high - speed turbine ) . both groups whose surface was completed with fine - grit diamond burs in high - speed turbine ( g3 and g5 ) presented the lowest microshear bond strength values , which were also statistically different from g1 . for both enamel and dentin , there was no statistical difference when comparing the groups gritted with the same brand of medium- and fine - grit burs and tips ( g2 or g3 ; g4 or g5 ; g6 or g7 ) . no correlation was observed between ra and microshear bond strength for enamel and dentin ( r = 0.00251 and 0.07486 , resp . ) bonding ceramic restorations to tooth structure rely on a number of factors , including the treatment of the ceramic surface , selection of a suitable resin luting agent , and appropriate treatment of prepared tooth structure [ 1416 ] . various cavity surface finishing procedures have been routinely used in the dental practice , resulting in different topographies , but little information is available regarding the bond strength of porcelains and glass - ceramics to enamel and dentin gritted with different diamond instruments . the preparation technique , the cavity surfaces characteristics , and the bond strength of ceramics to enamel and dentin are key factors to the clinical longevity of laminate veneers . whenever possible , laminate veneer preparation should be made meticulously and maintained completely in enamel . this is more often achieved in minimum thickness veneers . in these cases , while better adhesion to enamel is achieved , decreased resistance of the ceramic material due to the lower thickness is expected . thus , the technique of minimum intervention relies on a good bonding between both resin cement and enamel and resin cement and ceramics . on the other hand , the exposure of considerable amounts of dentin is sometimes inevitable during preparation , especially at the cervical and proximal areas . it is also worth considering that high failure rates of ceramic laminate veneers have been related to large exposed dentin surfaces [ 13 , 18 ] . ztrk et al . , demonstrated that the type of preparation surface had a significant effect on the shear bond strength of porcelain laminate veneers . when the tooth surfaces were compared , there was no significant difference between the groups bonded to enamel and enamel - dentin complex substrates . however , dentine groups exhibited lower bond strength values . this is in accordance with the present study , which demonstrated higher bond strength than obtained for dentine , irrespective of the surface finishing . all bond strength values obtained in the present study were above 30 mpa , which is in accordance with previous works . laminate veneer preparations are often finished with fine - grit diamond burs or with polishing discs . in theory , smoother surfaces would be more favorable to impression taking , but it has been described that higher bond strength can be obtained by increasing the roughness surface , since it enhances the surface area to be bonded . in the present study , the use of fine - grit burs in high - speed turbine showed lower surface roughness readings in enamel and dentin when compared to medium - grit burs . when cvd tips mounted in ultrasound were used , there was no significant difference in the roughness parameters for medium- and fine - grit tips . the roughness values for both cvd tips were similar to those of fine - grit burs in high - speed turbine . however , there was no correlation between the roughness values and the bond strength for enamel and dentin . it is expected that all the superficial dissimilarities produced by different preparation techniques would influence bond strength to dentin . within the dentin groups , according to the results of the present study , although microshear bond strength values of fine - grit diamond bur group tended to be lower than medium - grit diamond bur group , these variations were not significantly different ( table 3 ) . literature reports that differences in bond strength values in dentin surfaces prepared with different grit diamond burs are probably due to the difference of the smear layers created , which influences penetration of monomers . under sem examination a thick smear layer was produced , uniformly covering the dentin surface . on the other hand , dentin gritted with cvd tips in ultrasonic devices exhibited a relatively smooth surface , a thin smear layer , and the presence of opened dentin tubules in areas where the smear layer was absent . a two - step etch - and - rinse adhesive was used in the present study . it is well known that this type of adhesive removes the smear layer due to the previous etching with phosphoric acid . as mentioned above , the amount and quality of smear layer produced by different surface finishing procedures are different . thus , the results of the present study could be different if a smear layer - modifier dentin adhesive such as self - etching ones was employed . moreover , universal adhesives containing silane - coupling agents could have significant effects on bonding outcomes . recently , it was demonstrated that glass particle size of resin cements significantly influenced ceramic bond strength , while surface treatments showed a minor effect . in the present study a light - cured resin cement was used due to its easy handling and esthetic characteristics . regarding the surface roughness parameters analyzed , the majority of the studies report only ra values . however , ra alone may not be sufficient to discriminate among surfaces . under certain conditions , other parameters ( rq , ry , or rz ) may be important and should be used in addition to ra to properly describe the surface characteristics [ 12 , 14 ] . in the present study , ra , rz , and rq were determined , but the ranking of the groups was practically the same for the three parameters , for enamel and dentin . these materials include esthetic and high - strength ceramics , composite resins , and hybrid ceramics ( polymer - infiltrated ceramic - network materials ) . laminate veneers , porcelain , glass - ceramics , resin composites , and hybrid materials can be used with good aesthetical results . it is important to know the microstructural characteristics of new cad - cam materials ; and their bond strength to enamel and dentin should be investigated . in the present study , a new zirconia reinforced lithium silicate glass - ceramic was chosen because , to the authors ' knowledge , no studies were yet published on this material . the authors did not find information on the use of ultrasonic diamond tips for the preparation of ceramic laminate veneers . cvd diamond tips mounted in ultrasonic devices can be used to perform the finishing procedures on the finishing line of indirect restorations , particularly in ceramic laminate veneers in anterior teeth . it is well known that the subgingival dental preparation as well as retraction cords and hemostatic solutions may cause injury to gingival tissues . diamond burs produced by cvd technology may be used in ultrasonic handpieces ; thus the process is slower to avoid injury to the gingival tissues [ 4 , 5 ] , which would be advantageous when finishing the gingival margins . these characteristics would avoid gingival bleeding and damage , allowing a more reliable impression taking immediately after dental preparation . furthermore , based on the results of the present study , cvd tips showed good values for roughness parameters in enamel and dentin and higher values of bond strength . this is an in vitro study and some limitations need to be addressed , such as the number of specimens per group and the use of microshear bond strength test . a larger sample size could influence the results , lowering the standard deviations and assuring an adequate power to detect statistical significance . microtensile bond strength is usually regarded as a more reliable bond strength test ; however , in the present study , the microshear bond strength test was chosen because it also allowed small areas to be tested , is relatively easy to perform , and does not need sectioning procedures to obtain specimens , since these procedures may induce early microcracking within the specimens . within the limitations of this study , it can be concluded that the cavity surface finishing influenced the roughness parameters and the microshear bond strength of a glass - ceramic to enamel and dentin . medium - grit diamond burs in high - speed turbine showed the highest mean roughness values . fine - grit cvd tips in ultrasound presented the highest microshear bond strength values for both enamel and dentin . however , when comparing between the same manufacturer / product categories , there were no significant differences in the microshear bond strength .

objectives . this study evaluated the influence of cavity surface finishing with diamond burs of different grit mounted on high - speed turbine and ultrasound on the roughness and microshear bond strength ( mbs ) of a lithium silicate glass - ceramic to enamel and dentin . methods . enamel and dentin specimens were divided into seven groups , according to the type of surface finishing : 1200-grit sandpaper ( control ) , two different brands of medium - grit and fine - grit diamond burs in a high - speed turbine ; medium - grit and fine - grit cvd ( chemical vapor deposition ) tips in an ultrasonic device . roughness parameters ( n = 5 ) and msbs to a glass - ceramic ( n = 10 ) were determined . data were analyzed using anova and tukey 's test ( = 5% ) . results . control group showed lower mean roughness readings and groups that used medium - grit diamond burs showed the highest mean roughness values . regarding msbs , there was no statistical difference when comparing the groups gritted with the same brand of medium- and fine - grit burs and tips . conclusions . cavity surface finishing influenced the roughness parameters and msbs of a glass - ceramic to enamel and dentin . medium - grit diamond burs in high - speed turbine showed the highest mean roughness values . fine - grit cvd tips in ultrasound presented the highest msbs values for both enamel and dentin .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

people with advanced human immunodeficiency virus ( hiv ) are vulnerable to infections called opportunistic infections ( ois ) because organisms take advantage of the opportunity offered by a weakened immune system . since the beginning of the hiv epidemic , ois have been recognized as common complications of hiv infection . ois cause substantial morbidity and hospitalization , necessitate toxic and expensive therapies , and shorten the survival of people with hiv infection . a decrease in cd4 count is at least partially responsible for the profound immunodeficiency that leads to various ois in hiv - infected persons . the introduction of antiretroviral therapy ( art ) has dramatically reduced the incidence of oi among hiv - positive people who have received art ; however , around the world , millions of people living with hiv in resource - poor communities have no access to art . at present , the initiation of primary prophylactic therapies for ois is based chiefly on the absolute cd4 count , which has been shown to be an excellent predictor of the short term overall risk of developing acquired immunodeficiency syndrome ( aids ) among hiv - infected patients . the relative frequencies of specific opportunistic diseases may vary in different countries and even in different areas within the same country . human immunodeficiency virus causes progressive impairment of the body 's cellular immune system leading to increased susceptibility to tumours and infections , and the fatal condition is known as aids . the unique feature in the pathogenesis of hiv / aids is that the primary target cell for hiv is immune cells bearing the cd4 marker at their surface . with hiv infection , there will be a gradual decrease of human immune cells bearing cd4 antigen receptor , the most important being t helper cells ( cd4 t cells ) , b lymphocytes , macrophage and natural killer cells leading to development of wide varieties of ois that is , severe infections induced by agents that rarely cause serious diseases in immune competent individuals . in this way aids related mortality and morbidity , which is significantly higher in number as compared to other diseases , is actually due to ois rather than hiv itself . in the era of effective art , diagnosis and treatment of ois is an integral part of this treatment strategy because some ois regimens ( for e.g. , antituberculosis drugs ) and art regimen should not be administrated at the same time to prevent drug induced toxicity and mortality and morbidity due to immune reconstitution inflammatory syndrome . cd4 cell count is the best validated predictor of the likelihood of developing ois . besides , it has great utility in clinical consideration of hiv disease classification and aids definition , assessment of prognosis and designing of clinical trials , for e.g. , decision about initiation of art / prophylaxis . there are wide range of ois affecting different system , for example , respiratory tract infection , gastrointestinal tract infection , urinary tract infections , sexually transmitted infection and disseminated infection . once the standard value of cd4 count and the ois incidence rate at that stage is obtained , this can be used as a model for other resource limited settings ( where cd4 count facilities are not available ) to take decision about the initiation of art in relation to cd4 status were done in a local setting with limited sample size but specific studies aimed at making standard profile of different ois according to cd4 were lacking . hence , this study is conducted with a general objective to explore the relationship between cd4 level and different types of ois which may be helpful for the development of guidelines regarding the initiation and monitoring of art / prophylaxis . furthermore , the result of the findings will be helpful in the prognosis of different ois in hiv / aids patients . descriptive study was carried out during january 2013 to november 2013 at medicine out patient department , medicine wards and art centre of a tertiary care hospital . human immunodeficiency virus positive patients older than 18 years with ois were the study subjects . the exclusion criteria were - patients harbouring ois who are immune - suppressed because of causes other than hiv ; non - consenting patients ; seriously ill patients from whom data collection was impossible . total of 164 hiv positive patients with ois were considered for the study and the sampling technique adopted was non probability purposive sampling . primary data was collected from patients by interview technique and secondary data from case records after obtaining written informed consent . data was analyzed by computing proportions , mean and t - test ; by using microsoft excel and spss 20.0 trial version downloaded from www14.software.ibm.com . the current study revealed that the maximum number of patients who had ois were in the age group of 2837 years ( 45.7% ) , followed by the age group 1827 years ( 27.4% ) and 3847 years ( 21.3% ) . there was higher proportion of males ( 68.3% ) as compared to females ( 29.3% ) . the hiv patients with ois were less educated that is , 27.4% were illiterate and 39% had primary education . most of the patients , who harboured ois , were unskilled labourers ( 39% ) , followed by semiskilled ( 14.6% ) , skilled ( 10.9% ) business , professional and others constituted 35% . of all the cases maximum number of hiv positivity with ois were seen in the married group ( 54.3% ) , followed by people who were unmarried ( 20.7% ) , who were separated ( 17.7% ) and least among widows ( 7.3% ) . heterosexual mode of transmission was the commonest mode of transmission , accounting for 84.7% [ table 1 ] . sociodemographic characteristics of study subjects in the current study as depicted in table 2 , the most common symptoms at presentation were fever ( 78% ) , loss of appetite ( 76% ) , weight loss ( 74% ) , cough ( 58% ) , where as diarrhoea was seen in only 18%of the cases [ table 2 ] . distribution of study subjects based on symptoms among patients who presented with history of fever , 88.5% of them had fever of more than 1-month duration . among patients who presented with history of weight loss , 62.2% of them had loss of body weight of more than 10% . among patients who presented with history of diarrhoea , 83.3% of them had diarrhoea of more than 1-month duration . among patients who presented with history of cough , 91.4% of them had cough of more than 1-month duration . on general examination , the most common signs found were emaciated built ( 73% ) , fever ( 60% ) , tachycardia ( 94% ) , tachyopnea ( 62% ) and pallor ( 76% ) . genital lesions and skin lesions constituted 18% [ table 3 ] . among patients with ois , respiratory system was more commonly affected ( 56% ) followed by nervous system ( 26% ) , skin ( 15% ) , genitals ( 10% ) . only 56% of patients had abnormality in chorionic villus sampling and abdomen [ table 4 ] . it is evident from the present study that tb is the most of frequent ois accounting for 50% of all ois , followed by candidiasis in 49% of cases [ table 5 ] . distribution of study subjects based on clinical signs on general examination distribution of study subjects based on systemic examination distribution of study subjects based on type of opportunistic infections in this study , 65% of the patients with ois had cd4 t - cell counts below 200/l , where as there were none with counts above 500/l [ table 6 ] . low values were observed in promyelocytic leukemia ( 18.10/l ) , cytomegalovirus ( 18.5/l ) . statistically significant association was found between cd4 count and tb , candidiasis , cryptosporidiosis , isosporiosis , and strongyloidiasis [ table 7 ] . in the present study , majority of patients were in the age group of 28 - 37 years . it was observed that the frequency of oi was highest in the sexually active age group of society . this indicates a trend of young and productive generation being affected ; a reflection of the devastating effects india will face as the this work force is affected . the incidence of oi was significantly high in patients who were less educated and this directly indicates the impact of level of education on the transmission of the disease , as education is directly related to the level of awareness . the commonest mode of transmission was heterosexual ( 84.7% ) followed by others , as heterosexual transmission remains the commonest mode since other sexual practices being very uncommon in this part of the world . this study also found that a small proportion of transmission attributed to blood transfusion / needle prick as observed in other studies . in this study , it was found that fever was the most frequently occurring symptom present in 78% of the cases followed by loss of appetite , weight loss and cough . numerous ois occur in hiv infected patients due to down regulation of the immune system . in the present study , it was found that tb was the most frequent oi accounting for 50% of all infections followed by candidiasis in 49% of cases . similarly in a study of sharma and vajpayee , tb was most common oi followed by candidiasis , whereas contradictory to this in a study of patel , giri , and singh , candidiasis was the most common followed by tb . human immunodeficiency virus - tb co infection is a serious problem worldwide but especially of concern in india where background rates of tb is highest in the world . in india the mean cd4 count was low for tb in our study compared to western data . candidiasis in our study occurred at a wide range of cd4 levels ; but mean cd4 count was189.07 . compared to our study , studies by moore et al . and crow et al . the mean cd4 count for cryptosporidiosis , isosporidiosis , and strongyloidosis in our study was very similar to the mean cd4 count of moore et al . in our set up most frequent ois are accounted by tb and candidiasis and there is a direct correlation between the values of cd4 count and the severity of the ois , hence indicating the level of immunity and the severity of the disease .

background : human immunodeficiency virus ( hiv ) virus , causative agent in acquired immunodeficiency syndrome , is fast becoming a major threat in the indian subcontinent , with an estimated 3.7 million persons being infected with hiv . hiv infection is complicated by various opportunistic infections ( ois ) such as tuberculosis ( tb ) , candidiasis , herpes zoster , pneumocystis jirvoceii , cytomegalovirus ( cmv ) etc . , this study carried out to know the clinical profile of hiv patients with ois.methods:a case series study was carried out at a tertiary care hospital in bellary , karnataka , india . a hospital based case series study was conducted among 164 hiv patients with ois admitted to various wards as well as attending outpatient department at vijayanagara institute of medical sciences hospital , bellary during jan 2013 to nov 2013 . both primary and secondary data was collected to gather information on clinical profile . the statistical tests used were descriptive statistics and independent t test.results:among 164 patients , 29.3% were females and 68.3% males . high proportions of patients were observed in 28 - 37 years of age group and heterosexual route was the most common mode of transmission . tb ( 50% ) is the most frequent oi followed by candidiasis ( 49% ) , pneumocystis ( 16% ) and others . the mean cd4 cell count in tb was 237.02/ml and in candidiasis 189.07/ml . low values were observed in promyelocytic leukemia ( 18.10/ml ) , cmv ( 18.5/ml ) and in toxoplasmosis ( 73.1/ml).conclusions : respiratory system was the most common system involved by ois and most of patients with ois had cd4 t cell count below 200/ml , whereas there were no patients in the study with counts above 500/ml .

I M R D C

as a scientist , administrator , and diplomat , david p. rall pioneered the effort to identify and understand the elements that make up the human environment and their consequences for human health . as an intellectual and aggressive activist , he educated scientists , governments , and the world community to the critical need to address the existence of environmental agents and their consequences for human health . as a leader he marshalled some of the best minds and hearts of his time to the cause of world health through a safe and clean environment . and as a visionary he provided the goals of environmental health science and the direction to guide both current and future generations . his death on september 28 brought to a close a chapter in the evolution of our understanding of the interconnectedness of human health and the environment , a chapter he was largely responsible for writing.imagespa538-a

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a 29 year old male was admitted to keimyung university hospital in 1987 with a continous substernal chest pain of three days duration . he had been previously admitted in because of pain , chills , a tingling sensation and raynaud s phenomenon of the left hand and both feet . at that time peripheral arterial mapping indicated that the left brachial and radial arterial pulsations were weaker than the right , and the popliteal and dorsalis pedis arterial pulsations were absent in both . the laboratory findings included wbc , 12,800/cu mm with 25% eosinophils ( 3,000/cu mm ) , hematocrit , 45% ; ast , 17 iu/1 ; aso titer , 125 u ; c - reactive protein , negative ; cryoglobulin and cryofibrinogen , negative ; serum ige level , zero ; hbs antigen , negative . the electrocardiogram taken on admission , revealed a normal sinus rhythm with no abnormal q waves and st segments . 1 ) showed a segmental luminal narrowing of the profunda brachii and brachial arteries with thin , tree root shaped collaterals . the femoral angiogram revealed luminal narrowing and a corrugated appearance of the left popliteal artery and prominent sural artery , which resembled a spider leg or vine tendrill . buerger s disease was easily confirmed on the basis of the clinical features and angiographic findings . six months prior to readmission , he began smoking again , and three months later , anterior chest discomfort following heavy exercise or consumption of alcohol was noted . after 3 days of episodes of severe substernal chest pain which continued for several hours and did not subside even with rest , he presented at keimyung university hospital to an evaluation of the chest pain . upon admission the patient s vital sign were blood pressure : 130/80 mmhg , body temperature : 36.8c , and pulse rate 78/minitue . the peripheral arterial pulsations on both sides of the upper and lower extremities were symmetric , but the pulsations of the popliteal and dorsalis pedis arteries , which were absent on the first admission , were present but weak , the laboratory results included wbc , 6,300/cu mm with eosinophils 14% ( 910/cu mm ) ; hematocrit , 39.3% ; esr , 20 mm / hour ; creatinine kinase , 84.0 u / l ; asparate transaminase , 15.4 u / l ; lactic dehydrogenase , 158.4 u / l ; total cholesterol , 109 mg / dl ; total protein , 7.2 mg / dl and uric acid 5.9 mg / dl . 2 ) displayed a sinus rhythm with abnormal q wavese and an elevation of more than 1 mm in the st segments in lead i , avl and v2 - 5 . two weeks after the substernal chest pain episode , left ventriculography and coronary angiography were performed , and the left ventriculogram revealed hypokinetic or akinetic movement in the anterior region . 3 ) in the right and left anterior oblique projections demonstrated partial segmental occlusion of the proximal left anterior descending artery ( 42% of the luminal area ) , complete occlusion of the first diagonal brach of the left anterior descending artery in the distal portion , and irregular and tortous contour of the right coronary artery without obvious luminal narrowing . conservative management included the administration of nitrate , beta - blocker and calcium channel blocker . leo buerger first described thromboangitis obliterans ( buerger s disease ) as a progressive peripheral insufficiency occuring primarily in young male smokers , many authors have reported on this unique clinicopathologic disease entity . this disease has a geographic and ethnic prevalence , especially among indians , jews and orientals . in korea , buerger s disease is a much more prevalent peripheral vascular occlusive disease than arteriosclerotic obliterans . although there have been occasional reports of involvement of the mesenteric and cerebral arteries and rarely of the coronary arteries , myocardial infarction , as a clinical feature , is an extremely rare event in buerger s disease . however , shionoya et al , reported that on autosy a patient buerger s disease showed atheromatous changes in the coronary and abdominal aorta , and also that patients with buerger s disease were susceptible to the arteriosclerotic process . our patient s electrocardiomgram showed an extensive anterior wall infarction , and the coronary angiogram revealed a partially occluded proximal portion of the left anterior descending artery , further proof of an anterior myocardial infarction . it is widely accepted that a myocardial infarction is the result of either a totally or critically occluded ( less than 25% of the luminal area ) coronary artery . recently , several mechanisms have been proposed as possible cause of the myocardial infarction in patients with normal or near normal coronary arteries . in 1983 , rosenblatt and selzer suggested three possible mechanisms to explain such a discrepancy ; 1 ) a myocardial infarction is produced by a coronary arterial spasm ; 2 ) coronary lesions may cause myocardial infarctions , but their presence is not noted on a subsequent coronary angiogram ; and 3 ) an occlusive lesion , present at the time of infarction , has disappeared due to recanalization or lysis of the thrombus . coronary arteriographic studies in our patient revealed a 42% luminal narrowing of the left anterior descending artery , which was greater than the critical level of acute myocardial infarction . thus we suspected that other mechanisms , such as coronary vasospasm or recanalization , might be the cause of a transmural infarction with arteriosclerosis . another finding which supports the suggestion of a coronary vasospasm , as a cause of an acute myocardial infarction , is raynaud s phenomenon which was observed in the patient prior to the myocardial infarction . ciraulo et al , reported that migrain or raynaud s phenomenon was more frequently associated with coronary vasospasm and resulted in myocardial infarction in patients with normal coronary arteries . based on these reports , we sugges that the coronary vasospastic phenomenon was involved in this case . the etiology of buerger s disease , though still obscure , is correlated with cigarette smoking . most of the patients cited in the literature were cigarette smokers who experienced a slowdown in the progression of disease inwhen they stopped smoking . in 1983 , adar et al reported that patients with buerger s disease exhibited a cellular sensitivity to human type i or type iii antigens ( or both ) and developed antibodies to anticoagulants , suggesting that immunologic factors were involved in the etiology of the disease . other aspects of the disease are the genetic predisposition and the noted prevalence of hla - a9 and hla - b5 in many affected persons and of hla - a9 and hla - w10 in japanese patients . in 1985 , ferguson et al , reported a case of buerger s disease associated with idiopathic hypereosinophilia and suggested that eosinophilia might be involved in the pathogenesis of buerger s disease . they proposed a wherein long term smoking incited an allergic reaction with hypereosinophilia in some patients . it is interesting to note that on both admission , our patient presented hypereosinophilia with a normal ige level and other immunologic data , but these results might not be consistent with ferguson s report . the treatment of buerger s disease is rather simple & specific ; the patients should stop smoking . when our patient stopped smoking and followed conservative management for the myocardial infarction , his chest pain subsided . in conclusion , a myocardial infarction is a possibility in buerger s disease due to coronary vasospasm and coronary arteriosclerosis .

a twenty - nine year old male smoker with a three year history of buerger s disease was admitted with excruciating precordial chest pain . the electrocardiogram indicated an anterior transmural infarction , and he also exhibited hypereosinophilia.a coronary angiogram disclosed a partial segmental occlusion of the left anterior descending artery at the proximal portion . he was discharged without any complications after conservative managment .

REPORT OF A CASE COMMENTS

temporal arteritis / giant cell arteritis ( gca ) is a systemic immune - mediated vasculitis first described by horton et al . that affects medium - sized and large - sized arteries , which include main branches of the aorta , its primary and auxiliary arteries , superficial temporal , ophthalmic , posterior ciliary and vertebral arteries . it is commonly seen in older women more than 50 years ( m : f ratio 2:5 ) . vision loss due to ischemia of the optic nerves may result from temporal arteritis secondary to vascular occlusion . since vision loss is permanent , temporal arteritis can drastically reduce the prevalence of vision loss associated with gca ( giant cell arteritis)/temporal arteritis . additional life - threatening conditions like mi , aneurysm of the aorta , infarction of the intestine , renal insufficiency , pulmonary embolism , transient ischemic attacks and stroke may be related to temporal arteritis / gca ( giant cell arteritis ) . to avoid further complications , early diagnosis and appropriate management are essential . temporal arteritis has a wide spectrum of clinical manifestation related to both systemic inflammation and ischemia . the most prevalent signs and symptoms include temporal headache , jaw claudication , pmr ( polymyalgia rheumatica ) , scalp tenderness , and constitutional syndrome ( asthenia , anorexia , with appetite loss ) . orofacial manifestations of temporal arteritis include trismus , throat pain during chewing , changes in tongue sensation , tongue claudication , odontongenic pain , dysphagia , dysarthria , submandibular mass , lip and chin numbness , macroglossia , glossitis , lip and tongue necrosis , and facial swelling . the present article discusses a rare case of temporal arteritis , rheumatoid arthritis with extra cranial manifestation ( interstitial lung disease ) presenting as pulo - periodontol pathology . a 75-year - old male patient came to department of periodontics , army college of dental sciences with pain in the upper right quadrant since 2 weeks . intraoral examination revealed periodontal pockets in 16 , 17 region and radiographic examination revealed a horizontal bone loss in 16 , 17 region and furcation involvement of 16 with periapical radiolucency [ figure 1 ] . endodontic treatment for 16 was decided as the first line of treatment followed by periodontal treatment . the patient gave a history of visit to ent specialist 1 week prior with same complaint but ent examination was non - diagnostic . five days later , the pain increased drastically , wherein the patient described the onset of pain as sudden and pointed at the right ear / right tmj area and right temple regions as the sites of pain . the pain was described as pressing , pulsating in quality , continuous and gradually worsening . the patient also complained of cough , breathlessness and weakness in the shoulder and legs . the patient complained initially about blurring of vision and later partial loss of vision in the right eye . the patient was referred to an ophthalmologist who suspected temporal arteritis and was sent for temporal artery biopsy . orthopantomogram showing bone loss and furcation involvement in 16 and 17 since the patient also complained about cough , breathlessness , weakness in the shoulder and legs , blood tests like cbp ( complete blood picture ) , esr ( erythrocyte sedimentation rate ) , sgpt ( alanine amino transferase ) , sgot ( aspartate amino transferase ) , serum creatinine , plasma glucose , crp ( c - reactive protein ) , and thyroid function tests were done . the tests revealed elevated esr 70 mm / hr , crp- 3.6 , sgot levels - 172 , rheumatoid factor assay-119 iu / ml , and x - ray of knee revealed osteoarthritic changes . ct of lung and pulmonary function tests with spirometry were done which showed interstitial lung disease [ figure 2 ] . diagnosis of temporal arteritis was confirmed along with rheumatoid arthritis and interstitial lung disease ( ild ) . ct scan of chest showing diffuse ground glass opacity in bilateral lung fields with few areas of sparing linear soft tissue specimen of the right temporal artery was taken for biopsy , which revealed thickening [ figure 3a ] and presence of nodular projections in the intima with discontinuous endothelium [ figure 3b ] , variable myxoid and hyaline degeneration of the thickened wall in the sub intimal region [ figure 3c ] . ( a ) h and e 4 showing cross section of temporal artery a - showing the thickened artery wall b - showing the artery lumen ( b ) h and e 10 a- nodular projections of tunica intima into the artery lumen b - inflammatory cells in tunica media ( c ) h and e 40 a- hyaline degeneration in the subintimal layer b- subintimal layer showing inflammatory cells ( d ) h and e 4 showing the longitudinal section of temporal artery a- thickening of intima , media , and adventitia b- with three layers tunica intima , media , and adventitia since the patient also complained about cough , breathlessness , weakness in the shoulder and legs , blood tests like cbp ( complete blood picture ) , esr ( erythrocyte sedimentation rate ) , sgpt ( alanine amino transferase ) , sgot ( aspartate amino transferase ) , serum creatinine , plasma glucose , crp ( c - reactive protein ) , and thyroid function tests were done . the tests revealed elevated esr 70 mm / hr , crp- 3.6 , sgot levels - 172 , rheumatoid factor assay-119 iu / ml , and x - ray of knee revealed osteoarthritic changes . ct of lung and pulmonary function tests with spirometry were done which showed interstitial lung disease [ figure 2 ] . diagnosis of temporal arteritis was confirmed along with rheumatoid arthritis and interstitial lung disease ( ild ) . ct scan of chest showing diffuse ground glass opacity in bilateral lung fields with few areas of sparing linear soft tissue specimen of the right temporal artery was taken for biopsy , which revealed thickening [ figure 3a ] and presence of nodular projections in the intima with discontinuous endothelium [ figure 3b ] , variable myxoid and hyaline degeneration of the thickened wall in the sub intimal region [ figure 3c ] . ( a ) h and e 4 showing cross section of temporal artery a - showing the thickened artery wall b - showing the artery lumen ( b ) h and e 10 a- nodular projections of tunica intima into the artery lumen b - inflammatory cells in tunica media ( c ) h and e 40 a- hyaline degeneration in the subintimal layer b- subintimal layer showing inflammatory cells ( d ) h and e 4 showing the longitudinal section of temporal artery a- thickening of intima , media , and adventitia b- with three layers tunica intima , media , and adventitia since temporal arteritis is associated with serious systemic complications , management was done by a team of specialists comprising neurologist , ophthalmologist and rheumatologist . the goals of treatment were to halt progression of vision loss in the affected eye and prevent involvement of the other eye or other regions . an initial dose of 40 - 60 mg prednisone per day was started for the patient . the initial dose of prednisone was given for 4 weeks until all reversible signs and symptoms resolved and acute phase reactants crp and esr were normalized . the decision to taper the steroid dose was based on the regular assessment of clinical symptoms , signs , esr and c - reactive protein levels . dose tapering was done each week or every 2 weeks by a maximum of 10% of the total daily dose . the daily dose of prednisone was reduced by 5 mg every 2 - 4 weeks to about 25 mg / day . the range of taper was about 2.5 mg / day every 2 - 4 weeks until a dose of 10 mg / day was achieved . tapering below 10 mg complications of long - term steroid therapy include osteoporosis , osteoporosis - related fractures , avascular necrosis of the hip , diabetes mellitus , infections , gastrointestinal bleeding and cataract . to prevent complications of steroid therapy , supplements of calcium ( 500 mg / day ) and vitamin d ( 60,000 iu / week ) were given . to prevent gastrointestinal complications , rabeprazole 20 mg was given once daily on empty stomach . to wean the patient from steroid therapy , methotrexate ( 2.5 - 5 mg / day ) and aspirin ( 81 mg / day ) were given . patients with temporal arteritis may seek dental consultation because the first , and sometimes the only symptom is pain in the jaw or ear when chewing ( jaw claudication ) . it can easily be mistaken for tmj dysfunction , myofascial pain syndrome ( mfps ) , acute alveolar abscess of the molar teeth and sinusitis and in the present case as endo - perio lesion . the pain described by the patient should be differentiated from odontongenic pain to avoid unnecessary dental treatment . guttenberg et al . described a report where inappropriate , ineffective endodontic surgery was performed in temporal arteritis case , which mimicked dental pain . the pain in temporal arteritis is localized to the anatomic area of the artery and radiated down to the ear and teeth , which is usually unilateral and will frequently cause scalp tenderness in the ipsilateral occipital area . hence , common signs and symptoms of temporal arteritis should be differentiated from headache , ent pathology , and dental pathology . in 40 to 50% of cases , polymyalgia rheumatica is a common inflammatory syndrome in elderly patient and has a diverse clinical profile that often overlaps with other rheumatic and inflammatory conditions and hence proper diagnosis should be done based on diagnostic criteria . criteria for diagnosis of polymyalgia rheumatica includes age > 65 years , esr over 40 mm / hr , bilateral upper arm tenderness , morning stiffness of more than 1 hour , onset of illness less than 2 weeks , and depression and/or weight loss . over one third of patients with temporal arteritis have polymyalgia rheumatica at presentation . some patients may have both polymyalgia rheumatica and temporal arteritis simultaneously ; others evolve from one condition to the other . the patient was diagnosed initially with polymyalgia rheumatica but since rheumatoid factor assay was found to be 119 iu / ml , later the diagnosis was changed to rheumatoid arthritis . in 1990 , the american college of rheumatology ( acr ) developed a set of criteria that have been used to diagnose gca / temporal arteritis . the guidelines given by american college of rheumatology in 1990 are useful to diagnose temporal arteritis even today [ table 1 ] . the presence of any three of the five criteria given by american college of rheumatology permitted a diagnosis of temporal arteritis / giant cell arteritis with a sensitivity of 93.5% and a specificity of 91.2% based on a population of patients ( n = 807 ) with rheumatological disease . classification criteria for giant cell arteritis a case of temporal arteritis must be suspected in patients over the age of 50 years , presenting with pain of sudden onset . there are no specific clinical symptoms or novel laboratory markers for the definite diagnosis of temporal arteritis / giant cell arteritis ; however , the combination of clinic , laboratory tests , and histopathology comprise the basis for diagnosis . several studies have found that the sensitivity of crp alone is about 98% or higher for active temporal arteritis . but , it is also important to remember that a negative biopsy result may be found in up to 10 to 15% of all diagnosed cases . infiltration by mononuclear cells and concentration of multinucleated giant cells is around the inner half of the media , characteristically along the disrupted internal elastic lamina . it is important to remember that giant cells are present in about 50% of biopsy specimens and thus are not a necessary feature for histopathological confirmation of giant cell arteritis . apart from the routine findings in a temporal arteritis case , and investigations showed elevated esr and crp levels . there is an emerging evidence that il-6 is a more sensitive indicator of disease activity than esr or crp ; however , this test is not routinely available from most laboratories . the less invasive investigative techniques which show promise for the diagnosis and monitoring of temporal arteritis / giant cell arteritis ( particularly large vessel disease ) are ultrasonography , pet ( positron emission tomography ) , and mri ( magnetic resonance imaging ) . however , none of the above mentioned investigations is as valuable as temporal artery biopsy ( tab ) . after diagnosis , prompt treatment was planned with a team comprising of ophthalmologist , neurologist , and rheumatologist . the present case is a complicated giant cell arteritis ( evolving visual loss ) , which required high dose of prednisone of 40 - 60 mg , calcium and vit d supplements . the initial dose of prednisone was given for 4 weeks until all reversible signs and symptoms have resolved and acute phase reactants crp , esr are normalized . monitoring of the treatment was done by scheduled visits at 0 , 1 , 3 , 6 weeks and then at months - 3 , 6 , 9 , 12 . at each visit , full blood count , esr / crp was checked . because of long - term effects of steroid therapy which include diabetes , hypertension , secondary infections , and steroid related osteoporosis , prophylactic measures have to taken before starting the treatment . periodic monitoring has to be done to check for initiation of steroid - related complications . to control steroid - related complications , steroid sparing agents have been studied in the treatment of temporal arteritis like azathioprine , methotrexate , infliximab , and low - dose aspirin . this case also presented interstitial lung disease , which is a rare extra cranial manifestation of temporal arteritis . most patients with ild ( interstitial lung disease ) present with nonspecific respiratory complaint such as cough or dyspnea . the investigations which help in diagnosis are chest x - ray , pulmonary function test and high - resolution computed tomography . temporal arteritis should be considered in differential diagnosis of pain of odontogenic origin , especially in an elderly patient with complaints of toothache , jaw claudication and throat pain during chewing . therefore , a thorough case history that takes into account of patients age , all pain characteristics especially when pain radiates to the temple region , is mandatory for a correct differential diagnosis so as to prevent permanent complications like vision loss . since temporal arteritis is mostly associated with polymyalgia rheumatica , which is easily confused with other rheumatological conditions and also has extracranial manifestations , proper understanding of different medical conditions by the dental professional is paramount so as to prevent unnecessary dental treatment .

a 75-year - old male patient was planned for dental treatment due to pain of suspected pulpo - periodontal origin in relation to right maxillary first molar . careful evaluation revealed the pain to be non - odontogenic in nature and led to the diagnosis of temporal arteritis with rheumatoid arthritis along with interstitial lung disease ( ild ) . characteristic findings of temporal arteritis include headache , jaw claudication , visual loss , and constitutional symptoms ( malaise , fever , weight loss , loss of appetite ) . temporal artery biopsy ( tab ) remains the gold standard for diagnosis . additional diagnostic tests include blood tests ( esr , crp ) . this article reports and discusses how the orofacial manifestations can lead to misdiagnosis of temporal arteritis . hence , temporal arteritis should be included in the differential diagnosis of orofacial pain in the elderly especially to prevent complications like vision loss .

INTRODUCTION CASE REPORT Investigations Histopathology TREATMENT DISCUSSION

microbial keratitis is an infective process of the cornea with a potentially sight - threatening condition and serious visual impairments ( 1 ) . untreated or severe keratitis may lead to significant public health problems like perforation of cornea and endophthalmitis ( 2 , 3 ) . contact lenses are a major cause of microbial keratitis in the developed countries especially among young people with about 12% hospitalized cases requiring corneal transplantation ( 4 , 5 ) . contact lenses may reduce the epithelial barrier function by interfering with normal epithelial cell proliferation and differentiation . with the growth of soft contact lens wear , the incidence of this problem raised globally ( 3 , 6 ) . there are several differences in the incidence of contact lens keratitis ( clk ) across the world ( 7 ) . pseudomonas aeruginosa can adhere to the surface of the contact lens and colonize during wear and survive in contact lens storage cases . these processes maybe lead to corneal ulcers ; a severe infection can cause permanent blindness ( 8) . although corneal ulcer is a rare complication of contact lens wear , increasing in the number of lens wearers and the risk of blindness , provide important reasons to evaluate patients with contact lens induced corneal ulcers ( 9 , 10 ) . the number of contact lens wearers has dramatically increased in iran , particularly among young adults in recent years . therefore , the purpose of the present study was to evaluate the frequency and microbiological characteristic of clk in patients referred to the emergency department ( ed ) of teaching hospitals , babol , iran . study design and patients this is a cross - sectional study of all patients with contact lens induced corneal ulcers who were referred to the ed of teaching hospitals , babol , iran , from 2011 to 2013 . patients were excluded from the study with a previous history of anterior segment surgery , treatment for ocular surface disorders , use of any topical ocular medications , or noninfectious corneal ulcers include autoimmune , neurotropic , toxic , and marginal keratitis . an ophthalmologist examined patients with the slit - lamp and clinical features of them were noted ( including pain , redness , foreign body sensation , chemosis , epiphora , blurred vision , discomfort , photophobia , discharge , ocular redness and swelling ) . all patients wore soft contact lenses , either conventional daily or disposable extended wear contact lenses . the use of disinfection solution ( hydrogen peroxide formulations ) was also asked from patients and registered . clinical characteristics of patients with contact lens microbial keratitis antibiotic sensitivity and resistance pattern of microorganism isolated from corneal ulcers in patients with microbial keratitis sampling , culture , and susceptibility tests after a detailed ocular examination , all suspected infectious corneal ulcers were scraped for microbial culture and susceptibility studies initiated before treatment . after instillation of 0.5% proparacaine hydrochloride , two slides were prepared by an ophthalmologist for the direct microscopic examination using flame - sterilized kimura spatula or a sterile 21 gauge needle , from the leading edge and the bed of the ulcer . the obtained materials were spread onto labeled slides for gram and giemsa stains and also inoculated using cotton swab applicators onto the surfaces of blood agar , chocolate agar , and sabouraud agar plates ( for fungal infection assay ) . if bacterial growth was observed in the media , the culture considered as positive and the resistance to antibiotics determined by the mueller - hinton media , too . results were interpreted according to the guidelines of the national committee on clinical laboratory standards ( nccls ) ( 11 ) . any reduction in vision was tested using snellen letter charts and measured in comparison with the unaffected eye . however , when both eyes were affected , the amount of vision loss was calculated using the worse eye and a standard reference of 6/6 snellen acuity . similarly , if there was amblyopia in the unaffected eye , the standard reference of 6/6 was used for analogy . visual acuity was categorized as no light perception ( nlp ) , counting fingers ( cf ) , hand motion ( hm ) , loss of two or more lines , and no loss of vision . clk is defined as a supportive corneal infiltrate and overlying epithelial defect with the recent history of contact lens use , with or without hypopyon . the ulcer size gives an estimate area of the ulcer to calculate the analysis more easily as follow : size of ulcer = length breadth / mm . duration of hospitalization was referred to the length of stay in hospital . when the corneal healing was not associated with visual loss , excellent , in patients had < 2 lines loss of visual acuity as good ; and for loss 2 lines or patients underwent penetrating keratoplasty as poor . quantitative data were expressed as mean standard deviation and qualitative ones as frequency and percentage . 14 patients ( 17 eyes ) were recruited into the study who all of them were female . the patients age ranged from 16 - 37 years old ( with mean standard deviation 21.587.23 ) . the mean lag time between the onset of symptoms and the first time patients referred to the ed was 48 hours . the mean treatment duration was 297 days in outpatient and 9621 days in inpatient cases . keratitis involved the right eye in seven cases ( 41.2% ) and the left eye in five ones ( 29.4% ) . all 14 patients were contact lens wearers ; there were two cases ( 14.3% ) of therapeutic contact lens and 12 ( 85.7% ) of cosmetic lens users . four of the 12 ( 75% ) cosmetic contact lens wearers were using lenses of another person in time of the infectious event . all cosmetic contact lens wearers chose and wore their lenses without any consultation with ophthalmologists , and 10 ( 71.4% ) subjects applied no disinfection regimen of contact lenses . the most prevalent observed clinical signs were pain and redness that reported in all patients . table 2 indicates the antibiogram results that 71.4% of the microorganisms were sensitive to ciprofloxacin , whereas ceftazidime , imipenem , and meropenem considered as the second most effective antibiotics . treatment outcome was excellent in 23.5% , good in 47.1% , and poor in 29.4% of cases . the most prevalent observed clinical signs were pain and redness and the most common causative microorganism of clk was pseudomonas aeroginosa . treatment outcome was excellent in 23.5% , good in 47.1% , and poor in 29.4% of cases . all of patients were using soft contact lenses from three days to 20 years ( 12 ) . in a study by malaysian national eye database study group , 202 patients were notified to the contact - lens related microbial keratitis registry with a mean age of 26.7 years ( 71.8% female ) , during 2007 - 2008 , while all of them wore soft contact lens ( 13 ) . evaluation of 56 ulcerative keratitis subjects associated with contact lens wearing showed that contact lens - associated ulcers were seen in 86% of those wearing soft lenses ( 14 ) . conducted a descriptive study of 51 patients presented to the ophthalmology hospital in casablanca with contact lens - related microbial keratitis ; the mean age of patients was 22 years , with a sex ratio of 7.5 female to male ( 15 ) . in the present study all patients contacted with lens wearers and there were 2 and 12 cases of therapeutic contact lens and cosmetic lens users , respectively . the overnight and extended wear of contact lens is known as a risk factor for microbial keratitis ( 16 ) . the microorganisms found from the corneal scrapes were not part of the ocular flora and their presence demonstrated that the source of contamination was external ( 17 ) . it has been shown that some microorganisms including potentially pathogenic species can survive for hours on contact lenses and have harmful effects on the ocular surface ( 8) . here , it was found that pseudomonas is the dominant causative agent in contact lens- related microbial keratitis , accounting for more than 75% of the culture - positive specimens . previous studies revealed that pseudomonas aeruginosa is the most common microorganism in contact lens - related microbial keratitis , followed by gram - positive bacteria , fungi , and acanthamoeba ( 12 - 15 , 18 , 19 ) . pseudomonas aeruginosa was the most frequent isolated pathogen in 60% and 79.7% of clk cases of mela et al . and goh et al . study corneal bacterial cultures were positive in 47.8% of subjects pathogens found were staphylococcus aureus , pseudomonas aeruginosa , and acanthamba ( 15 ) . pseudomonas was the most common isolate , presented in 13 ( 23% ) of the 56 cases in galantine et al study ( 14 ) . staphylococcus species were the second most common pathogen in 11 ( 20% ) of the 56 cases . findings of the present study demonstrated on the importance of referral of all contact lens wearers with suspected corneal infection to ophthalmologists for further treatment . improper lens wear and care as well as the lack of awareness about the importance of aftercare visits have been identified as risk factors for the corneal ulcer among contact lens wearers . training and increasing the awareness of adequate lens care and disinfection practices of users , consulting with an ophthalmologist , and frequent replacement of contact lens storage cases would greatly help reducing the risk of microbial keratitis . improper lens wear and care as well as the lack of awareness about the importance of aftercare visits have been identified as potential risk factors for the corneal ulcer among contact lens wearers . training and increasing the awareness of adequate lens care and disinfection practices , consulting with an ophthalmologist , and frequent replacement of contact lens storage cases would greatly help reducing the risk of microbial keratitis .

introduction : microbial keratitis is an infective process of the cornea with a potentially and serious visual impairments . contact lenses are a major cause of microbial keratitis in the developed countries especially among young people . therefore , the purpose of the present study was to evaluate the frequency and microbiological characteristic of clk in patients referred to the emergency department ( ed ) of teaching hospitals , babol , iran . methods : this is a cross - sectional study of all patients with contact lens induced corneal ulcers admitted to the teaching hospitals of babol , iran , from 2011- 2013 . an ophthalmologist examined patients with the slit - lamp and clinical features of them were noted ( including pain , redness , foreign body sensation , chemosis , epiphora , blurred vision , discomfort , photophobia , discharge , ocular redness and swelling ) . all suspected infectious corneal ulcers were scraped for microbial culture and two slides were prepared . data were analyzed using spss software , version 18.0 . results : a total of 14 patients ( 17 eyes ) were recruited into the study ( 100% female ) . the patients age ranged from 16 - 37 years old ( mean age 21.587.23 years ) . the most prevalent observed clinical signs were pain and redness . three samples reported as sterile . the most common isolated causative organism was pseudomonas aeroginosa ( 78.6% ) , staphylococcus aureus 14.3% , and enterobacter 7.1% , respectively . treatment outcome was excellent in 23.5% , good in 47.1% , and poor in 29.4% of cases . conclusion : improper lens wear and care as well as the lack of awareness about the importance of aftercare visits have been identified as potential risk factors for the corneal ulcer among contact lens wearers . training and increasing the awareness of adequate lens care and disinfection practices , consulting with an ophthalmologist , and frequent replacement of contact lens storage cases would greatly help reducing the risk of microbial keratitis .

Introduction: Methods: Results: Discussion: Conclusion: Conflict of interest: Funding support: Authors contributions:

monoamine oxidase is a flavoprotein located at the outer membrane of mitochondria in neuronal , glial and other cells . it catalyzes oxidative deamination of monoamine neurotransmitters such as serotonin , norepinephrine and dopamine and hence is a target enzyme for antidepressant drugs . in addition , it is also responsible for the biotransformation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into 1-methyl-4-phenylpyridinium a parkinsonian producing neurotoxin . cloning and sequencing of mao - a and mao - b has great impact in understanding their molecular properties respectively . this has established the fact that both the enzymes are separate and share many similar properties such as covalent link between fad and cysteine residue , cys 406 in mao - a and cys 397 in mao - b , through an 8-(cysteinyl)-riboflavin . in spite of these similarities , therefore , design of specific inhibitors would lead to little or no side effects which most of existing inhibitors suffer from . mao - a has a higher affinity for serotonin and norepinephrine , while ; mao - b preferentially deaminates phenylethylamine and benzylamine . selective mao - a inhibitors are used in the treatment of neurological disorders such as depression , whereas the mao - b inhibitors are useful in the treatment of parkinson s and alzheimer s disease . in the light of these facts , we ventured into developing a pharmacophore for a set of 64 coumarin analogs , with close comparisons from structure based interactions . this is an integrated commercially available software package that generates pharmacophores , commonly referred to as hypotheses . it enables the use of structure and activity data for a set of lead compounds to create a hypothesis , thus characterizing the activity of the lead set . at the heart of the software is the hypogen algorithm that allows identification of hypotheses that are common to the active molecules in the training set but at the same time not present in the inactives . structures of 64 monoamine oxidase inhibitors of the training and test set were built and energy minimized using smart minimize module from the cerius . the catalyst model treats molecular structures as templates comprising chemical functions localized in space that will bind effectively with complementary functions on the respective binding proteins . the most relevant chemical features are extracted from a small set of compounds that cover a broad range of activity . the best searching procedure was applied to select representative conformers within 20 kcal / mol from the global minimum . the conformational model of the training set was used for hypothesis ( pharmacophore ) generation within catalyst , which aims to identify the best 3-dimensional arrangement of chemical functions explaining the activity variations among the compounds in the training set . the automatic generation procedure using the hypogen algorithm in catalyst was adopted for generation of the hypotheses . in order to obtain a reliable model , which adequately describes the interaction of ligands with high predictability , the method recommends a collection of training set with biological activity covering 45 orders of magnitude for the training set . the pharmacophore / hypotheses are described by a set of functional features such as hydrophobic , hydrogen - bond donor , hydrogen - bond acceptor , and positive and negative ionizable sites distributed over a 3d space . the statistical relevance of the obtained hypotheses is assessed on the basis of their cost relative to the null hypothesis and their correlation coefficient . pharmacophore generation was carried out with the 64 monoamine oxidase - a inhibitors ( table 1 ) by setting the default parameters in the automatic generation procedure in catalyst such as function weight 0.302 , mapping coefficient 0 , resolution 10 pm ( due to smaller size of the inhibitor set considered for the study ) , and activity uncertainty 3 . as uncertainty in the catalyst paradigm indicates an activity value lying somewhere in the interval from activity divided by to activity multiplied by . hypotheses approximating the pharmacophore of the mao - a inhibitors are described as a set of aromatic hydrophobic , hydrogen - bond acceptor , hydrogen bond acceptor lipid , positively and negatively ionizable sites distributed within a 3d space . the statistical relevance of various generated hypothesis is assessed on the basis of the cost relative to the null hypothesis and the correlation coefficients . these activities are derived from the best conformation generation mode of the conformers displaying the smallest root - mean - square ( rms ) deviations when projected onto the hypothesis . each feature ( e.g. , hydrogen - bond acceptor , hydrogen - bond donor , hydrophobic , positive ionizable group , etc . ) contributes equally to estimate the activity . similarly , each chemical feature in the hypogen pharmacophore requires a match to a corresponding ligand atom to be within the same distance ( tolerance ) . thus , two parameters such as the fit score and conformational energy costs are crucial for estimation of predicted activity of the compounds . the crystal structure of monoamine oxidase a with clorgyline ( scheme 1 ) was used in the study . docking was carried out using gold , which used the genetic algorithm ( ga ) with default values . docking was carried out using gold , which uses the genetic algorithm ( ga ) . for each of the 10 independent ga runs , a maximum number of 100,000 ga operations were performed on a set of five groups with a population size of 100 individuals . operator weights for crossover , mutation , and migration were set to 95 , 95 , and 10 , respectively . default cutoff values of 2.5 ( dh - x ) for hydrogen bonds and 4.0 for van der waals were employed . hydrophobic fitting points were calculated to facilitate the correct starting orientation of the ligand for docking by placing the hydrophobic ligand atoms appropriately in the corresponding areas of the active site . when the top three solutions attained root - mean - square deviation ( rmsd ) values within 1.5 , ga docking was terminated . the crystal structure of monoamine oxidase a with clorgyline ( scheme 1 ) was used in the study . docking was carried out using gold , which used the genetic algorithm ( ga ) with default values . docking was carried out using gold , which uses the genetic algorithm ( ga ) . for each of the 10 independent ga runs , a maximum number of 100,000 ga operations were performed on a set of five groups with a population size of 100 individuals . operator weights for crossover , mutation , and migration were set to 95 , 95 , and 10 , respectively . default cutoff values of 2.5 ( dh - x ) for hydrogen bonds and 4.0 for van der waals were employed . hydrophobic fitting points were calculated to facilitate the correct starting orientation of the ligand for docking by placing the hydrophobic ligand atoms appropriately in the corresponding areas of the active site . when the top three solutions attained root - mean - square deviation ( rmsd ) values within 1.5 , ga docking was terminated . we developed a hypogen 3d - qsar pharmacophore model for monoamine oxidase - a inhibitory activity from a set of 64 inhibitors ( table i ) . these compounds display a broad range of inhibitory activity against mao - a with experimental ic50 values ranging from 2.0 10 to 1.0 10 ( table ii ) . on the basis of the structure - activity relationships within this set of compounds , pharmacophore models of mao - the best model produced by catalyst consisted of the spatial arrangement of five functional groups : two hydrogen - bond acceptors , three hydrophobic groups ( figure 1 ) . the model shows a good correlation ( r = 0.95 ) between experimental and predicted inhibitory activity of the mao - a inhibitors of the training set . the catalyst procedure resulted in the generation of 10 alternative pharmacophores describing the mao - a inhibitory activity of the training set compounds . these pharmacophore models were then evaluated by using them to estimate the inhibitory activity of the training set compounds . the correlation between the estimated and experimental values ranges between 0.95 and 0.66 , and the rms value is 1.2 . the statistical significance of the pharmacophores ( hypotheses ) falls within the recommended range of values in catalyst . the difference between the fixed and the null cost is found to be 54.4 bits , indicating the robustness of the correlation . the cost difference between the first and the tenth hypothesis is 13.294 bits , closer to the fixed costs than the null costs . all these calculated cost differences were found to be well within the recommended acceptable limits in the cost analysis of the catalyst procedure . the best pharmacophore model is characterized by two hydrogen bond acceptor functions , three hydrophobic functions ( figure 1 ) and is also statistically the most relevant model . the estimated activity values along with the experimental ic50 values for mao - a inhibition are presented in table ii . experimentally determined ic50 values versus the calculated activities demonstrate a good correlation ( r = 0.95 ) within the range of uncertainty 3 , indicating a good predictive power of the model . the most potent compound in the training set , molecule 62 maps well to the functional features of the pharmacophore with all five features of the molecule mapped well with hypotheses generated , whereas the least potent members of the series , molecule 26 , maps poorly with the pharmacophore with only four of five features are mapped missing out the second acceptor feature ( figure 2a , b ) . inspection of figure 2b clearly shows that molecule 26 fails to map the second hydrogen bond acceptor feature of the pharmacophore . thus , it appears that the second hydrogen bond acceptor feature may be specific requirement for binding to mao - a . analysis of these test set molecules suggests that bulky substitutions in the 6 and 7 positions of the coumarin ring could be responsible for this deviation in their activity . estimated activities were calculated by scoring the pharmacophore model on the test set and comparing with the experimental ic50 values . the enrichment factor ideal for a set of 125 dataset of compounds that contain 24 monoamine oxidase specific inhibitors and 101 nonspecific inhibitors is 5.35 . of these 24 specific monoamine oxidase inhibitors the pharmacophore generated has picked 17 molecules and within 100 nonspecific inhibitors dataset , model has picked six molecules . hence enrichment factor calculated over this model generated to 125 dataset is around 4 . these numbers speak of evaluation of the model and ability to pick the mao specific inhibitors in the dataset . coumarin derivatives were docked into the active site of mao - a and docking analysis was performed ( figure 3 ) . it was observed that a interaction with phe 407 seems to be crucial in its bound ligand clorgyline as well as coumarin analogs docked . clorgyline methyl group has a ch interaction with phenyl ring of tyr 407 and chn interaction with n1 of fad ( cofactor ) . this aromatic sandwich with tyr 407 is important in stabilizing substrate within active site and is also crucial for mao - a catalytic activity . pharmacophore model generated has three hydrophobic points which also confirms that aromatic interactions to be very important for stabilizing interactions . santana et al . , in their recent report on the qsar of coumarin analogs confirms that hydrophobicity along with polarizability to be important physical factor . one of the highest active molecule 64 has a strong oho interaction between oh group of tyr 197 and oxygen of no2 , two weak cho and one chn interactions with tyr 197 and fad ( phenyl ch of ligand has a cho interaction with hydroxy oxygen of tyr 197 , chn with n1 of fad and another cho between oxygen of nitro group of the ligand with ch of fad ) . this molecule has very fewer interactions and perhaps the low activity can be attributed to this lack of h - bond interactions . coumarin derivatives were docked into the active site of mao - a and docking analysis was performed ( figure 3 ) . it was observed that a interaction with phe 407 seems to be crucial in its bound ligand clorgyline as well as coumarin analogs docked . clorgyline methyl group has a ch interaction with phenyl ring of tyr 407 and chn interaction with n1 of fad ( cofactor ) . this aromatic sandwich with tyr 407 is important in stabilizing substrate within active site and is also crucial for mao - a catalytic activity . pharmacophore model generated has three hydrophobic points which also confirms that aromatic interactions to be very important for stabilizing interactions . santana et al . , in their recent report on the qsar of coumarin analogs confirms that hydrophobicity along with polarizability to be important physical factor . one of the highest active molecule 64 has a strong oho interaction between oh group of tyr 197 and oxygen of no2 , two weak cho and one chn interactions with tyr 197 and fad ( phenyl ch of ligand has a cho interaction with hydroxy oxygen of tyr 197 , chn with n1 of fad and another cho between oxygen of nitro group of the ligand with ch of fad ) . this molecule has very fewer interactions and perhaps the low activity can be attributed to this lack of h - bond interactions . this study suggests that it would be difficult for the active site with 7 width to accommodate bigger molecules , in this case molecules 32 and 34 . this study partly tries to address the inactivity of the some of the coumarin analogs using analog based methods like pharmacophore hypothesis and structure based methods like docking . a detailed study on each class of compounds would throw light on the optimization aspect in a series of analogs and structural requirements in order to have optimum binding . understanding of topology and h - bond interactions aiding molecular recognition with receptor govern the biological activity of this set of molecules . following table contains the list of compounds and citation used as a database to find out enrichment factor ( measure of validation of the model ) .

flavoprotein monoamine oxidase is located on the outer membrane of mitochondria . it catalyzes oxidative deamination of monoamine neurotransmitters such as serotonin , norepinephrine and dopamine and hence is a target enzyme for antidepressant drugs . mao ( mono amine oxidase ) has two isoforms , namely mao - a and mao - b . mao - a isoform has higher affinity for serotonin and norepinephrine , while ; mao - b preferentially deaminates phenylethylamine and benzylamine . these important properties determine the clinical importance of mao inhibitors . selective mao - a inhibitors are used in the treatment of neurological disorders such as depression . in this article we have developed a hypogen pharmacophore for a set of 64 coumarin analogs and tried to analyze the intermolecular h - bonds with receptor structure .

Introduction Computational Method Docking Results and Discussion Enrichment factor analysis Docking Conclusion Supplementary Information

it is attributed to the production of melanin , a pigment that renders color to the skin and is also an integral part of a protective barrier against ultraviolet radiations from the sun . their presence in the oral cavity , especially in the gingiva and buccal mucosa is a fairly consistent and well - documented finding , especially in the dark complexioned individuals . histologically , the melanocyte is a highly differentiated entity , with a well - endowed cellular synthetic and secretory apparatus . they are characterized by the presence of intracellular granules , also called melanosomes , which actually are a product of the cell 's golgi apparatus . it is these granules , which on maturation release melanin , an endogenous pigment into the surrounding epithelial cells through a unique cytocrine mechanism . the pigmentation is discerned clinically when melanocytes aggregate in clusters of about 1 - 3m in size . the absence of obvious pigmentation in caucasians / fair - skinned people has been associated with the presence of premelanin within the cells . melanocytes are quite rare in odontogenic tumors , but all the same , it is not unheard of . the melanotic neuroectodermal tumor of infancy was initially considered to be the only pigmented jaw tumor in existence . this was until 1964 , when the first case of odontogenic origin , that of a pigmented gingival cyst was reported by grand and marcoah . odontogenic tumors that have exhibited a pigmented variant in their ranks include : adenomatoid odontogenic tumor ( aot ) , odontoma , ameloblastic fibroodontoma , ameloblastic odontoma and calcifying odontogenic cyst . the presence of melanocytes in these tumors have not been found to relate to the location of occurrence , gender predilection or biological behavior of these lesions . but how exactly do these cells make their way into the lesion is intriguing and certainly a worthy query . thirty - seven cases of odontogenic tumors showing the presence of predominantly extracellular pigmentation have been reported till date which includes 20 calcifying cystic odontogenic tumors ( ccots ) [ figures 1 and 2 ] , four aots , three ameloblastic fibroodontomas , three odontomas [ figure 3 ] , two odontoameloblastomas , two ameloblastomas and one each of ameloblastic fibrodentionoma ( afd ) , calcifying epithelial odontogenic tumor ( ceot ) , odontogenic fibroma and ameloblastic fibroma each as summarized in [ table 1 ] . the tumors show an increased predisposition to occur in the maxilla in females ( 75% ) and the mandible in males ( 64.3% ) [ figure 4 ] . ethnicity also seems to play a role in occurrence with 44.44% males and 46.15% females presenting with the tumor , being of japanese origin [ figure 5 ] . the epithelial lining of a cystic odontogenic tumors ( ccot ) showed basal palisaded cuboidal cells and suprabasal stellate cells . small focal calcifi cations within the epithelium can also be observed ( h&e stain , 100 ) . melanin pigments are detected within the epithelial cells , in the ghost cells and also lie freely within the extracellular connective tissue . ( masson fontana stain , 400 ) photomicrograph of odontogenic epithelial cell rests stained using masson fontana , exhibiting the presence of intracellular melanin pigmentation ( masson fontana , 100 ) bar graph showing the increased tendency for pigmented tumors to occur in the maxilla in females and the mandible in males bar diagram showing racial preponderance for occurrence of pigmented odontogenic tumors cases of pigmented odontogenic tumors reported till date one of the earlier theories , pertaining to pigmented tumors of the jaw , was the retinal anlage theory , put forth by halpert and petzer in 1947 . this theory was opposed by willis in 1958 , sighting a stark lack of anatomical relativity to the lesion and the unreasonable exclusion of a very much possible tag prefixes the origin of a diverse group of cells , which include ganglion cells , parts of autonomic nervous system , chromaffin cells , melanocytes , neurilemma , odontoblasts and choroidal cells . neural crests , as described by avery , function in the primary induction and formation of the tooth anlage . moreover , formative melanocytes have been found to be associated with odontoblasts on the surface of the dental papilla and have also been consistently isolated from the dental primordium . the movement of the neural crest cells has been illustrated most elegantly by thorogood ( 1988 ) through the fly paper model that explains their differential , yet accurate migration as an intricate balance of active movement and passive displacement . the synchronized migration of an otherwise close knit group of cells is attributed to the " push or pull " effect elicited by various molecules . this involves initial loss of intercellular adhesion , brought about by the slug proteins and the loss of n - cadherin , the glue that binds these cells together . this is followed by the action of rhob that induces changes which promote cell migration , further compounded by fibronectin , tenasin , laminin and other collagen molecules . the extent of cellular migration is curtailed by the action of eph proteins that ensure the precise localization of these cells . what the neural crest cells then differentiate into , is a product of the synchrony between molecular activity and interactions with the surrounding cells and the environment . molecules like the endothelin-3 and stem cell factor ( scf ) have been implicated in the formation and proliferation of melanocytes , respectively . though largely ambiguous , the reports of melanocytes forming part of the regular histology of odontogenic neoplasms could be associated with the following causes : firstly , the neural crest cells of different origins can intermingle at the same site , thus differentiating into their respective lineage cells on being stimulated . secondly , melanocytes are seen on the surface of the dental papilla and as the dentin laid down , the odontoblasts and melanocytes retract , but the pigment still remains in the processes that are entrapped between the newly formed dentin . this could be a probable explanation to the compelling finding of extracellular pigmentation occurring in tumors that present with " dentinoid "- like material as an integral part of their histology . thirdly , the dental lamina is a derivative of the oral ectoderm just like the neural crest cells , which could explain the strong tendency for melanocytes to aggregate around the dental lamina . all these factors compounded by the incomplete migration or aberrant differentiation of the neural crest cells offer a plausible explanation as to how melanocytes gain access to the odontogenic environment . the remnants of cells that contribute to the odontogenic apparatus , proliferate in most tumors , thus increasing the expressions of wingless neurotropin ( wnt ) signaling pathway and stem cell factor ( scf ) . these molecules in turn , are known to directly affect proliferation and differentiation of melanocytes , thus rendering a pigmented component to the tumoral histology . also , odontogenic lesions are believed to contain inactive melanocytes that may escape routine histopathological examination , but under certain conditions , they are activated to produce the pigmented variant of their respective lesions . melanocytes are quite rare in odontogenic tumors , but all the same , it is not unheard of . the melanotic neuroectodermal tumor of infancy was initially considered to be the only pigmented jaw tumor in existence . this was until 1964 , when the first case of odontogenic origin , that of a pigmented gingival cyst was reported by grand and marcoah . odontogenic tumors that have exhibited a pigmented variant in their ranks include : adenomatoid odontogenic tumor ( aot ) , odontoma , ameloblastic fibroodontoma , ameloblastic odontoma and calcifying odontogenic cyst . the presence of melanocytes in these tumors have not been found to relate to the location of occurrence , gender predilection or biological behavior of these lesions . but how exactly do these cells make their way into the lesion is intriguing and certainly a worthy query . thirty - seven cases of odontogenic tumors showing the presence of predominantly extracellular pigmentation have been reported till date which includes 20 calcifying cystic odontogenic tumors ( ccots ) [ figures 1 and 2 ] , four aots , three ameloblastic fibroodontomas , three odontomas [ figure 3 ] , two odontoameloblastomas , two ameloblastomas and one each of ameloblastic fibrodentionoma ( afd ) , calcifying epithelial odontogenic tumor ( ceot ) , odontogenic fibroma and ameloblastic fibroma each as summarized in [ table 1 ] . the tumors show an increased predisposition to occur in the maxilla in females ( 75% ) and the mandible in males ( 64.3% ) [ figure 4 ] . ethnicity also seems to play a role in occurrence with 44.44% males and 46.15% females presenting with the tumor , being of japanese origin [ figure 5 ] . the epithelial lining of a cystic odontogenic tumors ( ccot ) showed basal palisaded cuboidal cells and suprabasal stellate cells . small focal calcifi cations within the epithelium can also be observed ( h&e stain , 100 ) . melanin pigments are detected within the epithelial cells , in the ghost cells and also lie freely within the extracellular connective tissue . ( masson fontana stain , 400 ) photomicrograph of odontogenic epithelial cell rests stained using masson fontana , exhibiting the presence of intracellular melanin pigmentation ( masson fontana , 100 ) bar graph showing the increased tendency for pigmented tumors to occur in the maxilla in females and the mandible in males bar diagram showing racial preponderance for occurrence of pigmented odontogenic tumors cases of pigmented odontogenic tumors reported till date one of the earlier theories , pertaining to pigmented tumors of the jaw , was the retinal anlage theory , put forth by halpert and petzer in 1947 . this theory was opposed by willis in 1958 , sighting a stark lack of anatomical relativity to the lesion and the unreasonable exclusion of a very much possible tag prefixes the origin of a diverse group of cells , which include ganglion cells , parts of autonomic nervous system , chromaffin cells , melanocytes , neurilemma , odontoblasts and choroidal cells . neural crests , as described by avery , function in the primary induction and formation of the tooth anlage . moreover , formative melanocytes have been found to be associated with odontoblasts on the surface of the dental papilla and have also been consistently isolated from the dental primordium . the movement of the neural crest cells has been illustrated most elegantly by thorogood ( 1988 ) through the fly paper model that explains their differential , yet accurate migration as an intricate balance of active movement and passive displacement . the synchronized migration of an otherwise close knit group of cells is attributed to the " push or pull " effect elicited by various molecules . this involves initial loss of intercellular adhesion , brought about by the slug proteins and the loss of n - cadherin , the glue that binds these cells together . this is followed by the action of rhob that induces changes which promote cell migration , further compounded by fibronectin , tenasin , laminin and other collagen molecules . the extent of cellular migration is curtailed by the action of eph proteins that ensure the precise localization of these cells . what the neural crest cells then differentiate into , is a product of the synchrony between molecular activity and interactions with the surrounding cells and the environment . molecules like the endothelin-3 and stem cell factor ( scf ) have been implicated in the formation and proliferation of melanocytes , respectively . though largely ambiguous , the reports of melanocytes forming part of the regular histology of odontogenic neoplasms could be associated with the following causes : firstly , the neural crest cells of different origins can intermingle at the same site , thus differentiating into their respective lineage cells on being stimulated . secondly , melanocytes are seen on the surface of the dental papilla and as the dentin laid down , the odontoblasts and melanocytes retract , but the pigment still remains in the processes that are entrapped between the newly formed dentin . this could be a probable explanation to the compelling finding of extracellular pigmentation occurring in tumors that present with " dentinoid "- like material as an integral part of their histology . thirdly , the dental lamina is a derivative of the oral ectoderm just like the neural crest cells , which could explain the strong tendency for melanocytes to aggregate around the dental lamina . all these factors compounded by the incomplete migration or aberrant differentiation of the neural crest cells offer a plausible explanation as to how melanocytes gain access to the odontogenic environment . the remnants of cells that contribute to the odontogenic apparatus , proliferate in most tumors , thus increasing the expressions of wingless neurotropin ( wnt ) signaling pathway and stem cell factor ( scf ) . these molecules in turn , are known to directly affect proliferation and differentiation of melanocytes , thus rendering a pigmented component to the tumoral histology . also , odontogenic lesions are believed to contain inactive melanocytes that may escape routine histopathological examination , but under certain conditions , they are activated to produce the pigmented variant of their respective lesions . are rare in odontogenic tumors , and questions pertinent to their appearance and diagnostic relevance remain largely unanswered . astute observations of the histology , the presence of melanocytes and biological behavior of pigmented odontogenic lesions need to be made and documented , to facilitate an enhanced understanding of this feature and its possible prognostic implications if any .

melanocytes are neural crest derivatives that exhibit a ubiquitous presence in the epidermis . they determine the complexion of an individual and most importantly , provide a barrier against ultraviolet radiations from the sun . their presence in the oral cavity is a consistent finding , especially in the gingiva and buccal mucosa of the dark complexioned . melanocytes occasionally form a part of the histology of a variety of odontogenic cysts and tumors . how these cells make their way into the lesional tissue and the diagnostic relevance of their presence remains elusive . this write up attempts to trace the path melanocytes take to find themselves within odontogenic tumors and also offer possible explanations for the same .

INTRODUCTION Melanocytes and odontogenic tumors The fly paper model-Tracing footstep What causes melanocytes to be seen in odontogenic tumors CONCLUSION

open radical cystectomy ( orc ) is currently regarded as the gold standard for the management of muscle - invasive bladder cancer , extensive uncontrollable non - muscle - invasive cancer , and refractory carcinoma in situ ( cis ) . although recent development and refinement of surgical technique has improved postoperative patients ' quality of life and has reduced complications , orc is still associated with significant morbidity . laparoscopic radical cystectomy ( lrc ) is one of the minimally invasive options , and some reports have shown that oncological outcomes were equivalent to orc [ 3 - 5 ] . however , lrc does not yet have widespread use because of the technical difficulty of the procedure and long operation time . with the recent development of the da vinci robot system , robot - associated laparoscopic surgery has been applied to radical cystectomy . surgeons have begun to report small series of robot - assisted laparoscopic radical cystectomy ( rlrc ) [ 6 - 8 ] . these reports have demonstrated the surgical effectiveness of rlrc with advantages in blood loss , return of bowel function , and even hospital discharge . however , a large case of prospective randomized trials has not been reported . in this study , we present the short - term clinical and oncologic outcomes of our 17 consecutive cases of rlrc with pelvic lymph node dissection and extracorporeal urinary diversion . between october 2008 and november 2009 , a total of 17 consecutive patients underwent rlrc using the da vinci robot system ( intuitive surgical , sunnyvale , usa ) with four robot arms by a single surgeon . the patients included 5 women and 12 men with a mean age of 63.7 years ( range , 48 - 74 years ) . according to american society of anesthesiologists ( asa ) risk classification , 10 were asa score i and 7 were asa score ii . mean body mass index ( bmi ) was 22.6 kg / m ( range , 18.3 - 26.5 kg / m ) ( table 1 ) . all patients requiring cystectomy were offered the robot - assisted operation if they met the inclusion criteria . these consisted of patients with radiologically localized disease , no history of previous major lower abdominal pelvic surgery , no previous pelvic radiation , and ability to tolerate a steep trendelenburg position at the anesthetic assessment . all patients received mechanical bowel preparation ( using an osmotic laxative ) 2 days prior to surgery and a phosphate enema 8 hours prior to surgery . the 12 mm camera port was inserted in the midline 5 cm above the upper umbilical margin and two 8 mm robotic ports were placed 8 cm away from the umbilicus , along the line from the umbilicus to the anterior spine of the iliac crest ( asic ) bilaterally . an additional 8 mm robotic port for the fourth arm was placed 8 cm directly lateral from the right - sided robotic port . a 12 mm assistant port for retraction and stapling was placed 8 cm lateral from the left - side robotic port . a further 5 mm assistant port for suction and irrigation was placed on the left side between the camera port and the left robotic arm port ( fig . standard pelvic lymphadenectomies ( both obturator , external iliac ) were performed in all patients . all patients underwent extracorporeal urinary diversions ( 13 ileal conduits and 4 orthotopic neobladders by studer method ) and 5 - 7 cm lower midline incisions were made for specimen removal and urinary diversion . in case of an ileal conduit , uretero - ileal anastomosis was performed over 6 fr double j stents by using 4 - 0 pds suture , and the distal end of the conduit was fashioned as a stoma at the right robot arm port site . all orthotopic neobladder was performed by using the studer method and ureteral stents were used and brought out anteriorly through separate stab wounds . urethro - enteric anastomosis was then performed intracorporeally after redocking the robotic system ( fig . a jackson - pratt drain was placed in the pelvic cavity and around the uretero - enteric anastomosis site , respectively . the nasogastric tube was removed 4 days after surgery and oral liquids were started as tolerated . patients were reviewed at 4 weeks and checked by a renal ultrasound at 2 weeks after stent removal , by computed tomography scans at 3 and 6 months postoperatively , and then at 6-month intervals . at these visits , they had a clinical examination , assessment of hemoglobin , electrolytes , creatinine , chloride , bicarbonate , and urethral washing cytology . the mean total operative time was 379.1 minutes ( range , 330 - 460 minutes ) , including 32.6 minutes for pelvic lymph node dissection , 185.2 minutes for rlrc , and 159.4 minutes for urinary diversion . all patients underwent extracorporeal urinary diversions ( 13 ileal conduits and 4 orthotopic neobladders by the studer method ) , and there were no patients who underwent urethrectomy . the mean operative time for the ileal conduit ( ic ) group was significantly shorter than that for the orthotopic neobladder ( on ) group ( 371.0 minutes vs. 442.5 minutes , p=0.010 ) . the mean estimated blood loss was 215.3 ml ( range , 120 - 400 ml ) for the ic group and 195.0 ml ( range , 180 - 200 ml ) for the on group ( p=0.871 ) . the time to oral intake and time to ambulation were 5.0 days ( range , 4 - 8 days ) and 1.3 days ( range , 1 - 3 days ) , respectively . mean hospital stay was 20.7 days ( range , 11 - 41 days ) , including 18.2 days ( range , 11 - 41 days ) for the ic group and 27.5 days ( range , 17 - 40 days ) for the on group ( p=0.245 ) . the time to oral intake ( 5.1 vs. 4.5 days ) and time to ambulation ( 1.4 vs. 1.0 day ) were similar between the groups ( p>0.05 ) . perioperative complications occurred in 4 patients ( 23.5% ) , including 1 ileus , 1 acute renal failure , 1 acute pyelonephritis , and 1 urinary leakage . there were no significant differences in perioperative outcomes between the male and female groups ( table 3 ) . the pathologic reports showed urothelial cell carcinomas in all cases , and the pathologic stages were 1 tisn0m0 , 5 t1n0m0 , 1 t1n1m0 , 3 t2n0m0 , 5 t3n0m0 , and 2 t4n0m0 . the average number of retrieved lymph nodes was 5.9 ( range , 0 - 18 ) . one patient had lymph node metastasis and 1 of 3 nodes was involved by the tumor . the mean follow - up period for rlrc was 8.3 months ( range , 3 - 19 months ) , and there was no local recurrence or distant metastasis during the follow - up period . rlrc is in evolution and is a procedure that can combine the minimally invasive advantages of lrc with the technical advantages of robotics . apart from superior visualization and articulated instrumentation , the improved ergonomic position offered at the robotic console benefits the surgeon during prolonged procedures . in 2003 , menon et al first reported the feasibility of rlrc using the da vinci surgical system . the operative time ranged from 260 to 308 minutes depending on whether an ileal conduit or orthotopic neobladder was performed . blood loss was less than 150 ml and surgical margins were clear in all cases . guru et al reported their experience with rlrc and extracorporeal urinary diversion in 20 patients . the mean total operative time was 442 minutes , including 133 minutes for urinary diversion . the mean hospital stay was 10 days and the time to return to strenuous activity was 10 weeks . rhee et al compared 23 orc with 7 rlrc cases and found that although blood loss was lower for rlrc , 4 of 7 patients needed transfusion . the operative time was 638 minutes for rlrc compared with 507 minutes for orc and hospital stay was 11 and 13 days , respectively . pruthi and wallen also compared 20 men undergoing rlrc and extracorporeal urinary diversion with 24 matched men who underwent orc . the mean operative time for rlrc was 6.1 hours as opposed to 3.8 hours for orc . mean time to flatus and bowel movement was significantly shorter than in men undergoing orc . there were six postoperative complications ( 30% ) in five patients , including one rectal injury ( repaired intraoperatively ) , one postoperative hemorrhage requiring laparotomy , and one parastomal hernia requiring repair . in our initial experiences , the mean total operative time was 379.1 minutes ( range , 330 - 460 minutes ) , including 32.6 minutes for pelvic lymph node dissection , 185.2 minutes for rlrc , and 159.4 minutes for urinary diversion . these results were relative longer than that of orc , which was reported in another study . however , mean estimated blood loss was acceptable ( 210.5 ml ) and diet was usually resumed on postoperative day 4 or 5 . these results might be due to lower intraoperative bleeding and minimal bowel exposure and manipulation . although rlrc with complete intracorporeal urinary diversion has been reported , long operating times have prevented widespread adoption of this approach , and most units prefer an extracorporeal approach for urinary diversion . we also performed extracorporeal urinary diversion in all cases to reduce operative time , but in case of orthotopic neobladder , urethro - ileal anastomosis was performed intracorporeally using a robotic system for the surgeon 's convenience . the obvious goal of rlrc must be to maintain the oncologic standards of orc while reducing the associated morbidity . it is not clear whether pneumoperitoneum has an effect on the spread or recurrence of urothelial cell carcinoma in the medium to long term . it is interesting to note a report of port - site metastasis following rlrc for a pt3bg3 urothelial cell carcinoma of the bladder . it is hoped that this outcome will be a very rare phenomenon , which it appears to be with other types of laparoscopic surgery . although several studies have reported encouraging short - term oncologic outcomes of rlrc and our perioperative outcomes of rlrc were comparable to orc , the ultimate oncologic safety of rlrc is not yet clear . to adequately evaluate and validate this procedure , a randomized prospective trial is needed to compare orc , lrc , and rlrc in terms of operative , oncological , and functional outcomes . moreover , the feasibility of performing rlrc can not be separated from the costs of developing new surgical methods in the current economic environment . therefore , the cost / benefit ratio will require continuous review . although rlrc still has concerns about long operative time and high cost , and long - term follow - up is needed to establish oncologic equivalence with orc , robot - assisted surgery may provide benefits to patients including minimal blood loss , rapid recovery , and smaller surgical wounds . our initial clinical experience indicates that rlrc is a safe and feasible procedure that may be an alternative to the open technique .

purposerobot - assisted laparoscopic radical cystectomy ( rlrc ) is a new option for the treatment of muscle - invasive bladder cancer , and case series for rlrc have been increasing recently . we report our operative technique and initial experiences with rlrc with extracorporeal urinary diversion.materials and methodsbetween october 2008 and november 2009 , 17 consecutive patients with muscle - invasive bladder cancer underwent rlrc , pelvic lymph node dissection , and extracorporeal urinary diversion . urinary diversion included 13 ileal conduits and 4 orthotopic neobladders ( studer method ) . data were collected prospectively on patient demographics , intraoperative parameters , pathologic staging , and postoperative outcomes.resultsthe mean patient age was 63.7 years . the mean body mass index was 22.6 kg / m2 . no patients had a history of previous abdominal surgery . the mean operative time was 379.1 minutes , including 32.6 minutes for pelvic lymph node dissection , 185.2 minutes for rlrc , and 159.4 minutes for urinary diversion . the mean estimated blood loss was 210.5 ml . the mean hospital stay was 20.7 days and the mean time to oral intake and ambulation was 5.0 and 1.3 days , respectively . there were no major perioperative complications . the pathologic reports showed urothelial cell carcinomas in all cases.conclusionsour initial clinical experiences indicate that rlrc with pelvic lymph node dissection and extracorporeal urinary diversion is a safe and feasible procedure with minimal blood loss and rapid recovery . long - term follow up in a larger patient population is needed to determine the true oncological and functional benefit of this procedure .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

a 57-year - old woman visited the thoracic surgery department of the samsung medical center , seoul , for further evaluation of an abnormal finding on lung examination that had been incidentally detected by routine medical check - up . she denied any past medical history , and routine physical exams and laboratory tests were within normal limits . chest computed tomography ( ct ) identified a single 4-mm - sized vascular dilation in the anterior segment of left upper lobe ( fig . there was no abnormality in the rest of lung parenchyma . under the impression of pavf cut section of the specimen revealed a 0.5-cm - sized cystic lesion under the pleura . microscopically , the lesion consisted of irregularly shaped aneurysmal dilation of thick vein ( fig . additionally , elastic stain showed hypertrophic arteriopathy of pulmonary arterioles around the main aneurysmal vein ( fig . the patient was discharged without any post - operative complication . a 54-year - old woman presented with incidental abnormal finding on chest x - ray chest ct revealed a 38-mm - sized large aneurysmal sac in the right middle lobe ( fig . cut section of the specimen showed a thin - walled cyst with smooth whitish inner surface , measuring 38 mm in diameter ( fig . elastic stain revealed the lesion to be consisted of aneurysmal dilation of both artery and vein ( fig . he has had symptoms of cyanosis and clubbing of fingers since he was 4 years old and diagnosed with pavf from chest ct work up . he showed general cyanosis and complained of dizziness and general weakness , low extremities in particular . section of the specimen showed many engorged vessels with blood clots mainly in the posterobasal segment , measuring approximately 9 cm in extent ( fig . he was discharged with no specific postoperative complication . however , diffuse pavfs still remained in other lobes . he constantly suffered from general weakness and spo2 was still checked as 70% on average . a 36-year - old man was admitted to the thoracic surgery department for scheduled operation . although his symptom was relieved , post - treatment chest ct still revealed a multi - cystic lesion in the lateral side of right lung showing plump vascular supply through intercostal arteries or pulmonary arteries ( fig . it showed no segmental or lobar distribution . under the clinical impression of congenital cystic adenomatoid malformation ( ccam ) , cut section of the specimen revealed a multi - lobulated cystic lesion filled with blood , measuring 7 cm in extent ( fig . microscopic examination revealed that the cystic lining consisted of dilated thick vascular structure ( fig . the final pathologic diagnosis was given as fistulous arteriovenous malformation / hemangioma ( avmh ) . a 57-year - old woman visited the thoracic surgery department of the samsung medical center , seoul , for further evaluation of an abnormal finding on lung examination that had been incidentally detected by routine medical check - up . she denied any past medical history , and routine physical exams and laboratory tests were within normal limits . chest computed tomography ( ct ) identified a single 4-mm - sized vascular dilation in the anterior segment of left upper lobe ( fig . there was no abnormality in the rest of lung parenchyma . under the impression of pavf cut section of the specimen revealed a 0.5-cm - sized cystic lesion under the pleura . microscopically , the lesion consisted of irregularly shaped aneurysmal dilation of thick vein ( fig . additionally , elastic stain showed hypertrophic arteriopathy of pulmonary arterioles around the main aneurysmal vein ( fig . a 54-year - old woman presented with incidental abnormal finding on chest x - ray . chest ct revealed a 38-mm - sized large aneurysmal sac in the right middle lobe ( fig . cut section of the specimen showed a thin - walled cyst with smooth whitish inner surface , measuring 38 mm in diameter ( fig . elastic stain revealed the lesion to be consisted of aneurysmal dilation of both artery and vein ( fig . he has had symptoms of cyanosis and clubbing of fingers since he was 4 years old and diagnosed with pavf from chest ct work up . he showed general cyanosis and complained of dizziness and general weakness , low extremities in particular . cut section of the specimen showed many engorged vessels with blood clots mainly in the posterobasal segment , measuring approximately 9 cm in extent ( fig . he constantly suffered from general weakness and spo2 was still checked as 70% on average . a 36-year - old man was admitted to the thoracic surgery department for scheduled operation . although his symptom was relieved , post - treatment chest ct still revealed a multi - cystic lesion in the lateral side of right lung showing plump vascular supply through intercostal arteries or pulmonary arteries ( fig . it showed no segmental or lobar distribution . under the clinical impression of congenital cystic adenomatoid malformation ( ccam ) , cut section of the specimen revealed a multi - lobulated cystic lesion filled with blood , measuring 7 cm in extent ( fig . microscopic examination revealed that the cystic lining consisted of dilated thick vascular structure ( fig . the final pathologic diagnosis was given as fistulous arteriovenous malformation / hemangioma ( avmh ) . pavf is described as abnormally dilated vessels that provide a right - to - left shunt between pulmonary artery and pulmonary vein and is divided into simple and complex type . the literature review showed that abnormal communications between pulmonary arteries and veins have been given various names including pavf , arteriovenous aneurysm , and arteriovenous hemangioma [ 1 - 3 ] . while pavfs have been often described as pulmonary arteriovenous malformation in several reports [ 1 - 5 ] , we suggest that pulmonary avmh should be used preferably to describe tumorous hemangioma lesion and pavf should be used preferably to describe abnormal vascular communication . pavfs are divided into simple and complex type , and the former is more common . simple pavfs are those of a well - defined peripheral nodule and may be rounded or multi - lobulated . histologically , simple type presents as a single aneurysmal sac and contains a single segmental artery feeding the malformation . complex pavfs involve multiple abnormal vessels and consist of one or more lobulated venous sacs of variable size supplied by more than one feeding artery , often arising from adjacent segmental pulmonary artery branches . two patients ( cases 1 and 2 ) presented with solitary aneurysmal vascular lesion , which corresponded to simple pavf . these incidental lesions did not cause any clinical symptom and surgical removal was successfully performed . on the other hand , case 3 presented with diffuse vascular lesion causing arteriovenous shunt and was consistent with the diagnosis of complex pavf . lastly , case 4 showed a localized multi - lobular vascular lesion grossly mimicking pulmonary sequestration or ccam . the histology of case 4 was unusual and also different from cases 1 , 2 , and 3 in that it had no definite aneurysmal sac or shunt formation . while avmh is an acquired tumor - like condition characterized by complex thick - walled vessels , it was not certain whether case 4 was a tumorous condition of avmh or not . however , we concluded that the terminology of avmh would represent the histologic characteristics of case 4 the best in that there was definite localized overgrowth of thick vessels . although it did not cause serious hemodynamic or pulmonary symptoms , his episodes of pneumonia may probably be related to this lesion . while most pavf patients are known to have hht , patients in our series did not . pavfs are not easily diagnosed routinely , due to its rarity and its unspecific findings on routine examinations . ct is generally considered the reference standard investigation for diagnosing pavfs . in terms of treatment , the four rare cases of surgically resected pavfs described herein show various spectrums of clinical and histologic features . they can present as either localized or diffuse , and they can involve either thin or thick blood vessels .

pulmonary arteriovenous fistula ( pavf ) is abnormally dilated vessels that provide a right - to - left shunt between pulmonary artery and pulmonary vein and is clinically divided into simple and complex type . here , we report four cases of surgically resected sporadic pavfs presenting various clinical and histologic spectrums . cases 1 ( a 57-old - female ) and 2 ( a 54-old - female ) presented as incidentally identified single aneurysmal fistulas and the lesions were surgically removed without complication . on the other hand , case 3 ( an 11-old - male ) showed diffuse dilated vascular sacs involving both lungs and caused severe hemodynamic and pulmonary dysfunction . embolization and surgical resection of the main lesion failed to relieve the symptoms . case 4 ( a 36-old - male ) had a localized multiloculated cyst clinically mimicking congenital cystic adenomatoid malformation . microscopically , the lesion consisted of dilated thick vessels , consistent with the diagnosis of fistulous arteriovenous malformation / hemangioma .

CASE REPORTS Case 1 Case 2 Case 3 Case 4 DISCUSSION

in order to select dna fragments of interest to be purified , these should be resolved in agarose or acrylamide gels gel stained with ethidium bromide by electrophoresis using 0.5x tbe buffer ( tris borate , edta ) at 120 volts for 1 hour . for this , ~ 700 ng of plasmid pproex - gdmre11s ( 6000-bp ) previously digested with ndei and hindiii was used to release a 900-bp fragment ( 560 ng of plasmid and 84 ng of fragment ) . the electroeluter tank ( figure 1 ) should be filled with 0.5x tbe equally distributed in each side of the mid plateau taking care of not spill it onto the mid plateau . the selected band ( or bands ) is cut out of the gel , and placed in sample chamber as close as possible to the v channel ( one slice per well ) . running buffer should be added to each chamber ; just enough to cover the gel slice . each v channel used must be flushed with a pasteur pipette to eliminate any air bubble trapped . then 100 ul of 10 m nh4 acetate ( to facilitate visualization a small amount of bromophenol blue is added , just enough to color it ) are gently added inside the v channel . the lid should be closed gently to prevent any resuspension of the high salt cushion . when electrolution is completed , 400 ul are removed from v - channel , and placed in a microfuge tube to be precipitated with 2 volumes of cold ethanol and glycogen ( recommended to improve dna recovery ) at 4 c for 1 hour or overnight . the recovery obtained was 75% ( 63 ng recovered when 84 ng were placed on the v - channel ) . anod an cathode are indicated on each side of tank , dna contained in gel sliced ( illustrated as a black square ) will migrate towards cathode due to its negative charge . then it will be trapped in the salt cushion ( represented as an inverted black triangle ) located in the v - channel . the duration of the run will depend highly on the size of the fragments , normally for fragments up to 20 kb 50 minutes to 1 hour is enough , while for small fragments uv light monitoring every 10 minutes is required to prevent the fragment to go through the salt cushion and consequently to the anodal buffer chamber decreasing recovery . it is important to make sure that when you set your power supply at 100 volts , there is a current at least of 10 mamp . the dna band can be monitored by using a uv - hand lamp and detect when this abandons the gel slice . this procedure allows the purification of dna or rna fragments of different sizes with good recovery ( ~80% ) to be radiolabeled , digested by restriction enzymes , processed by modifying enzymes , etc .

purified dna fragments are used for different purposes in molecular biology and they can be prepared by several procedures . most of them require a previous electrophoresis of the dna fragments in order to separate the band of interest . then , this band is excised out from an agarose or acrylamide gel and purified by using either : binding and elution from glass or silica particles , deae - cellulose membranes , " crush and soak method " , electroelution or very often expensive commercial purification kits . thus , selecting a method will depend mostly of what is available in the laboratory . the electroelution procedure allows one to purify very clean dna to be used in a large number of applications ( sequencing , radiolabeling , enzymatic restriction , enzymatic modification , cloning etc ) . this procedure consists in placing dna band - containing agarose or acrylamide slices into sample wells of the electroeluter , then applying current will make the dna fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation .

Protocol Discussion Disclosures

endoscopic retrograde cholangiopancreatography ( ercp ) is a well - established modality in the management of pancreaticobiliary conditions . the procedure carries with it several well - recognised complications , as well as other much less common ones such as splenic injury , which can be associated with significant morbidity or mortality . since the first reported case of post - ercp splenic injury in 1989 , there have been a total of only 19 cases reported to date in the english , german and spanish literature [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] . herein , we report on a patient who had a peri - splenic haematoma following ercp and was successfully managed conservatively . we also review in detail all of the published cases to better understand this rare complication . a 59-year - old woman presented to the hospital with progressive right upper quadrant abdominal pain for 7 days accompanied by profound jaundice . prior to this admission , she had presented to another hospital 7 months previously with similar symptoms and was diagnosed with choledocholithiasis . she underwent an ercp with sphincterotomy and stenting of the common bile duct but did not return for a planned cholecystectomy and removal of stent . her past medical history included untreated hepatitis c without cirrhosis , intravenous drug use , alcohol abuse and a previous laparotomy for ovarian cystectomy . her regular medications included methadone 80 mg daily and escitalopram 40 mg daily . at presentation , her blood pressure was 104/70 mm hg and all other vital signs were within normal limits . her liver function tests were elevated : bilirubin 120 mol / l ( normal , < 20 mol / l ) , alkaline phosphatase 1,490 u / l ( normal , 30110 u / l ) , gamma - glutamyl transferase 850 u / l ( normal , < 38 u / l ) , alanine transaminase 196 u / l ( normal , < 34 u / l ) and aspartate aminotransferase 145 u / l ( normal , a liver ultrasound demonstrated cholelithiasis with evidence of intrahepatic and proximal extrahepatic bile duct dilatation . she also underwent liver magnetic resonance imaging with a magnetic resonance cholangiopancreatography , which revealed a large impacted stone in the neck of the gallbladder causing obstruction of the common hepatic duct , just proximal to the previously inserted stent , associated with extensive pre - stenotic , intra- and extrahepatic ductal dilatation which was in keeping with mirizzi syndrome . the stent was removed with a basket and the common bile duct was cannulated easily through the pre - existing sphincterotomy . the cholangiogram confirmed tight stenosis in the upper third of the main bile duct in keeping with magnetic resonance cholangiopancreatography findings . the procedure was technically difficult requiring a sustained long scope position to achieve biliary cannulation and stent placement . approximately 4 h after the procedure , routine nursing observations identified newly developed hypotension with a blood pressure of 83/50 mm hg and a heart rate of 70 beats / min . the patient was otherwise asymptomatic , afebrile and did not complain of any abdominal pain . physical examination did not reveal any abdominal tenderness or signs of peritonitis and there was no evidence of gastrointestinal bleeding or overt dehydration . following the administration of 500 ml of intravenous colloid ( 4% albumin ) , her systolic blood pressure stabilised at 103 mm hg which was similar to that on admission . on the next morning , routine blood tests showed a haemoglobin level of 57 g / l ) . platelet count and coagulation studies were unchanged and within normal limits . she remained haemodynamically stable and physical examination was again unremarkable , and without evidence of gastrointestinal or any other obvious source of bleeding . given that the ercp and stent exchange were uneventful and performed through a pre - existing sphincterotomy , a gastrointestinal bleeding source was felt to be unlikely and an urgent abdominal and pelvic computed tomography ( ct ) scan was organised . the ct showed a peri - splenic haematoma and moderate volume of hyperdense abdominal free fluid , consistent with a haemoperitoneum ( fig . 1 ) . considering she was haemodynamically stable , a decision was made for conservative management including a blood transfusion of 3 units of packed cells . in the next 48 h , the patient made an uneventful recovery and was subsequently discharged from hospital with a plan for an eventual elective cholecystectomy . she was followed up as an outpatient 3 weeks later ; she was clinically well with normal - range haemoglobin and bilirubin . since 1968 , when ercp was first introduced by mccune et al . , there has been significant advancement and increased utility of this procedure for diagnostic and now mainly therapeutic purposes in managing pancreaticobiliary conditions . common complications of this procedure as reported by the american society for gastrointestinal endoscopy ( asge ) in 2012 include pancreatitis ( approx . 3.5% ) , haemorrhage ( especially following sphincterotomy ; 1.3% ) , cholangitis ( 1% or less ) , cardiopulmonary complications ( 1% ) , perforation ( 0.10.6% ) and overall mortality ( 0.33% ) . splenic - related injuries have always been considered a rare but recognised complication of endoscopy , owing to the close anatomical proximity of the spleen to the stomach and colon . other reported rare complications of ercp include liver laceration and disruption of the transverse mesocolon with resultant colonic ischaemia [ 1 , 2 ] . prior to 1975 , 2 large case series comprising over 12,000 patients who underwent colonoscopy and endoscopy did not find a single case of splenic injury . as of 2014 , weaver et al . reported that there were over 60 cases of splenic injury following colonoscopy but only 13 reports of a similar injury after ercp in the literature . the first case of splenic injury following ercp was reported by trondsen et al . in 1989 . a female patient admitted with acute gallstone pancreatitis was found to have suffered a complete avulsion of the splenic capsule necessitating splenectomy approximately 15 h after ercp . since then , similar cases have been reported over the following 27 years albeit of differing patient and procedural characteristics . the types of splenic injuries that occur are limited , and the proposed mechanism of injury and predisposing factors have had little differing opinion among the authors , as will be discussed below . at the time of this report , a total of 19 cases of ercp - related splenic injury were found in the english , german , and spanish literature ( summarised in table 1 ) . these published cases included 11 female and 8 male patients with a median age of 57 years ( range , 3382 years ) . the exact mechanism(s ) of injury to the spleen during ercp remains unresolved ; however , excessive scope - related direct , traction or shear forces appear most likely causative . as the side - viewing duodenoscope is usually advanced into a loop ( long ) position in order to reach the second part of the duodenum , the scope has to be torqued to the right and withdrawn partially ( short position ) to bring the ampulla into a satisfactory position for cannulation . this manoeuvre causes initial bowing of the endoscope within the stomach , likely translating into direct forces towards surrounding organs including the spleen , followed by torsion on the greater curvature of the stomach . adjacent viscera that are anatomically attached , such as the spleen , could then be injured by traction forces , manifesting in the form of splenic capsular tears or vascular avulsion [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ] . a sustained long scope position , as in our case , there are at least 3 predisposing factors that have been postulated to lead to splenic injury during ercp ( table 2 ) . decreased movement between the spleen and adjacent structures , excess traction on the gastrosplenic ligament and direct scope - induced trauma have been hypothesised [ 3 , 13 ] . five cases involved patients with either known or newly diagnosed ( intraoperative ) chronic pancreatitis , and it has been suggested that calcification and fibrosis of the attaching ligaments preclude any compensatory mobility between the surrounding organs [ 2 , 3 , 4 , 15 , 16 ] . adhesions from prior abdominal surgery , as in our case , were also found to be associated with splenic injuries for the same reason [ 2 , 12 , 13 , 17 ] . in fact , given the intimate proximity between the spleen and tail of pancreas , acute and post - ercp pancreatitis is increasingly recognised as a risk factor for splenic parenchymal injuries . technically difficult cases due to altered anatomy or other patient factors may result in prolonged procedure time and more scope manipulation , thereby increasing the risk of excessive ligamentous tension and injury . stomach inflation itself , in the opinion of the authors of at least 2 cases , may well render the highest ( and shortest ) short gastric vessels vulnerable to avulsion injury [ 2 , 14 ] . alternatively , direct splenic trauma by the duodenoscope is a possibility , which often results in injury to the medial or hilar surface of the spleen . it is worth noting that splenic injuries have occurred even in non - technically difficult procedures , as highlighted by wu and katon and chavalitdhamrong et al . . of the 19 cases of post - ercp splenic injuries , 10 procedures were performed without difficulty , 7 were difficult , and in 2 it was unreported . in addition , although therapeutic ercp historically carries a higher morbidity and mortality than diagnostic ercp [ 1 , 21 ] , the difference was not significantly demonstrated in our review . signs and symptoms that should alert a clinician to a possible splenic injury are not specific and overlap considerably with significantly more common complications of ercp , such as pancreatitis , perforation and post - sphincterotomy bleeding . the typical presentation may include abdominal pain with or without signs of peritonitis ( often left upper quadrant and epigastric ) , hypotension and/or tachycardia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ] . fever was found in only 1 case who represented 10 days later in the setting of a previously known splenic haematoma that became secondarily infected from a skin source and turned into an abscess . it must also be emphasised that there is a considerably variable time interval between developing symptoms and index ercp . while someone who complains of acute abdominal pain minutes after the procedure would immediately raise suspicion of a related complication , delayed presentations of up to 6 days have been reported and may be overlooked as an unrelated issue . although in 5 out of 19 cases , the patient was taken directly to theatre for emergency laparotomy based on strong clinical suspicion alone , an abdominal ct scan is still considered the test of choice to investigate any suspected severe complication [ 23 , 24 ] . alternatively , abdominal ultrasound has also been used in 2 cases which revealed findings that supported the indication for surgery [ 5 , 13 ] . common pathologic findings on ct include moderate to large volume of free fluid ( haemoperitoneum ) , subcapsular or peri - splenic haematoma , and splenic laceration . often the exact diagnosis can only be ascertained intraoperatively , as in cases due to avulsion of blood vessels and splenic capsular tears . management of the patient who has likely suffered a post - ercp splenic injury does not always entail a laparotomy . in this review , indeed , management should be tailored to the patient 's clinical state and with regard to their haemodynamic stability . trauma guidelines should also be followed as per any blunt splenic trauma , as the management is similar despite differing mechanisms of injury . early involvement of the surgical team is prudent for any ercp - related complication , particularly for splenic injury . a surgical approach is usually indicated in patients who experience haemodynamic compromise despite adequate resuscitation , while those who are clinically stable may well be observed and treated conservatively by an experienced clinician . in select cases , a splenorrhaphy rather than a splenectomy may be considered at laparotomy for stable patients with an identifiable splenic injury that is repairable [ 3 , 10 ] . more recently , splenic artery embolisation has emerged as a useful adjunct to non - operative management and is increasingly used in trauma settings . the patient in our case underwent a technically difficult but successful ercp with stenting for biliary obstruction , and subsequently experienced an episode of hypotension with no abdominal pain and a drop in haemoglobin the next day without any other concerning features . ercp - related gastrointestinal bleeding appeared unlikely given that biliary cannulation and stenting was performed through a pre - existing sphincterotomy . the ct showed a peri - splenic haematoma with haemoperitoneum , which we suspect was due to splenic capsular injury , likely associated with forces from a sustained long scope position required to successfully perform ercp and stent placement . , splenic injury with splenic haematoma or haemorrhage should be recognised as a rare but clinically significant complication of ercp . clinical suspicion should be raised if the procedure has been difficult , requiring a sustained long position of the duodenoscope . symptoms may include initial asymptomatic post - procedural hypotension , left upper quadrant pain , and/or markedly reduced haemoglobin with no haematemesis or melaena . consideration should also be given for including the rare risk of splenic injury in the consent information for ercp .

splenic injury following endoscopy is a rare but potentially fatal complication . while this has been found to occur more frequently after colonoscopy , splenic injury following endoscopic retrograde cholangiopancreatography ( ercp ) remains highly uncommon since its first reported case in 1989 . indeed , there have been only 19 such cases reported in the english , german , and spanish literature collectively over the past 27 years . we report on a 59-year - old woman who developed a peri - splenic haematoma diagnosed on abdominal computed tomography the day following ercp and stenting for mirizzi syndrome . the patient was treated conservatively and made a full recovery . we reviewed all cases of post - ercp splenic injuries reported to date and discuss the published opinions on the likely mechanism of injury , predisposing factors , presenting features , investigation , and treatment options . ultimately , patient outcome relies on clinical suspicion of this rare complication following ercp .

Introduction Case Report Discussion Statement of Ethics Disclosure Statement

the outbreak was reported in july 2010 in bom jesus do tocantins county ( 52 60s , 483636w ) , par state , in the amazon region of brazil ( figure 1 , appendix ) . bom jesus do tocantins county ( black ) in par state ( gray ) , brazil , where an outbreak of bovine vaccinia occurred in 2010 . dairy cattle and workers were affected . however , the source ( index case ) of this outbreak was not identified . at the study site , 44 lactating dairy cows became sick and had painful vesicular lesions on teats , udders , and inner thighs that rapidly progressed to ulcerative lesions and scabs ( figure 2 , panels b and d ) . two animals had extensive necrosis because of secondary infections , which led to loss of teats . lesions were also observed on lips , muzzles , oral cavities , and tongues of calves ( figure 2 , panel d ) . three dairy workers became sick during the outbreak after direct contact with sick animals and had typical orthopoxvirus ( opv ) lesions on their hands , forearms , and abdomen ( figure 2 , panel a ) . pain in the lesion region , fever , and fatigue were also reported by sick patients . b and d ) typical lesions on teats and udder of a dairy cow infected by vacv at different stages , ranging from ulceration to scabs . c ) lesions on a calf s muzzle probably caused by vacv infection during suckling . we collected 4 scabs and 44 serum samples from the 44 sick animals and 3 serum samples from the 3 dairy workers . serum samples were tested by using 50% plaque reduction neutralization tests as described ( 8) . neutralizing antibodies were detected in 40 ( 90.0% ) bovine and in 3 ( 100% ) human samples , and titers ranged from 20 to 640 neutralizing units / ml . supernatants were diluted 1:100 in phosphate - buffered saline and used in a nested pcr specific for the c11r viral growth factor gene as described ( 9,10 ) . we isolated virus from 1 of the samples that was positive for viral growth factor by nested pcr . negative results for vacv by pcr and virus isolation might have been caused by loss of virus titers and dna degradation during sample transportation . after a typical poxvirus cytopathic effect was observed , virus was plaque purified and placed on vero cell monolayers for viral amplification . this new vacv isolate was named par virus ( parv ) . to investigate the relationship between parv and bav , virus gene a56r ( hemagglutinin ) was amplified and sequenced ( 11 ) . the a56r gene is traditionally used for phylogenetic analysis . in addition , parv a26l ( a - type inclusion body ) was also sequenced ( 12 ) . the pcr fragments obtained were directly sequenced in both orientations and in triplicate by using a mega - bace 1000 sequencer ) ( ge healthcare , little chalfont , uk ) . sequences were aligned with opv sequences from genbank by using clustalw ( www.ncbi.nlm.nih.gov/pmc/articles/pmc308517 ) , and alignments were manually checked with mega version 4.0 software ( arizona state university , phoenix , az , usa ) . optimal alignment of the a56r gene showed high identity among parv and several group 1 vacv - br isolates ( average identity 99.8% ) , including vacv - to ca ( gu322359 ) ( identity 99.9% ) , an amplicon obtained from blood of an amazon monkey in tocantins state , brazil , in 2002 ( figure 3 ) . parv also showed a signature deletion of 18 nt that was also present in a56r sequences of other group 1 vacv - br isolates phylogenetic trees based on orthopoxvirus nucleotide sequences of a56r ( a ) and a26l ( b ) genes of vaccinia virus ( vacv ) , par state , brazil . phylogenetic analysis showed that parv ( black dots ) clustered in the vacv - br - g1 clade and that bean58058 virus ( bav ) clustered in the vacv - br - g2 clade . trees were constructed by using the neighbor - joining method , the tamura - nei model of nucleotide substitutions , and bootstrap of 1,000 replicates in mega version 4.0 software ( arizona state university , phoenix , az , usa ) . in panel gp2v , guarani p2 virus ; murv , muriae virus ; ctgv , cantagalo virus ; carv , carangola virus ; marv , mariana virus ; arav , araatuba virus ; hspv , horsepox virus ; gp1v , guarani p1 virus ; mpxv , monkeypox virus ; pstv , passatempo ; cpxv , cowpox virus ; varv , variola virus . phylogenetic trees of the a56r ( figure 3 , panel a ) or a26l ( figure 3 , panel b ) genes were constructed by using the neighbor - joining method , 1,000 bootstrap replicates , and the tamura 3-parameter model ( mega version 4 ) . parv sequences clustered with several group 1 vacv - brs isolated during several bovine vaccinia outbreaks in brazil . in both trees , parv clustered in group 1 vacv - br , whereas bav clustered in group 2 . our results confirm circulation of a new group 1 vacv - br isolate in par state in the amazon region of brazil in the same location where bav ( group 2 vacv - br ) was isolated ( 6,7 ) . in recent years , bovine vaccinia outbreaks in southeastern brazil rapidly spread to neighboring states ( 1 ) . epidemiologic studies suggest that movement of sick humans and the animal trade are the main causes of this circulation ( 1 ) . the relevance of vacv circulation in the context of bovine vaccinia outbreaks has been discussed ( 13 ) . several isolates belong to group 1 , which is most commonly isolated from sick cattle or cow milkers ; some isolates were detected in peridomestic rodents and wild monkeys ( 8,13 ) . therefore , although our data demonstrated that parv does not cluster with bav , it is not possible to phylogenetically define which group 1 isolate specifically generated parv or caused the outbreak because of limited number of available gene sequences from vacv - br isolates . nevertheless , we believe the presence of this new isolate in par state likely resulted from virus spread from tocantins , maranho , or mato grosso , 3 neighboring states of par state , which had bovine vaccinia outbreaks in recent years , and not from reemergence of bav ( 1 ) . although group 2 vacv - br isolates , including bav , have not been detected in the amazon region of brazil in recent years , we believe that these viruses may be silently circulating or associated with bovine vaccinia outbreaks . as in other regions in brazil , vacv outbreaks are underreported in the amazon region because of its large size and the natural complexity involved in surveillance of zoonotic diseases . our results reinforce the need for studies on vacv diversity and its transmission chain , which would be useful for the amazon region in brazil .

in 2010 , vaccinia virus caused an outbreak of bovine vaccinia that affected dairy cattle and rural workers in par state , brazil . genetic analyses identified the virus as distinct from bean58058 vaccinia virus ( identified in 1960s ) and from smallpox vaccine virus strains . these findings suggest spread of autochthonous group 1 vaccinia virus in this region .

The Study Conclusions

hepatocellular carcinoma ( hcc ) is the most common primary malignancy of the liver , the sixth most common cancer ( 749,000 new cases each year ) , and the third cause of cancer - related death worldwide . in the western world , most cases of hcc develop within an established background of chronic liver disease and portal hypertension ( 70%90% of all patients ) . liver resection is only possible in selected cases due to the high incidence of morbidity and mortality in patients with cirrhosis and elevated portal pressures . liver transplantation ( lt ) has become the treatment of choice for patients with hcc and end - stage liver disease , as it has the advantage of eradicating the tumor and the premalignant cirrhotic liver . recurrence after lt ranges from 8% to 15% when a specific criterion for selection of patients is used . surgical resection and ablation therapies have been associated with much higher rates of recurrence . after milan criteria were established ( single nodule less than 5 cm or 3 nodules less than 3 cm ) , excellent results have been reported with survival in the range of 60%70% at 5 years [ 3 , 4 ] . nonetheless , shortage of organ donors is increasing the waiting time and consequently leading to 30%40% dropout per year because of tumor progression . therefore , the practice of treating hcc patients with locoregional therapies before lt , as they are waiting to be transplanted , has become standard in most centers . we reviewed the literature on the use of locoregional therapies prior to liver transplantation and analyzed patients undergoing transplantation for hcc in our institution with emphasis on bridging therapy . they encompass a broad range of modalities including radiofrequency ablation ( rfa ) , percutaneous ethanol injection ( pei ) , transarterial chemoembolization ( tace ) , liver resection , and microwave ablation [ 7 , 8 ] . the most significant problem in patients with hcc on the waiting list is the possibility of tumor progression . for this reason , most centers started to use locoregional or neoadjuvant therapies to control tumor growth in patients while waiting . although bridging therapies using ablation , tace , resection , or combination treatments have been used by different transplant centers worldwide , the real impact and indication of any type of neoadjuvant treatments are still in debate . some authors propose that patients with hcc waiting for more than 3 to 6 months should be treated [ 9 , 10 ] . various studies have suggested that treatment of hcc prior to lt in patients with a waiting time less than 6 months is not associated with an impact in patient survival or tumor recurrence and raises the question of cost effectiveness of treatment . the overall risk of dropout in patients with diagnosis of hcc waiting for liver transplantation has been reported in the range of 15% to 30% at one year . new studies have reported that a lower incidence of dropout in the range of 0% to 25% may be related to the use of neoadjuvant therapies . however , locoregional or neoadjuvant treatments prior to liver transplantation have been used to reduce tumor burden if patients are considered to be outside criteria for transplantation in a strategy called downstaging . the group from paris , france , at l'hopital paul brousse , initially recommended this strategy in 1997 . they observed higher rates of survival in tace responders than in nonresponders in an analysis of patients with more than three nodules or nodules greater than 3 cm . furthermore , several prospective studies have reported good patient survival compared to patients undergoing lt without prior intervention . the use of radiofrequency ablation ( rfa ) for the treatment of liver tumors started in the early 1990s both in europe and in the usa . radiofrequency ablation ( rfa ) is a form of locoregional therapy that utilizes a high - frequency alternating current using a probe inserted into the tumor . the radiofrequency waves are converted into thermal energy within the conducting tissue , destroying the tumor . early experiences reported high risk of seeding , making rfa not an appealing treatment in patients while waiting to be transplanted . however , in the last few years , a well - conducted cohort study demonstrated that seeding is a rare event . percutaneous ethanol injection was used for several years in asia and then in europe and the usa to treat hcc with excellent results in patients with small tumors and was usually limited to less than 3 - 4 lesions . pei was used for many years as treatment and more recently as bridging therapy in patients waiting for liver transplantation . other authors previously published that pei can produce similar amounts of tumor necrosis although it requires a significant additional number of sessions compared to rfa . for that reason , most centers in the usa use rfa as the preferred method of ablative therapy to treat or as neoadjuvant treatment in patients with hcc . demonstrated in a markov model that ablation in patients awaiting liver transplantation had a possible gain in life expectancy and cost per year of life saved . several studies [ 7 , 1519 ] validated the efficacy of rfa as the most promising therapy for bridging patients to transplantation . patients with single nodules treated with rfa showed drop - out rates ranging from 0% to 21% , while historical nontreated controls showed drop - out rates at 1 year of 30% . it has less posttreatment discomfort and simple percutaneous access , and it can be applied in patients with mild - moderate to severe liver dysfunction . ethanol injection could still be recommended in cases where radiofrequency ablation is not technically feasible such as lesions located close to large vascular structures where heat sinking effect can be observed ( around 10%15% ) . other therapies such as microwave ablation have been used in some centers , but they are still under investigation . irreversible electroporation is another ablation technique that has been used in patients with tumors abutting major vascular structures where heat sink and collateral damage must be avoided . the role of this technique as neoadjuvant treatment in hcc patients awaiting transplantation is still to be determined . first , application of chemotherapeutic agents is usually mixed with lipiodol as a vehicle into the feeding vessels of the tumor . lipiodol is an oily contrast used for lymphographic studies and is selectively retained within the tumor , raising the exposure of neoplastic cells to chemotherapy . second , the feeding artery is occluded by microparticles inducing ischemia and a prolonged exposure to the chemotherapeutic agent . hepatic artery obstruction is usually achieved by gelfoam particles , but not polyvinyl alcohol ( pva ) , starch microspheres , metallic coils , and autologous blood clots . drug - eluting beads ( deb ) are a novel system consisting of pva beads ( 500700 m ) that are specifically designed to release chemotherapy at a slow rate . survival of patients with advanced hcc not suitable for radical therapies treated with tace is improved compared with best supportive care . side effects range from the postembolization syndrome up to hepatic insufficiency , which is very rare . the main purpose of tace as a bridge to transplantation is to reach local tumor control until a donor organ becomes available . tace is the preferred single - treatment modality in downstaging protocols , especially for multifocal tumors . but combined modalities of tace , rfa , pei , and resection seem to downstage patients more effectively than tace alone . tace is the most commonly used form of neoadjuvant therapy , alone or in combination with ablation / resection , in patients listed for lt or included in a program of downstaging . the use of external beam radiation therapy in hcc treatment has been limited by the low radiation tolerance of the nontumoral cirrhotic liver . transarterial radioembolization ( tare ) instead has been recently used in the management of hcc not suitable for curative treatment with similar indications as those of tace . radioembolization consists of infusion of radioactive substances including microspheres containing yttrium-90 ( y90 ) , iodine-131-iodized oil , or similar agents into the hepatic artery . it represents an interesting alternative , as it appears to induce a more efficient decrease in tumor size , with a shorter time to response ( 4.2 versus 10.9 months ) . in addition , tare could be performed in cases of portal vein thrombosis , a relative contraindication to tace . there are no randomized control trials available to assess the real place of this treatment modality in hcc patients or as a bridge to transplantation . resection and transplantation achieve the best overall outcomes in well - selected candidates ( 5-year survival of 60%80% ) and compete as the first options in patients with early tumors . resection is the first - line treatment option for patients with solitary tumors and very well - preserved liver function , defined as normal bilirubin with either hepatic venous pressure gradient 10 mmh or platelet count 100,000 . while selected patients with cirrhosis are best treated with resection , patients with esld and portal hypertension are at increased risk of morbidity and mortality compared to noncirrhotic counterparts . for these reasons , only 20%30% of patients with cirrhosis , portal hypertension , and hcc are candidates for resection . selection of the ideal candidate requires an adequate assessment of the liver functional reserve , tumor extension , and risk of postoperative complications and mortality . several different systems have been used to try to address risk of morbidity and mortality after resection in patients with portal hypertension and cirrhosis . more sophisticated measurements such as indocyanine green retention rate at 15 min ( icg15 ) and hepatic venous pressure gradient ( hvpg ) as direct measurements of relevant portal hypertension are utilized [ 31 , 32 ] . low platelet count has been confirmed as a strong independent predictor of mortality in patients with hcc and cirrhosis [ 22 , 33 ] . resection can be used as a therapy for hcc prior to lt in different settings . initially , resection can be used as a first - line treatment for patients with small hcc and preserved liver function . also resection could help refine the selection process for lt according to detailed pathological examination of the tumor and the surrounding liver parenchyma . it could help in selection of candidates for lt in patients with tumors slightly outside the milan criteria but with histological features of good prognosis or in denying lt in patients within the milan criteria but with histological features of poor prognosis such as undetected macrovascular invasion . several groups considered surgical resection as one of the bridging treatment modalities prior to lt . other groups consider liver resection as one of the options for downstaging patients with tumors outside the transplantation criteria . in these instances , the patients could be reassessed , staged , and retreated accordingly . there are several centers in the usa and europe trying to use different types of neoadjuvant therapy to treat hcc to decrease tumor burden to fulfill usually milan criteria and then perform liver transplantation . the type of therapy that should be used and the upper limit of tumor size that should be downstaged are not clear . some authors have recommended putting the candidate on hold until downstaging by local ablation and/or chemoembolization is achieved and maintained ( disease stability ) for a period of at least three months . there is not a single randomized control trial or a large cohort study available on patients consistently treated and properly followed . various prospective small studies suggested that downstaging tumors to reach milan criteria using rfa and/or tace achieves 5-year survival rates similar to those within the conventional criteria after lt . presented a prospective study providing validation of a downstaging protocol , in which patients , initially excluded by the conventional transplant criteria , undergoing successful downstaging obtained satisfactory survival after lt . still efficacy of downstaging in patients exceeding conventional criteria is strictly related to tumor size and number at presentation . the bigger the tumor bulk , the lower the efficacy of downstaging in terms of tumor response . considering the existing information , downstaging of patients beyond milan criteria still remains controversial , and criteria for selecting candidates are not well established . chapman et al . considered most patients beyond milan criteria and did not exclude a priori any patients . they reported comparable outcomes in these 17 patients to those in patients within milan criteria . yao et al . restricted downstaging eligibility criteria to one lesion > 5 cm and up to 8 cm ; 2 to 3 tumors with at least one lesion > 3 cm but not exceeding 5 cm , with total tumor diameter up to 8 cm ; or 4 to 5 lesions with none > 3 cm with total tumor diameter less than 8 cm using these criteria as an upper limit for downstaging , 57.4% ( 35/61 ) of their patients were successfully downstaged and transplanted . prospectively analyzed patients with a single hcc 5 cm to 6 cm , 2 lesions 5 cm , or less than 6 hcc lesions 4 cm and sum diameter 12 cm , and they achieved a 67% transplantation rate ( 32/48 patients ) . in the transplant center at the university of kentucky , the strategy for management of hcc has evolved with a multimodality algorithm approach similar to that adopted in other centers . we retrospectively analyzed our experience of 96 consecutive patients undergoing transplantation for hcc from september 1999 to september 2011 . as expected , hepatitis c ( hcv ) was the most common indication in this group ( 48 patients , 50% ) , followed by alcoholic liver disease ( 40 patients , 41.7% ) . among the 96 patients who underwent lt , 31 patients were identified as having incidental hcc on the explanted livers , undetected on preoperative imaging ( table 1 ) . the mean diameter of the greatest lesion was 2.3 cm , ranging from 0.7 to 7.6 cm . intention - to - treat survival , all 96 patients were followed up from the time of listing to death or last followup . overall survival rates of 89.2% , 74.1% , and 62.1% at one , three , and five years were observed , respectively . as expected , patients with vascular invasion had significantly worse survival compared to those patients with tumors without vascular invasion ( p < 0.005 ) . rates at one , three , and five years for patients without vascular invasion were 90.9% , 77.6% , and 66.1% , respectively , while for patients with vascular invasion survival was considerably lower ; 71.6% , 61.7% , and 46.3% ( figures 1 and 2 ) . we compared patient characteristics in individuals undergoing bridging therapy with those without preoperative treatment ( table 2 ) . patients characteristics were similar in both groups other than tumor size greater than 3 cm that was more common in patients in the bt group ( p < 0.005 ) . nineteen liver transplant candidates representing 29.2% of known hcc cases were treated with locoregional therapies ( table 2 ) . of those patients undergoing neoadjuvant treatments , 10 were treated with rfa ( 15.4% ) , 8 with tace ( 12.3% ) , 1 with resection ( 1.5% ) , and 1 with rfa / tace combination ( 1.5% ) . survival and recurrence rates were similar in treated patients versus nontreated individuals ( p = ns ) . liver transplantation represents the best curative option for hcc and cirrhosis as it has the advantage of removing the tumor and treating the underlying disease . despite that it has been reported an approximate doubling time for these cancers of around 6 months . as a consequence , tumors can grow beyond conventional criteria , increasing the possibility of microvascular invasion and occult metastasis and further worsening the patient condition . recent advances in imaging techniques , especially with the use of liver - specific contrast mri , are making diagnosis of hcc and quantification of the amount of disease more accurate . gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid ( gd - eob - dtpa ) is a liver - specific magnetic resonance imaging contrast agent that has up to 50% hepatobiliary excretion in the normal liver . after intravenous injection , gd - eob - dtpa distributes into the vascular and extravascular spaces during the arterial , portal venous and late dynamic phases and then progressively into the hepatocytes and bile ducts during the hepatobiliary phase . the role of gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid - enhanced magnetic resonance imaging ( eob - mri ) in the management of hcc is still to be determined . however , most centers are now using eovist when regular dynamic ct / mri images do not show conclusive findings . selection criteria for liver transplantation in hcc patients are still controversial [ 22 , 4043 ] . although initial lt results in nonselected patients with hcc were discouraging , subsequent series proved that better results could be achieved by employing defined selection criteria . the criteria proposed by mazzaferro et al . in 1996 are used as a basic stratification tool by numerous transplantation centers worldwide . using these criteria , a patient with a single tumor measuring 5 cm or less , or three or fewer nodules each smaller than 3 cm , would be a candidate for lt . excellent 5-year survival rates in the range of 60%70% or higher have been reported when these criteria are followed . subsequently , the university of california at san francisco reported 57% survival for patients with hcc who exceeded milan criteria but were within the limits of their expanded criteria , including patients with solitary lesions less than 6.5 cm in size or up to three tumors with the largest not more than 4.5 cm and a combined tumor diameter of not more than 8 cm . the tnm criteria by marsh et al . at the university of pittsburgh are based on hcc characteristics including microvascular or macrovascular invasion , lobar distribution , tumor size , and lymph node involvement , and they seemed to provide a more clear - cut discrimination with respect to post - olt survival for each tnm tumor stage ( i to iv ) . yao et al . compared the previous three proposed criteria and supported a modest expansion of the tumor size limits of the milan criteria ( the ucsf criteria ) while still preserving acceptable survival after olt . the expanded criteria offered the benefits of olt to about 20% of our patients who would have otherwise been excluded from olt under the more restrictive milan criteria . the ucsf criteria also confer an advantage over the pittsburgh criteria , which require information on microvascular invasion that is difficult to ascertain preoperatively without the attendant risks of biopsy . candidacy is now decided based on size and number of lesions ( milan , ucsf criteria , etc . ) . however , there are other important predictors of recurrence and patient survival that have not been included in the current selection system such as afp / afpl3% levels , tumor differentiation , and microvascular invasion . lens culinaris agglutinin - reactive afp ( afp - l3 ) is an isoform of afp that is very specific for screening and diagnosis of hcc in the background of cirrhosis or hepatitis . unfortunately , there has been large heterogeneity on the results of multiple studies when assessing afp - l3% and afp in the same population . our group reported that tumor size and afp were strongly related to the presence of microvascular invasion ( mvi ) on the explanted liver . afp in conjunction with other markers such as des - gamma - carboxyprothrombin and afp - l3 has been associated with tumor burden as well . because of the relationship with tumor burden and outcomes , several centers are now using highly elevated levels of afp to rule out patients for liver transplantation . in 2012 , duvoux et al . published that levels of afp greater than 1000 u / dl are associated with high risk of recurrence and suggested that afp should be incorporated to the milan criteria in order to select patients for liver transplantation . based on unos data , the 3 most common indications for liver transplantation in the usa are hepatitis c , alcoholic liver disease , and hepatocellular carcinoma . interestingly , in 2010 , hcc is already the second most common indication for transplantation , second only to hepatitis c. a significant amount of patients with hcc have concomitant hcv infection which could compromise outcomes in this subgroup of patients . reinfection of the liver graft by hcv is constant , and the natural history of hcv recurrence is accelerated compared with nontransplanted patients when serum hcv rna remains detectable at lt . approximately 20% to 30% of patients will develop cirrhosis within 5 years after lt . the best strategy to prevent recurrence of hcv treatment before the development of injury to the graft in the early phase is currently not recommended because studies have shown that it is difficult to initiate antiviral therapy with ifn during the postoperative period and that it has a poor efficacy with remarkable side effects such as bacterial infections , hematological toxicity , and rejections , which lead to dose reduction or discontinuation of treatment . hcv therapy should be initiated in the presence of severe and rapid progression of fibrosis with a higher risk of graft loss , especially in the setting of cholestatic hepatitis . current regimens included antiviral therapy with peg - ifn / rbv , and several studies have shown that a sustained virological response is achieved in 8%45% . three different systematic reviews of peg - ifn / rbv after lt showed that the svr rate is around 30% [ 5153 ] . the duration of therapy is usually 48 weeks , and therapy is influence by several factors which influences the prognosis before and during therapy such as viral genotype , donor age , baseline viral load , il28b donor and recipient , absence of prior antiviral therapy , severity of baseline fibrosis , adherence to therapy , duration of therapy , rapid virological response , and early virological response . due to the development of new drugs for the treatment of hcv infection , most experts believe that treatment of hcv recurrence after lt will change in the next few years . the role of triple therapy using peg - ifn / rbv plus protease inhibitors is not clear . recently published the results of a multicenter study using triple therapy with telaprevir in hcv recurrence after lt reporting increased sustained viral response rates exceeding those with standard treatment with peg - ifn / rbv alone . these results must be balanced with high rates of adverse events including increased risk of readmissions , kidney dysfunction , and death . the role of these treatment combinations in the lt setting is still to be determined . the timing and adequacy of hcc treatment before liver transplantation to control the disease are unclear . new minimally invasive strategies are being implemented to decrease tumor progression in patients with diagnosis of hcc while waiting to be transplanted . the appropriate treatment alternative that should be used as neoadjuvant therapy prior to lt such as liver resection , radiofrequency ablation ( rfa ) , transarterial chemoembolization ( tace ) , or combination remains under debate . considering the strength and existing evidence , it is recommended to treat patients waiting for transplant with local ablation and/or chemoembolization when waiting time is estimated to exceed 6 months . furthermore , several cohort studies and a preliminary analysis of large registries suggest that bridging strategies with locoregional therapy are likely to be beneficial for patients waiting for 6 months or longer . the recommendation of bridging therapy is more important in unos t2 hcc patients to decrease dropout rates and achieve good posttransplant outcomes [ 55 , 56 ] . a markov - based cost - effectiveness analysis by llovet et al . pointed out the benefits of neoadjuvant therapies when waiting time exceeded 6 months . in our center , the decision to use bridging therapy is done on an individual basis by a multidisciplinary team integrated by hepatologist , radiologist , interventional radiologists , oncologist , radiation oncologists , and transplant surgeons . we usually recommend tace and/or rfa in patients with unos stage 2 hcc prior to lt if 6 months or longer waiting time is expected in rapidly growing tumors or in large lesions close to the upper size limits of the milan criteria . based on the international consensus conference held in december 2010 in zurich , switzerland , specific recommendations regarding downstaging can not be properly made due to the lack of evidence , and further research is needed . although adjuvant therapy is currently used in most centers in the usa and europe , further investigation is needed to determine the efficacy and timing of neoadjuvant treatment modalities for hcc in patients awaiting liver transplantation .

hepatocellular carcinoma ( hcc ) is the most common primary malignancy of the liver accounting for 7% of all cancers worldwide . most cases of hcc develop within an established background of chronic liver disease . for that reason , liver resection is only possible in selected patients . liver transplantation has become the treatment of choice in patients with hcc , end - stage liver disease , and significant portal hypertension . shortage of organ donors has resulted in overall increase of waiting list time with increased risk of dropout due to tumor progression . neoadjuvant therapies have emerged as an alternative to control tumor growth in patients while waiting . the aim of this study is to review the literature on the role of bridging therapy and downstaging prior to liver transplantation in patients with hcc . we are also presenting our single - center experience of 96 patients undergoing transplantation for hcc with and without bridging therapy .

1. Introduction 2. Locoregional Therapies as a Bridge to Liver Transplantation 3. Downstaging 4. Our Experience 5. Discussion

lung cancer remains the most lethal cancer , with over 160,000 annual deaths in the usa alone.1 over the past decade , the discovery of driver mutations has changed the landscape for the treatment of non - small - cell lung cancer ( nsclc).2 targeted therapies against epidermal growth factor receptor ( egfr ) or anaplastic lymphoma kinase ( alk ) have now been approved by the us food and drug administration as part of the standard first - line treatment of nsclc . despite good initial responses , most patients develop resistance within 812 months and have disease progression . hsp90 is a chaperone protein assisting other cellular proteins to fold properly , and stabilizes them against oxidative and heat stress , as well as helping with protein degradation.3 hsp90 is a weak atpase with a very rapid turnover rate ( 0.1/minute in humans ) . unlike alk and egfr nsclc that is driven by oncogenic mutations , the activity of hsp90 is regulated by the binding of cochaperone molecules that induce conformational change in the hsp90 . to date , over 20 cochaperone molecules have been identified.4 recent insights from the cancer genome atlas revealed that multiple malignancies overexpress or possess mutant kinases that depend on the hsp90chaperone complex.5 hsp90 has been shown to stabilize various signaling molecules , such as pi3k and akt proteins , thus inhibiting cellular apoptosis in cancerous cells ( figure 1).6 it also appears that hsp90 can act as a protector of unstable protein by - products of dna mutations , such as v - src and mutant forms of p53.7 relevant to nsclc , mutant egfr,8 erbb2,9 met,10 mutant braf,11 and the eml4alk translocation product12 are all hsp90-dependent proteins , the degradation of which leads to loss of tumor - cell viability in the corresponding adenocarcinoma subset . expression of hsp90 has thus been shown to correlate with a worse clinical prognosis and to be correlated with resistance to chemo- and radiotherapy.13 the first class of hsp90 inhibitors to be characterized were the benzoquinone ansamycins , including geldanamycin and its derivatives 17-allylamino-17-demethoxygeldanamycin ( 17-aag ) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin ( 17-dmag).14 they were derivatives of geldanamycin , which is an ansamycin antibiotic derived from a streptomyces sp . , and block atp binding to the active site of hsp90 . however , the clinical application of these drugs was limited by their poor pharmacokinetics and dynamics , including poor solubility , formulation problems , and potential multidrug efflux.15 as single agents , these molecules have shown only limited efficacy , thus pointing toward better results in combination therapy.16 in an effort to overcome these limitations , several second - generation synthetic hsp90 inhibitors representing multiple drug classes are currently under development . the goal of this review is to present the data supporting the use of hsp90 inhibitors in nsclc and to give an overview of the ongoing clinical trials involving new - generation hsp90 inhibitors . ganetespib , also known as sta-9090 , is a resorcinol derivative that binds the atp - binding pocket of hsp90 and leads to its inhibition . ganetespib acts by inducing g2/m cell - cycle phase arrest , resulting in apoptosis within 48 hours of treatment . it has also been shown to induce cell arrest in both erlotinib - sensitive and erlotinib - resistant cell lines , including h1975 with the t790 m gatekeeper mutation.17 compared to the older generation 17-aag , ganetespib has greater potency and potential efficacy against several nsclc subsets , including those harboring egfr or erbb2 mutations.18 treatment with ganetespib resulted in decreased downstream signaling through the pi3k akt mtor and raf mek erk pathways . in alk - rearranged nsclc cell lines , ganetespib induced loss of eml4alk expression and depletion of multiple oncogenic signaling proteins in alk - driven nsclc cells , leading to greater in vitro potency , superior antitumor efficacy , and prolonged animal survival compared with crizotinib monotherapy.19,20 ganetespib also overcame multiple forms of crizotinib resistance , including secondary alk mutations . cancer cells driven by alk amplification and oncogenic rearrangements of the ros1 and ret kinase genes were also sensitive to ganetespib exposure . hsp90 inhibition with ganetespib has also resulted in decreased viability of kras - mutated cell lines by impacting downstream signaling through the mtor and mek pathways.21 it was superior to both azd6244 , an mek inhibitor , and bez235 , a pi3k / mtor inhibitor when used as monotherapy , and also helped sensitize kras - mutated cell lines to cisplatin , pemetrexed , gemcitabine , and docetaxel . hsp90 inhibitors have been shown to exert synergistic activity when combined with taxanes ( paclitaxel and docetaxel ) , another g2/m cell - cycle arrest inhibitor . this combination has resulted in synergistic toxicity in both cell lines and animal xenograft models , resulting in more tumor regression than with taxanes alone.22 in the first published multicenter phase ii trial , ganetespib as monotherapy was assessed in previously treated patients with nsclc with specific genotypic subtypes.23 a total of 99 patients were enrolled in three cohorts : cohort a ( n=15 , mutant egfr ) , b ( n=17 , mutant kras ) , and c ( n=66 , no egfr or kras mutations ) . patients were treated with 200 mg / m ganetespib by intravenous infusion once - weekly for 3 weeks followed by 1 week of rest until disease progression . the primary end point of progression - free survival ( pfs ) at 16 weeks was achieved in 13.3% of egfr - mutated patients , 5.9% of kras - mutated patients , and 19.7% of wild - type patients ; 4% of patients achieved partial response , all harboring the alk translocation . eight ( 8.1% ) patients experienced treatment - related serious adverse events , two of which ( cardiac arrest and renal failure ) resulted in death . the most common other adverse effects were diarrhea , fatigue , nausea , and anorexia . given early success in preclinical studies as combination therapy with taxanes , ganetespib was assessed in a phase i trial in combination with docetaxel . the study , with 27 patients , defined the maximum tolerated dose ( mtd ) of ganetespib at 150 mg / m on days 1 and 15 of each 21-day cycle.24 the dose - limiting toxicities were febrile neutropenia and grade 4 neutropenia . this combination was further assessed in the phase ii galaxy-1 trial comparing docetaxel to docetaxel plus ganetespib:25,26 225 patients with advanced nsclc , one prior systemic therapy , and eastern cooperative oncology group performance status of 0/1 were included . docetaxel was given at 75 mg / m on day 1 of a 3-week cycle . in the experimental arm , docetaxel was given on day 1 and ganetespib at 150 mg / m on days 1 and 15 . patients were stratified by performance status , time since advanced disease diagnosis ( 6 months vs > 6 months ) , baseline lactate dehydrogenase ( ldh ) ( elevated or normal ) , or smoking status . the rationale for including patients with elevated ldh was the correlation with tumor hypoxia in these patients attributable to hif-1 , a client protein of hsp90 . after 72 patients were enrolled , nonadenocarcinoma patients were excluded , given the lack of efficacy and excess bleeding risk . for the combination vs the monotherapy arm , the median number of cycles delivered was five vs four , and grade 3/4 adverse events were neutropenia 38% vs 37% , fatigue 4% vs 3% , anemia 7% vs 6% , diarrhea 3% vs 0% , and fever with neutropenia 8% vs 2% . in this study , the overall survival hazard ratio ( hr ) was 0.69 ( 95% confidence interval [ ci ] 0.480.99 , p=0.093 ) , the pfs hr was 0.70 ( 95% ci 0.530.94 , p=0.012 ) , and the overall response rate ( orr ) was 15% vs 11% , all favoring the combination arm . for patients that were enrolled > 6 months after diagnosis of advanced nsclc ( n=175 , 69% ) , a prespecified stratification factor , the overall survival hr was 0.41 ( 95% ci 0.250.67 , p=0.0009 ) , the pfs hr was 0.47 ( 95% ci 0.320.69 , p=0.0005 ) , and the orr was 16% vs 12% . given positive results from this study , multiple phase iii trials are under way to assess the efficacy of ganetespib in other adenocarcinomas , including breast cancer . auy922 is a second - generation nongeldanamycin hsp90 inhibitor that is an isoxazole derivative . in preclinical studies , it has been shown to have clinical activity in lung cancer cells with met- and akl - mediated resistance.27 auy922 treatment effectively suppressed proliferation and induced cell death in both resistant cell lines by downregulating egfr , met , and axl expression , which led to decreased akt - pathway activation . auy922 has also been shown to act as a radiosensitizer to cell lines with acquired resistance to egfr inhibitors.2830 these cell - line studies have been replicated in animals with nsclc xenograft tumors with met- and axl - mediated resistance.27 a total of 101 patients were enrolled in the first - in - human dose - escalation study of auy922 in patients with solid tumors.31 the mtd was not reached ; however , because of concerns regarding visual toxicity , dose escalation beyond 70 mg / m was not attempted . dose - limiting toxicities occurred in eight patients , and included diarrhea , asthenia / fatigue , anorexia , atrial flutter , and visual symptoms . at 70 mg / m , the auy922 concentration achieved was consistent with active concentrations in a range of xenograft models , and that dose was recommended to be taken forward in phase ib and phase ii studies . all 101 patients were evaluable for a clinical response , and of these , none had a complete or partial response by adapted response evaluation criteria in solid tumors criteria . the first phase ii trial evaluated auy922 in 112 patients with nsclc , 61% of which had received at least three lines of therapy.32 patients were stratified in to four groups : egfr - mutated , kras - mutated , alk - rearranged , and wild - type nsclc . mean duration of exposure was 9 weeks . in a preliminary report presented at the american society of clinical oncology 2012 conference , partial responses were seen in 13 out of 101 ( 13% ) patients , two out of eight ( 25% ) alk - rearranged patients , six out of 33 ( 18% ) egfr - mutated patients , four out of 30 ( 13% ) wild - type patients , zero out of 26 ( 0% ) kras - mutated patients , and one out of four ( 25% ) patients of unknown status . in alk - rearranged patients , responses were seen in crizotinib - nave patients , and stable disease was seen with tumor shrinkage in crizotinib - resistant patients . the most frequent adverse events were diarrhea ( 73% ) , visual disturbances ( 71% ) , and nausea ( 43% ) . these data were updated at the 2013 american society of clinical oncology annual meeting , with a focus on egfr - mutated patients . data for 16 patients with acquired egfr tyrosine - kinase inhibitor ( tki ) resistance were presented , seven having tested positive for egfr t790 m at rebiopsy . the orr was two out of 16 ( 13% ) , and both patients with partial responses had the gatekeeper mutation . another phase ii trial looked at using auy922 in patients with acquired resistance to erlotinib;33 25 patients were evaluated , and the median time to tki resistance was 11 months . adverse events reported in 20% of patients were diarrhea , fatigue , myalgia , nausea , mucositis , and night blindness ; 68% ( 17 out of 25 ) experienced night blindness ( grade 12 only ) , and three patients came off the study due to eye - related toxicity . auy922 has also been combined with trastuzumab in patients with her2-amplified or her2-mutated nsclc;34 55 patients with her2 amplification , 34 patients with her2 overexpression , and seven patients with her2 mutation were treated . retaspimycin or ipi-504 is a water - soluble derivate of 17-aag that causes less liver toxicity and has shown activity in nsclc . in preclinical studies , great responses have been obtained with retaspimycin , especially in alk - rearranged cell lines . treatment of h3122 alk - rearranged cell lines with retaspimycin resulted in degradation of the fusion protein in <3 hours , making it a much more sensitive target than egfr or her2.12 the first multicenter phase ii study of retaspimycin enrolled 76 patients with egfr - mutated lung cancer that had progressed on tki therapy.35 the orr was a disappointing five out of 76 ( 7% ) in the overall study population , 10% in patients with egfr wild - type , and 4% in patient with egfr mutations , with a median pfs of 2.86 months . however , among three patients with alk rearrangements , two had partial responses and had prolonged stable disease of over 7 months duration . retaspimycin has also been evaluated in combination with taxanes in patients with metastatic nsclc in an expansion of a phase ib trial;36 23 patients with pathologically confirmed metastatic nsclc , all having received one to two prior chemotherapy regimens without prior docetaxel , were enrolled . docetaxel 75 mg / m iv was given once every 3 weeks , while retaspimycin 300 mg / m was administered intravenously once per week . six out of the 23 ( 26% ) patients had a partial response , including three of seven patients with squamous cell carcinoma . the most common reported adverse effects were fatigue , diarrhea , neutropenia , and anemia . it has been shown to have effect in nsclc cell lines , as well as mouse xenograft models.37 its long duration of action has enabled once - weekly dosing.38 at13387 has also shown clinical efficacy in alk - rearranged cell lines , as well as mouse xenograft models injected with those cells.39 in the first - in - human phase i dose - escalation study , at13387 was evaluated in 62 patients with advanced solid tumors . the drug was administered in both a twice - weekly and once - weekly regimen . one dose - limiting visual disturbance occurred at 120 mg / m twice weekly , thus establishing the mtd for this regimen . for the once - weekly regimen , no formal dose - limiting toxicity occurred ; however , multiple moderately severe toxicities , including diarrhea , nausea , vomiting , fatigue , and systemic infusion reactions , led to selection of 260 mg / m as the recommended phase ii dose . the hsp90 inhibitors are a diverse group of molecules with a wide range of activity in solid tumors , with ongoing trials presented in table 2 . they can also act as chemo- and radiosensitizers , as well as help circumvent acquired resistance to standard targeted therapies . the advantage of using hsp90 inhibitors is their ability to target multiple oncogenes at the same time . the galaxy-2 study ( nct01798485 ) , the first phase iii trial of hsp90 inhibitors in nsclc , is ongoing . a previous study reported single amino acid mutations in the hsp90 molecule associated with in vivo resistance to hsp90 inhibitors.40 further studies are required to identify additional predictive biomarkers of sensitivity and resistance to hsp90 inhibitors in humans .

lung cancer remains the most lethal cancer , with over 160,000 annual deaths in the usa alone . over the past decade , the discovery of driver mutations has changed the landscape for the treatment of non - small - cell lung cancer ( nsclc ) . targeted therapies against epidermal growth factor receptor ( egfr ) or anaplastic lymphoma kinase ( alk ) have now been approved by the food and drug administration as part of the standard first - line treatment of nsclc . despite good initial responses , most patients develop resistance within 812 months and have disease progression .

Introduction Ganetespib (STA-9090) AUY922 Retaspimycin (IPI-504) AT13387 Conclusion

aphasia , impaired communication ability that usually occurs in patients with left middle cerebral artery ( lmca ) stroke , is associated with high mortality , significant motor impairment , and severe limitations in social participation [ 15 ] . while 1/3 of new strokes have aphasia as one of its symptoms , the progress of aphasia rehabilitation has not matched the great strides in developing acute treatment strategies that , in some patients , decreased or eliminated the stroke - related deficits . further , the development of post - stroke aphasia treatments lags behind the development of therapies that alleviate motor deficits in patients with history of stroke such as constraint - induced motor therapy , mental practice , electrical stimulation of the affected extremity or neurostimulation . the current aphasia therapy approaches are based largely on compensatory strategies or repetitive training of lost functions rather than function restoration . while these therapies may lead to improvements in some patients , many continue to be aphasic despite interventions . further , while language recovery > 1 year after stroke is thought to be less likely , studies showed that improvements even in patients with chronic aphasia are possible when they are provided with an intervention . these patients need additional restorative therapies that will enable them to return to society as productive members . the question raised in this study is whether some form of repetitive transcranial magnetic stimulation ( rtms ) may be one of those interventions . tms is a noninvasive method of stimulating neurons by inducing weak electric currents by electromagnetic induction . when applied in repetitive paradigms , synaptic plasticity can be altered to transiently increase or decrease localized cortical activities . changes in the neuronal networks induced by tms can persist well beyond the actual period of stimulation with improved language functions observed in some excitatory rtms studies . but , the safety and efficacy of the excitatory rtms applied directly to the stroke / peri - stroke areas is unclear . further , early reports of rtms - related seizures have led to the development of safety rules regarding stimulation frequency , intensity , train duration and intertrain interval . neuroimaging studies have revealed that post - stroke reorganization of language functions may be related to increased cortical involvement of the non - dominant or dominant cortical areas [ 2022 ] . unfortunately , these discoveries have thus far not contributed to the development of specific aphasia treatments . therefore , the primary goals of this study were to determine whether excitatory repetitive transcranial magnetic stimulation with fmri guidance ( neuronavigated rtms ; nertms ) applied directly to the language area identified by fmri is safe and whether it might facilitate rehabilitation from post - stroke aphasia and improve patient outcomes . we hypothesized that nertms will have positive impact on language skills evaluated with aphasia battery . this exploratory study was approved by the institutional review board and all subjects provided written informed consent . the inclusion criteria were : 1 . all subjects were right - handed prior to stroke , as determined by an edinburgh handedness inventory ( ehi ) score 50 . fmri procedures at 4 t varian unity inova whole body mri / mrs scanner have been previously described in detail . briefly , participants were oriented to the equipment and engaged in explicit practice of the tasks ; they were allowed to enter the scanner only after expressing complete understanding of all procedures . first , an alignment scan was performed for head position adjustments so that the ac - pc reference line was as close as possible to the vertical axis of the scanner . a high resolution , t1-weighted 3-d mdeft ( modified driven equilibrium fourier transform ) anatomical image was obtained using the following parameters : tr / te=13.1/6 ms , fov=25.619.219.2 cm , flip angle=22 , and voxel dimensions=111 mm . finally , t2 * -weighted functional images were obtained using the following parameters : tr / te=3000/30 ms , fov=25.625.6 cm , matrix=6464 pixels , number of slices=30 , slice thickness=4 mm , and flip angle=75. for language localization we used a highly reliable semantic decision / tone decision ( sdtd ) task [ 20,2528 ] . briefly , subjects performed two different alternating conditions , the control condition ( tones recognition ) and the active condition ( semantic recognition / association ) , starting with the control condition . in the tone decision task , subjects heard brief sequences of four to seven low- ( 500 hz ) and high - pitch ( 750 hz ) tones every 3.75 seconds and responded with a non - dominant hand button press for any sequence containing two high - pitched tones . in the semantic task , subjects heard spoken english nouns designating animals every 3.75 seconds and responded with a non - dominant hand button press to stimuli that meet two criteria : lives in the united states and is commonly used by humans the contrast between the tone discrimination task and the semantic task results in an activation pattern that is inherent only to semantic , word - form , and phoneme processing , all of which are part of the language system . each subject performed this task twice during each scanning session and the runs were concatenated for processing . the fmri image post - processing was performed using software developed in the imaging research center at the cincinnati children s hospital medical center in the idl software environment ( idl 7.0 ; research systems inc . , boulder , co ) . as previously , hamming - filtering preceded data reconstruction and the geometric distortion correction using the multi - echo reference method . data were then co - registered to further reduce the effects of motion artifact using a previously developed pyramid co - registration algorithm . a general linear model ( glm ) was used to process the data with a set of cosine basis functions used to minimize artifacts due to signal drift from aliased respiratory and cardiac effects . finally , z - score maps were computed on a pixel - by - pixel basis and transformed into talairach space for composite mapping . data in native space were utilized to localize left - hemispheric language centers for subsequent rtms administration . to parameterize the fmri data , we used previously generated regions of interest ( rois ) from 49 healthy controls , to calculate the laterality indices ( li ) in the frontal and temporo - parietal brain regions ( see figure 1 in szaflarski et . these primary rois include the lateral inferior and middle frontal region that corresponds to broca s area and the lateral / posterior temporal and parietal region that corresponds to wernicke s area ; these rois were superimposed on the corresponding right hemispheric homologues ( right hemispheric rois mirrored the left hemispheric ones ) . we also combined both rois into one global language roi . next , to avoid biases induced by arbitrary thresholding schemes , we determined the mean value of the t - statistics for all voxels within each rois ; the number of voxels above the mean for each roi was entered in the following formula to calculate the lateralization index : li = ( nlnr)/(nl+nr ) , where nl and nr represent the number of voxels ( left and right , respectively ) above the mean t for each roi . this calculation method produces li ranging from 1 ( left lateralized ) to 1 ( right lateralized ) . a battery of neuropsychological measures was obtained on the day of fmri or within 1 week of initiating the nertms and again , within 1 week of completing the nertms protocol on the same day as the follow - up fmri . the battery included 1 ) boston naming test ( bnt ) , 2 ) controlled oral word association test ( cowat ) , 3 ) semantic fluency test ( sft ) , 4 ) complex ideation subtest from the boston diagnostic aphasia examination ( bdae ) and 5 ) peabody picture vocabulary test iv ( ppvt iv ) . for cowat , sft , and ppvt iv , all subjects also completed communicative abilities log ( mini - cal ) , a subjective , self - administered measure of communicative abilities . single - pulse tms was performed to establish resting motor threshold ( rmt ) and active motor threshold ( amt ) with a magstim 200 stimulator connected through a bistim module to a 70 mm figure-8 coil ( magstim co. , wales , uk ) . surface electromyography ( emg ) leads were placed over the first dorsal interosseous ( fdi ) muscle of both hands . the coil was then placed over the primary motor cortex in the right and left hemisphere at the optimal site for obtaining a mep in the fdi . after the rmt and amt were determined , itbs was performed using magstim rapid2 ( magstim co. , wales , uk ) with intensity set at 80% of amt obtained from the right hemisphere . motor threshold values from the left hemisphere were too high due to the lmca stroke ; therefore , these values were not used to set the rtms intensity . the figure-8 coil was positioned tangentially to the skull , with the handle parallel to the sagittal axis pointing occipitally . itbs consisted of bursts of three pulses at 50 hz given every 200 milliseconds in two second trains , repeated every 10 seconds over 200 seconds for a total of 600 pulses . montreal , canada ) was used for neuronavigation to guide rtms stimulation to the fmri - identified residual left hemispheric broca s area of each individual patient . this technology also enabled reliable three - dimensionally precise reapplication of rtms throughout the study . subjects received one session of itbs each day for 10 consecutive days from monday through friday . data were initially characterized using descriptive statistics ( means and standard deviations or frequencies and percentages as appropriate ) . next , the pre-/post - rtms aphasia testing and fmri lateralization indices were compared using the wilcoxon signed - rank test . all data management and analyses were performed using spss version 18.0 ( spss , chicago , il , usa ) . this exploratory study was approved by the institutional review board and all subjects provided written informed consent . the inclusion criteria were : 1 . all subjects were right - handed prior to stroke , as determined by an edinburgh handedness inventory ( ehi ) score 50 . fmri procedures at 4 t varian unity inova whole body mri / mrs scanner have been previously described in detail . briefly , participants were oriented to the equipment and engaged in explicit practice of the tasks ; they were allowed to enter the scanner only after expressing complete understanding of all procedures . first , an alignment scan was performed for head position adjustments so that the ac - pc reference line was as close as possible to the vertical axis of the scanner . a high resolution , t1-weighted 3-d mdeft ( modified driven equilibrium fourier transform ) anatomical image was obtained using the following parameters : tr / te=13.1/6 ms , fov=25.619.219.2 cm , flip angle=22 , and voxel dimensions=111 mm . finally , t2 * -weighted functional images were obtained using the following parameters : tr / te=3000/30 ms , fov=25.625.6 cm , matrix=6464 pixels , number of slices=30 , slice thickness=4 mm , and flip angle=75. for language localization we used a highly reliable semantic decision / tone decision ( sdtd ) task [ 20,2528 ] . briefly , subjects performed two different alternating conditions , the control condition ( tones recognition ) and the active condition ( semantic recognition / association ) , starting with the control condition . in the tone decision task , subjects heard brief sequences of four to seven low- ( 500 hz ) and high - pitch ( 750 hz ) tones every 3.75 seconds and responded with a non - dominant hand button press for any sequence containing two high - pitched tones . in the semantic task , subjects heard spoken english nouns designating animals every 3.75 seconds and responded with a non - dominant hand button press to stimuli that meet two criteria : lives in the united states and is commonly used by humans the contrast between the tone discrimination task and the semantic task results in an activation pattern that is inherent only to semantic , word - form , and phoneme processing , all of which are part of the language system . each subject performed this task twice during each scanning session and the runs were concatenated for processing . the fmri image post - processing was performed using software developed in the imaging research center at the cincinnati children s hospital medical center in the idl software environment ( idl 7.0 ; research systems inc . , boulder , co ) . as previously , hamming - filtering preceded data reconstruction and the geometric distortion correction using the multi - echo reference method . data were then co - registered to further reduce the effects of motion artifact using a previously developed pyramid co - registration algorithm . a general linear model ( glm ) was used to process the data with a set of cosine basis functions used to minimize artifacts due to signal drift from aliased respiratory and cardiac effects . finally , z - score maps were computed on a pixel - by - pixel basis and transformed into talairach space for composite mapping . data in native space were utilized to localize left - hemispheric language centers for subsequent rtms administration . to parameterize the fmri data , we used previously generated regions of interest ( rois ) from 49 healthy controls , to calculate the laterality indices ( li ) in the frontal and temporo - parietal brain regions ( see figure 1 in szaflarski et . these primary rois include the lateral inferior and middle frontal region that corresponds to broca s area and the lateral / posterior temporal and parietal region that corresponds to wernicke s area ; these rois were superimposed on the corresponding right hemispheric homologues ( right hemispheric rois mirrored the left hemispheric ones ) . we also combined both rois into one global language roi . next , to avoid biases induced by arbitrary thresholding schemes , we determined the mean value of the t - statistics for all voxels within each rois ; the number of voxels above the mean for each roi was entered in the following formula to calculate the lateralization index : li = ( nlnr)/(nl+nr ) , where nl and nr represent the number of voxels ( left and right , respectively ) above the mean t for each roi . this calculation method produces li ranging from 1 ( left lateralized ) to 1 ( right lateralized ) . a battery of neuropsychological measures was obtained on the day of fmri or within 1 week of initiating the nertms and again , within 1 week of completing the nertms protocol on the same day as the follow - up fmri . the battery included 1 ) boston naming test ( bnt ) , 2 ) controlled oral word association test ( cowat ) , 3 ) semantic fluency test ( sft ) , 4 ) complex ideation subtest from the boston diagnostic aphasia examination ( bdae ) and 5 ) peabody picture vocabulary test iv ( ppvt iv ) . for cowat , sft , and ppvt iv , different forms of the tests were administered pre-/post - nertms . all subjects also completed communicative abilities log ( mini - cal ) , a subjective , self - administered measure of communicative abilities . single - pulse tms was performed to establish resting motor threshold ( rmt ) and active motor threshold ( amt ) with a magstim 200 stimulator connected through a bistim module to a 70 mm figure-8 coil ( magstim co. , wales , uk ) . surface electromyography ( emg ) leads were placed over the first dorsal interosseous ( fdi ) muscle of both hands . the coil was then placed over the primary motor cortex in the right and left hemisphere at the optimal site for obtaining a mep in the fdi . after the rmt and amt were determined , itbs was performed using magstim rapid2 ( magstim co. , wales , uk ) with intensity set at 80% of amt obtained from the right hemisphere . motor threshold values from the left hemisphere were too high due to the lmca stroke ; therefore , these values were not used to set the rtms intensity . the figure-8 coil was positioned tangentially to the skull , with the handle parallel to the sagittal axis pointing occipitally . itbs consisted of bursts of three pulses at 50 hz given every 200 milliseconds in two second trains , repeated every 10 seconds over 200 seconds for a total of 600 pulses . montreal , canada ) was used for neuronavigation to guide rtms stimulation to the fmri - identified residual left hemispheric broca s area of each individual patient . this technology also enabled reliable three - dimensionally precise reapplication of rtms throughout the study . subjects received one session of itbs each day for 10 consecutive days from monday through friday . data were initially characterized using descriptive statistics ( means and standard deviations or frequencies and percentages as appropriate ) . next , the pre-/post - rtms aphasia testing and fmri lateralization indices were compared using the wilcoxon signed - rank test . all data management and analyses were performed using spss version 18.0 ( spss , chicago , il , usa ) . eight subjects fulfilled the inclusion criteria and completed all study procedures ( table 1 ) ; additional 3 subjects signed the informed consent but were later excluded due to metallic artifact ( 2 ) and history of a single seizure at the time of stroke ( 1 ) . all subjects were 1 year since the aphasia - producing stroke and were not engaged in any specific aphasia rehabilitation . aphasia testing showed trends towards improvement with all performed tests except for cowat ( table 1 ) ; improvements in semantic fluency were significant ( p=0.028 ) and mini - cal showed trend towards post - rtms communication improvements ( p=0.075 ) . clearly noted are individual activations that served as a target for nertms ( enclosed in circles ) . there does not appear to be any overlap between the areas of activation and the location of the stroke . pre - rtms scans showed areas of increased activation for the tone decision baseline in the right hemisphere and low - level activations predominantly in the left hemisphere . post - rtms group images showed clear overall increase in the bold signal changes in response to the intervention . further , pre-/post - rtms contrasts showed significant overall increases in the bold signal with apparent shifts to the left hemisphere ( p=0.025 for broca s ; p=0.036 for wernicke s ; p=0.018 for the global roi ) . while the pre- and post - rtms locations of the bold signal changes are similar and include language areas typical for this task ( table 2 ) , post - rtms images showed shifts in activations predominantly to the left hemispheric head regions ( figure 3 ) . in this exploratory study we evaluated the safety and efficacy of the neuronavigated excitatory rtms ( nertms ) applied to the identified by fmri frontal language areas as a tool for post - stroke aphasia rehabilitation . despite a relatively small sample size ( n=8 ) , we were able to show significant improvements in language function after 2 weeks of stimulation that were associated with significant shifts of fmri signal to the affected hemisphere ; patients also tended to report subjective improvements in language skills after therapy completion ( p=0.075 ) . these results are very encouraging and support further testing of nertms as a post - stroke aphasia rehabilitation tool . in general , rtms involves repeated brain stimulation at regular frequencies ; it is a noninvasive method of exciting or inhibiting neurons through repeated induction of weak electric currents in the brain via rapidly changing magnetic fields . if used with appropriate intensity , brain activity can be modulated using rtms without discomfort or ill effects . the cortical plasticity induced by rtms and its therapeutic effects are thought to be primarily related to synaptic plasticity ; short- and long - term modifications in neural communication mediated by post - synaptic nmda receptors in which rtms releases mg blockade allowing ca entering the post - synaptic cell and inducing long - term potentiation . the rtms stimulation area is usually very focal and the changes induced by rtms can persist well beyond the duration of the stimulation . we speculate that the efficacy of the nertms observed in this study is related to the excitatory effect on the peri - stroke area and to the induction of long - term potentiation in this area . further , we are encouraged by the fact that nert - ms induced changes in the entire semantic decision network ( figure 2 ) and not only in the left frontal ( stimulated ) area . this finding further supports the notion that the effect of nertms on a single area , if associated with improvement(s ) , may correspond to both local and remote changes in the relevant network , and , therefore , improvements in all language - related skills . while a further discussion about the localization of the pre-/post - rtms bold signal changes is beyond the scope of this manuscript , of importance is that the post - rtms increases are seen in brain regions typically active during this fmri task left inferior frontal and left temporo - parietal regions . certainly , the left frontal bold signal increase may be related to the direct stimulation of that area in the course of therapy ; this increase is in agreement with the improvements on an expressive language measure ( sft ) and subjective verbal output reported by the patients ( cal ) . the bold signal increase in the left temporo - parietal region ( the area that is typically responsible for lexical retrieval and word meaning ) was observed even though this region was not directly stimulated with rtms , and is also consistent with the observed improvements on the semantic fluency test ( sft ) . in healthy controls , inhibitory rtms may decrease motor and cognitive performance in the stimulated area . in patients with post - stroke aphasia caused by lmca stroke , inhibitory rtms applied to the unaffected , non - dominant right - hemispheric language homologues improved the left - hemispheric language functions . rtms - evoked inhibition of redundant language circuits in the non - dominant hemisphere may release the damaged , dominant language networks allowing them to participate in post - stroke recovery . these findings support the hypothesis that the residual left - hemispheric network(s ) may be more important or effective for post - stroke language processing when not negatively influenced by the redundant non - dominant ( less effective ) circuitry . in contrast to the inhibitory rtms , excitatory rtms has been shown to facilitate language processing via increased short- and long - term cortical excitability ; rtms applied to the left - hemispheric speech area facilitated naming in healthy controls or patients with alzheimer s disease or primary progressive aphasia . in general , as a treatment , rtms has been shown in numerous pilot studies to be efficacious in various neurological and psychiatric conditions and is now fda - cleared for the treatment of depression . the findings from the above studies combined with our findings suggest that the application of nertms to the damaged by stroke residual language circuits in the dominant - hemisphere may indeed lead to improved language skill as observed in our preliminary study . our original hypothesis that stimulating the left hemispheric language areas with nertms would produce aphasia improvements is further supported by the finding of increased post - nertms bold signal activation ( when compared to the pre - rtms fmri ) in the dominant - hemisphere language circuits and decreased involvement of the non - dominant for language hemisphere after the course of therapy as observed with fmri . post - stroke neuroimaging studies that have evaluated recovery from aphasia in adults with unilateral lesions show evidence of cortical reorganization and migration of language functions to the non - dominant hemisphere after a dominant hemisphere insult . interestingly , one study that showed such a redistribution pattern using pet and fmri language tasks also found negative association between increased non - dominant inferior frontal gyrus activation and recovery after an ischemic stroke . the best recovery was observed in patients with peri - infarct activation on the fmri and pet studies . ( 2007 ) applied inhibitory rtms to the non - dominant language circuits and found improved post - stroke aphasia recovery in patients with preserved left - hemispheric language centers ; recovery was independent of the recruitment of the non - dominant homologues . a recent study showed that the fmri activation may be dependent on the phase of post - stroke recovery with early right - hemispheric upregulation of the fmri signal changes correlating with language recovery and late consolidation of the activation in the left - hemispheric language centers . this and other studies support the presence of preexisting language pathways in both , the dominant and non - dominant hemispheres and suggest that in health the circuitry in the non - dominant hemisphere may be inhibited by the active circuitry in the dominant hemisphere ; when the preferred pathway is interrupted ( as in a stroke ) , the non - dominant circuitry is uninhibited , hence activated . these studies suggest that increased reliance on language circuits in the dominant hemisphere supports higher levels of recovery from aphasia after stroke . as hypothesized , in this study nertms applied to the identified by fmri language areas facilitated the increased reliance on these areas for post - stroke aphasia recovery . early reports of possible seizures associated with tms led over 10 years ago to the development of safety rules regarding stimulation frequency , intensity , train duration and intertrain interval . multiple subsequent studies of rtms have demonstrated this to be a safe technique for use in studies of stroke and even in epilepsy . in healthy adult studies , tbs has been well tolerated . however , there is one report of a seizure in a healthy subject caused by continuous tbs stimulation with intensity set at approximately 100% of the resting motor threshold , a threshold far exceeding our protocol . our results also demonstrate the safety of the nertms protocol ; no adverse reactions were observed in our pilot group of stroke patients despite 10 daily 200 seconds long nertms ( itbs ) sessions ; subjects reported no discomfort and the subjective assessment of pre-/post - rtms language skills ( mini - cal ) indicated the patients felt the treatments they received were beneficial . therefore we are confident in proposing nertms as a safe and potentially beneficial intervention in patients with chronic aphasia after stroke . the shortcomings of this study include small sample size , lack of sham treatment , un - blinded application of rtms and only short - term follow - up . nevertheless , we were able to show improved language skills and shifts of fmri activations to the left hemisphere in response to the neuronavigated rtms . in this study , we provide preliminary evidence that fmri - guided , excitatory rtms applied to the affected broca s area improves language skills in patients with chronic post - stroke aphasia . we also show that these linguistic gains are associated with stronger language lateralization to the dominant left hemisphere . finally , in addition to the objective gains , patients tend to report improvements in their language skills after the intervention . therefore , the used in this study rtms protocol appears to be safe and should be further tested in blinded studies assessing its short- and long - term safety and efficacy for post - stroke aphasia rehabilitation .

summarybackgroundaphasia affects 1/3 of stroke patients with improvements noted only in some of them . the goal of this exploratory study was to provide preliminary evidence regarding safety and efficacy of fmri - guided excitatory repetitive transcranial magnetic stimulation ( rtms ) applied to the residual left - hemispheric broca s area for chronic aphasia treatment.material/methodswe enrolled 8 patients with moderate or severe aphasia > 1 year after lmca stroke . linguistic battery was administered pre-/post - rtms ; a semantic decision / tone decision ( sdtd ) fmri task was used to localize left - hemispheric broca s area . rtms protocol consisted of 10 daily treatments of 200 seconds each using an excitatory stimulation protocol called intermittent theta burst stimulation ( itbs ) . coil placement was targeted individually to the left brocas.results6/8 patients showed significant pre-/post - rtms improvements in semantic fluency ( p=0.028 ) ; they were able to generate more appropriate words when prompted with a semantic category . pre-/post - rtms fmri maps showed increases in left fronto - temporo - parietal language networks with a significant left - hemispheric shift in the left frontal ( p=0.025 ) , left temporo - parietal ( p=0.038 ) regions and global language li ( p=0.018 ) . patients tended to report subjective improvement on communicative activities log ( mini - cal ; p=0.075 ) . none of the subjects reported ill effects of rtms.conclusionsfmri-guided , excitatory rtms applied to the affected broca s area improved language skills in patients with chronic post - stroke aphasia ; these improvements correlated with increased language lateralization to the left hemisphere . this rtms protocol appears to be safe and should be further tested in blinded studies assessing its short- and long - term safety / efficacy for post - stroke aphasia rehabilitation .

Background Material and Methods Subjects Functional MRI task and scanning procedures Functional MRI data post-processing and analyses Neuropsychological testing of aphasia Neuronavigated excitatory repetitive transcranial magnetic stimulation (nerTMS) Data analyses Results Discussion Conclusions

breast cancer ( bc ) is a malignant tumor that originates from healthy mammary gland cells . bc is most frequent among women aged between 50 and 70 , 1 - 3 . it affects one in eight women on average and is the most common type of cancer among women , 1 - 3 . about one million new cases of bc are diagnosed each year worldwide , representing about a third of new cases of all female cancers in industrialized countries and 15% of new cases in developing countries . with the improvement of treatments , the survival rate of women diagnosed with bc has increased by 2% a year during the last twenty years , 1 - 3 . however , despite this improvement , the mortality due to bc remains significant among women with about 20 women over 100 000 dying from this disease each year worldwide , 1 - 3 . while bc stages i ( localized tumors of less than 2 cm ) and ii ( tumors of 2 to 5 cm without nodes ) can be cured relatively simply using surgery resulting in a mortality rate of less than 50% , those of grades iii ( infiltrating tumors without metastases ) and iv ( presence of metastases ) are much harder to treat , usually require heavy treatments often involving a combination of surgery , chemotherapy and radiotherapy , and result in a mortality rate of more than 50% , 1 - 3 . firstly , bc arises because of several risks factors , such as aging , disease ( hyperplasia ) , a diet with a high intake of saturated fat , overweight , excessive alcohol consumption , exposure to ionizing radiation , the consumption of oral contraceptives , hormone replacement therapy , 1 - 3 . the first method , which can result in a decrease in the bc mortality rate , lies in the control of these risk factors , for example by making the women way of life healthier . secondly , the bc mortality rate can be decreased by improving bc diagnosis , 1 - 3 . the latter needs to be carried out frequently , accurately and early enough , i. e. possibly before stages iii and iv of bc have been reached . it yields controversial results due to the difficulty for the women to detect a tumor by themselves . instead , it is usually recommended that women should consult a trained doctor who carries out a clinical exam to detect a tumor inside the breast . the main and most reliable technique used for bc detection is mammography . with this technique , an image of the inside of the breast is obtained using x - rays and breast abnormalities are looked for . in the united states , 70% of women above 40 years old have had a mammography during the last two years , 4 . one of the main problems with mammography is the significant percentage of overdiagnosis ( ~ 10% ) , 5 , which leads women to be diagnosed with bc and treated whereas their condition is not life threatening and in some cases will never cause any symptoms . the third way to decrease bc mortality consists in improving bc treatments . indeed , it has shown its efficacy , specifically for treatments of bc of stages iii and iv for which the mortality rate is high and efficient treatments are lacking . the purpose of this review is to highlight the positive impacts as well as drawbacks of three different types of thermotherapies , hyperthermia , thermoablation and magnetic hyperthermia for bc treatment . ( general information regarding the different types of thermotherapies is provided in table 1 ; the advantages of magnetic hyperthermia compared with other types of thermotherapies are summarized in table 2 ) . surgery is usually divided into conservative surgery , which results in the removal of the tumor without that of the entire breast , usually designated as lumpectomy or partial mastectomy , and ' ' non conservative '' surgery , usually designated as total mastectomy , which involves heavier surgery with the removal of the whole breast . there is a need to develop new techniques that can replace total mastectomy , which is used in 30% of all bc surgeries , 1 - 3 , essentially to limit the risk of metastases , to preserve woman breasts . radiotherapy ( also called radiation therapy ) is commonly used for patients that have either previously been treated with conservative surgery or possess metastases or lymph nodes that appear at an advanced stage of bc ( typically stage iii or iv ) , 1 - 3 . two different types of radiotherapy can be carried out , external radiotherapy , which uses a generator that directs high energy radiations ( x - rays , gamma rays or electrons ) towards the breast tumor or brachytherapy , in which the radiation is generated using a radioactive material introduced inside the breast . briefly , the mechanism of tumor destruction by radiations involves the ionization of biological tissues , generating free radicals , which interact with biological material , cells and dna and yield tumor cell death . despite of the improvement of radiation therapy , for example with the development of the intrabeam equipment , 6 , or mammosite , 7 , a critical issue remains to be addressed : the possibility of the destruction of bc cells without inducing toxicity for healthy tissues . a therapeutic window has been identified , which corresponds to an exposure of the patient to a radiation dose of 60 to 80 gy , 1 - 3 . although such doses result in the destruction of cancer cells with a relatively high probability of 40 to 100 % , they do not completely prevent undesirable side effects that can occur with a probability of 20 to 90 % , 1 - 3 . in order to minimize these side effects , lower doses of radiation are required , which can be obtained by sensitizing the tumor cells , using for example hyperthermia , 8 . in chemotherapy , drugs , which are usually administered intravenously to act on the whole body , are either used to prevent the development of metastasis or to reduce tumor sizes . the main drawback of chemotherapy is due to the nonspecific targeting of tumor cells , which results in side effects such as hair losses or women overweight . hormone therapy works by reducing the activity of female hormones , estrogen and progesterone , which stimulate the growth of hormone sensitive tumors contained in 80% of women diagnosed with bc , 1 - 3 . hormone therapies either use drugs , x - rays or surgery to stop the production of female hormones by the ovaries . as for chemotherapy , it can cause a number of side effects such as menopause , osteoporosis or hair losses . targeted therapy combined with immunotherapy , 9 , is used to treat bc that overexpresses the her-2 ( human epidermal growth factor receptor 2 ) gene , which promotes the growth of bc cells . drugs such as herceptin ( trastuzumab ) , which is an antibody , specifically target her-2 and destroy the bc cells overexpressing her-2 through the stimulation of the immune system , 10 . her-2 is however overexpressed only in a limited percentage of women ( 20 % ) , restricting the use of this therapy to only a portion of the women diagnosed with bc , 1 - 3 . triple negative bcs that represent ~20% of all bc cases do not express her-2 and grow without the support of the hormones estrogen and progesterone . cryotherapy , also called cryosurgery , uses extreme cold to freeze the water present in bc cells . water , turned to ice crystals , along with the cold itself , destroys the cancer cells . it is too early to make conclusions regarding the efficacy of cryotherapy , since many of the results of studies involving this approach are still pending , 11 . finally , two reasons for the development of new bc therapies such as thermotherapies can be underlined : bc is the most important cancer among women , for indications such as local regional breast cancer recurrence , the treatments described above are inefficient and bc often results in the death of the patient . bc is the most important cancer among women , for indications such as local regional breast cancer recurrence , the treatments described above are inefficient and bc often results in the death of the patient . in hyperthermia , the temperature of the whole or part of the organism is raised to typical temperatures of 40 - 43 c , 12 . in this case , the antitumor activity is generated by heat . the latter produces ( i ) cytotoxic effects , yielding the denaturation of cytoplasmic and membrane tumor proteins , ( ii ) a decreases in blood flow , impairing oxygen and nutrient supply of the tumor and inducing tumor acidosis , 12 , 13 and ( iii ) the activation of heat shock proteins , such as hsp27 , hsp70 and hsp90 , which participate in the destruction of breast tumors through complex and still poorly understood mechanisms , 14 , 15 . it is commonly used as an adjuvant and combined with other types of bc treatments such as chemotherapy , radiotherapy , targeted therapy and cryotherapy , 16 - 19 . hyperthermia is combined with radiations to either treat localized recurrent bc of advanced stages 20 , 21 , 23 - 27 or , more rarely , primary tumors , 22 . in this case , patients first receive a dose of radiation of typically 8 to 32 gy for recurrent breast tumors that have previously been treated by radiotherapy 20 , 21 , 24 or a dose of 60 - 70 gy for superficial breast tumors treated for the first time , 22 . it is usually not possible to exceed these radiation doses without causing life threatening conditions for the patients . it does however happen , specifically for bc of advanced stages , that these radiation doses are not sufficient to completely eradicate bc . in this case , hyperthermia can be carried out after radiations , for example by increasing the tumor temperature to 40 - 44 c for about an hour , 20 - 23 . the efficacy of a treatment combining radiotherapy and hyperthermia was demonstrated in a series of clinical trials , which showed that the percentage of complete bc remission increases from 44% , 20 , or 41% , 23 , using radiotherapy alone to 70% , 20 , or 59% , 23 , using radiotherapy and hyperthermia . hyperthermia can also be combined with chemotherapy , for example to treat metastatic breast tumors . it was shown that combining whole or part body hyperthermia with chemotherapy increases the efficacy of chemotherapy , 29 . a few studies also report the use of hyperthermia combined with both radiotherapy and chemotherapy for the treatment of recurrent localized recurrent bc , 31 , 32 , 33 . for a patient with local metastases , where radiotherapy and chemotherapy were inefficient , the addition of hyperthermia at 38 - 41 c led to the disappearance of the tumor , 31 . in two other studies , this combination of treatments using drugs such as epirubicin and ifosfamide , 32 , or liposomal doxorubicin , 33 , led to 20% total remission of recurrent localized bc , 33 . for example , a system was used for drug delivery , which contained thermosensitive liposomes with her-2 targeting molecules located at their surface , 30 . this thermosensitive liposome delivered the antitumor drugs that it contained specifically at the breast tumor location after an increase of the tumor temperature to 39 - 41 c , which enabled the release of the drugs specifically to the breast tumors . the efficacy of this combined treatment was shown for the treatment of a large metastatic breast tumor of 12 cm in diameter , which could not successfully be treated by cryosurgery alone but disappeared completely when cryosurgery was combined with hyperthermia , 34 . hyperthermia was generated in several different ways using a microwave applicator operating at 433 mhz 18 , 21 , 24 , a sigma 60 applicator for part body hyperthermia , 26 , or a radiant system for whole body hyperthermia , 28 . with a radiant system , a temperature of 42 c can be maintained throughout the whole body during an hour . other hyperthermia systems include the aquatherm and iratherm systems in which heating is induced by far - infrared radiations , which heat the blood under the skin and gradually heat the whole body in 60 - 90 minutes . in thermal ablation , the tumor temperature is raised above 43 c , resulting in larger cytotoxic effects than in hyperthermia and in the direct destruction of the breast tumor . since thermal ablation alone is usually sufficient to destroy breast tumor tissues , thermoablation enables to treat localized breast tumors of small sizes ( 2 to 6 cm ) , 35 - 37 , 39 - 47 and more rarely metastatic liver tumors originating from bc , 38 . during the treatment , the tumor temperature is typically raised to 90 - 95 c during 10 to 15 minutes using radiofrequencies , 35,37 , or to 46 - 50 during 60 seconds using microwaves , 38 , to 60 - 90 c during one to two hours using high intensity focused ultrasound ( hifu ) , 44 , 45 , or to 48 c during 30 minutes using interstitial laser thermotherapy ( ilt or litt ) , 48 . the heat can be produced by different types of equipment , such as an rf 460 monopolar electrosurgical generator from rita medical system , 35 , 37 , a jc200 from chongqing haifu technology , 42 , 43 , a microwave coagulator operating at 2.45 ghz , 38 , or a yag : nd pulsed laser , 48 , for radiofrequency , hifu , microwave and litt thermotherapies , respectively . magnetic resonance temperature imaging ( mrti ) , ultrasound or ultrasonography can be used to measure the tumor temperature during the treatment and to try to prevent overheating of healthy tissues , 44 . mrti provides an estimate of the temperature by measuring the molecular diffusion coefficient , the longitudinal relaxation time t1 or the proton resonance frequency of tissue water , which depends on water , 49 . ultrasounds and ultrasonography measure the echo shifts due to changes in tissue thermal expansion and speed of sound , the variation in the attenuation coefficient or the change in backscattered energy from tissue inhomogeneity , 50 . for example , using microwaves , it was possible to destroy 100 % of breast tumors , 38 . clinical trials on bc involving hifu also led to the complete ( or almost complete ) disappearance of breast tumors , 42 . however , these thermotherapies possess a drawback , which comes from the high temperatures , which are needed to achieve high efficacy . without a method to predict and measure accurately these temperatures , in magnetic hyperthermia , nanoparticles are administrated ( or sent ) to tumors and heated under the application of an alternating magnetic field . the advantages of magnetic hyperthermia compared with the other heating techniques described above come from : ( i ) , the cellular internalization of the nanoparticles under the application of an alternating magnetic field , which yields more efficient tumor destruction than extracellular heating usually achieved with the other heating methods , 57 - 60 , ( ii ) , the possibility to target the tumors , for example by attaching to the nanoparticles molecules that specifically recognize the cancer cells and , ( iii ) , the fine temperature tuning that can be achieved by accurately selecting the strength or intensity of the applied alternating magnetic field and the quantity of nanoparticles administered , ( iv ) , the very localized heat that shall prevent damaging healthy tissues surrounding the tumor . two different types of nanoparticles have been tested for magnetic hyperthermia treatment of bc , chemically synthesized nanoparticles , which are the most frequently studied , and biologically produced nanoparticles , synthesized by magnetotactic bacteria , called magnetosomes . usually , chemically synthesized nanoparticles used to carry out magnetic hyperthermia on bc are small , typically less than 20 nm , made of maghemite or magnetite and superparamagnetic , which means that they possess a thermally unstable magnetic moment , 51 - 56 . magnetic hyperthermia has successfully been tested on breast tumors xeno - grafted under the skin of mice . for that , 1 to 2 mg of nanoparticles , 51 - 53 , have been administered either intravenously , 51 , 52 , or directly to tumors , 53 , of typical sizes 100 to 200 mm , 51 , 52 . specific targeting of the tumors was achieved by using 111inch antibody , 51 , 52 , or anti - her2 molecules attached either to the nanoparticles , 53 - 55 , or to the liposomes containing the nanoparticles , 56 . in order to generate heat , the nanoparticles were exposed to an alternating magnetic field of strength 12 - 130 mt and frequency of 118 - 360 khz during 20 - 30 minutes , 51 - 56 . the efficacy of these preclinical treatments was revealed by the disappearance of the tumors , 51 - 56 . clinical trials using magnetic hyperthermia are ongoing at the university of nagoya to treat bc . figure 1 shows the different steps , which may be involved in the treatment of bc using magnetic hyperthermia . mri may be used to visualize the nanoparticles during and after the administration of the nanoparticle suspension and to verify the correct location of the nanoparticles in the tumor before and during the treatment . after that , the patient may be positioned inside an instrument that generates an alternating magnetic field and heats the nanoparticles contained in the tumor . after treatment , the variation of the tumor size may be followed by mri . in order to increase the amount of heat delivered , nanoparticles synthesized by magnetotactic bacteria , called magnetosomes , which are usually larger , with average sizes lying between 40 and 60 nm , better crystallized and which possess a more stable magnetic moment than chemically synthesized nanoparticles , 57 - 65 , have been tested preclinically for bc treatment , 57 - 60 . figure 2 illustrates the experimental protocol , which was used during the treatment of the mice . shortly , 100 l of a suspension containing 1 mg of chains of magnetosomes , extracted from magnetotactic bacteria and mixed in water , was administered to mda - mb-231 breast tumors xeno - grafted under the skin of mice . the mice were then exposed three times during twenty minutes to an alternating magnetic field of average field strength 20 mt and frequency 198 khz . the treatment led to the complete disappearance of the tumor in several mice 30 days after the treatment as shown in figure 2 , 57 . for the mice , which were treated in the same way , but with a suspension containing 1 mg of chemically synthesized nanoparticles instead of the magnetosome , the treatment was inefficient and the tumor was still there 30 days following the beginning of the treatment as shown in figure 2 . it was therefore demonstrated that the magnetic hyperthermia treatment of bc was more efficient using magnetosomes than two types of chemically synthesized nanoparticles ( spion covered with peg or citrate molecules ) both in vivo on mda - mb231 tumors and in vitro on mda - mb231 cells and hella cells , 57 . the physical mechanisms , procedures , pre - preclinical and clinical data , available on each of the different types of thermotherapy are summarized in table 1 . in conclusion , two types of thermotherapies , hyperthermia that is usually combined with other commonly used bc treatments , and thermoablation , which is often used alone , have been described . the results obtained with an emerging thermotherapy , called magnetic hyperthermia , in which magnetic nanoparticles are heated inside tumors under the application of an alternating magnetic field , have also been presented . the advantages and drawbacks of magnetic hyperthermia compared with the other thermotherapies , hyperthermia may essentially be used for the treatment of recurrent localized breast tumor of stages iii and iv , while thermoablation may be carried out on small localized tumors . since hyperthermia and thermoablation have both shown efficacy , there is no reason why they should not be used more frequently for the treatment of these indications . magnetic hyperthermia is a promising thermotherapy , but research still needs to be carried out on several aspects such as the specific targeting of the tumor by the nanoparticles , the control of the heat released by the nanoparticles and the nanoparticle toxicity in order to be able to use it routinely for bc treatment . the cost of breast cancer is about 30 billion $ a year , with 30% and 50% of this cost being attributed to productivity loss and medical costs respectively . the loss in productivity and medical costs can be minimized by developing new , improved and less expensive treatments , such as those described in this review . pubmed , medline , google , google scholar and clinicaltrials.gov were used for the literature search with keywords such as : thermotherapy , hyperthermia , thermoablation , magnetic hyperthermia , breast cancer , breast tumor .

in this article , the use of different types of thermotherapies to treat breast cancer is reviewed . while hyperthermia is most commonly used as an adjuvant in combination with radiotherapy , chemotherapy , targeted therapy or cryotherapy to enhance the therapeutic effect of these therapies , thermoablation is usually carried out alone to eradicate small breast tumors . a recently developed thermotherapy , called magnetic hyperthermia , which involves localized heating of nanoparticles under the application of an alternating magnetic field , is also presented . the advantages and drawbacks of these different thermotherapies are highlighted .

Introduction Treatments commonly used to treat breast cancer BC treatments involving hyperthermia Treatments of breast cancer by thermoablation Magnetic hyperthermia Conclusion Methods used for the literature search

anaesthetic management of a pregnant patient with an intracranial space occupying lesion ( sol ) requires modification of neuroanaesthetic and obstetric practices , which have competing clinical goals to achieve the optimal safety of both mother and foetus . maternal alterations during pregnancy may complicate the anaesthetic management and increase monitoring requirements for safety of both mother and foetus . neuroanaesthesia for the pregnant patient is required infrequently , as overall frequency for non - obstetric surgery during pregnancy is low and surgery for intracranial sol during pregnancy is rare . intracranial tuberculomas are clinically and radio graphically indistinguishable from enhancing neoplastic lesions . tuberculosis ( tb ) of central nervous system accounts for about 5% of extra pulmonary cases and manifests as meningitis or uncommonly as tuberculoma . a 22-year - old woman , gravida2 para1 , 28 weeks pregnant , weighing 56 kg presented with headache , right hemi paresis and focal seizures in the right upper limb . magnetic resonance imaging ( mri ) brain revealed a hyper intense mass in the left fronto - parietal region with peri - lesional oedema , mass effect and midline shift to the right . mri brain [ figure 1 ] showed a hyper intense mass in the left fronto - parietal region with peri - lesional oedema . mr spectroscopy showed grossly elevated choline and reduced n - acetylaspartate and creatinine levels within the lesion . as the patient was 28 weeks pregnant with signs and symptoms of raised intracranial pressure ( icp ) with progressive neurological deficits , not manageable with drugs , elective craniotomy was planned for decompression and excision of the sol . treatment was started with dexamethasone 8 mg bd , carbamazepine 200 mg bd , to reduce icp and to prevent seizures . pre - anaesthetic evaluation was normal and haematological and biochemical parameters were within normal limits . patient was pre - medicated with tab.ranitidine 150 mg and tab.metoclopramide 10 mg previous night and on morning of surgery as prophylaxis against aspiration . in the operating theatre , initial monitoring consisted of electrocardiogram , pulse oximeter , non - invasive blood pressure and capnography . two large bore [ 16 g ] peripheral intravenous ( iv ) cannulae were secured . patient was slightly tilted to the left and a wedge placed under the right buttock to avoid aorto - caval compression . after pre - oxygenation , modified rapid sequence induction and intubation was performed using thiopentone 5 mg / kg , fentanyl 2 g / kg , lignocaine 1 mg / kg and rocuronium 1 mg / kg . re - inforced cuffed endotracheal tube size 7 was used and an intra - arterial line was secured . anaesthesia was maintained with isoflurane 0.7 mac in oxygen and air , vecuronium and intermittent boluses of morphine . additional monitoring included urinary catheter , oesophageal temperature probe and arterial blood pressure ( bp ) . magnetic resonance imaging brain showing a hyper intense mass in the left fronto - parietal region with peri - lesional oedema antibiotic prophylaxis with ceftriaxone 1 g , and dexamethasone 8 mg iv , and mannitol 0.25 g / kg ( iv infusion after skin incision ) were administered . patient was haemodynamically stable throughout the procedure , with systolic bp between 100 and 120 mm hg , heart rate 6080 beats / min . spo2 ( 98100% ) , etco2 ( 2834 mm hg ) and body temperature ( 3637c ) were maintained . total duration of procedure was 4 h , during which 2000 ml of crystalloids was infused . estimated blood loss was 400 ml and urine output was maintained at > 1 ml / kg / h . intra - operative arterial blood gas analysis showed values as below : ph 7.42 , pco2 32.6 mm hg , po2 239.9 mm hg , hco3 23.0 mmol / l , o2 sat 99.6% . at the end of the procedure , patient was extubated fully awake , with no new neurological deficits , in the operating theatre and shifted to intensive care unit for recovery and observation . after a steady recovery , she was shifted to the ward 48 h after surgery . the histopathology examination report [ hpe ] revealed features of granulomatous encephalitis , consistent with tuberculoma . patient was started on antituberculous therapy [ att ] in consultation with obstetric team and was discharged from hospital . at 38 week of gestation , patient delivered a healthy male baby weighing 2.8 kg by normal vaginal delivery . evidence based recommendations for neuroanaesthetic management in pregnancy are sparse and so planning and decision making must be made largely on general principles of neurosurgical and obstetric anaesthesia . the foetus may be compromised indirectly by maternal hypotension , uterine artery vasoconstriction , maternal hypoxaemia and acid - base changes . indeed any change in maternal physiology that reduces utero - placental perfusion compromises foetal gas exchange . the adverse foetal effects of drugs administered in the perioperative period need to be carefully considered . all the drugs administered in our case belonged to either food and drug administration ( fda ) pregnancy category b ( animal studies fail to demonstrate risk to foetus ) or category c ( potential benefits may warrant use of drugs in pregnant women despite potential risks ) . ranitidine , metoclopramide , lignocaine , ceftriaxone and glycopyrrolate are included in fda category b. mannitol , dexamethasone , thiopentone , fentanyl , morphine , rocuronium , vecuronium , isoflurane and neostigmine are included in fda category c. foetal heart rate monitoring may help in detecting foetal hypoxia and metabolic acidosis which may result in tissue damage or foetal death . after 16 week of gestation , continuous fhr monitoring may help in early detection of foetal hypoxia in the peri - operative period . in our case , we used continuous fhr monitoring intraoperatively with external cardiotocography . the anaesthesiologist must understand the physiological changes of pregnancy , their implications , and the specific risks of anaesthesia during pregnancy . prophylactic tocolytics aim to stop uterine contractions and to temporarily delay delivery with the objective of improving neonatal outcome by preventing preterm labour . however , they are controversial as they have considerable maternal side effects and their efficacy in non - obstetric surgery is not established . our patient was afebrile without pulmonary manifestations of tb , a normal chest x - ray and was hiv negative . intracranial tuberculomas are thought to arise when tubercles in the brain parenchyma enlarge without rupturing into the subarachnoid space . a high index of suspicion of tuberculoma is warranted in situations like focal or generalized seizures , focal neurological deficits and in patients with symptoms and signs of raised intra - cranial tension such as headache , vomiting and papilloedema . pre - operatively , based on the mri and mr spectroscopic features and clinical presentation , the diagnosis was a tumour , possibly glioma . however , there are limitations of mr spectroscopy for the diagnosis of intracranial sol as it may not be able to distinguish brain tumours such as glioma from inflammatory lesions such as tuberculomas and may be inconclusive in tuberculomas . during surgery , however , the hpe report clearly suggested it was an intracranial tuberculoma and the patient responded well to att . cerebral tuberculoma should be considered in the differential diagnosis of sol in a pregnant woman presenting with signs and symptoms of raised icp , especially in women coming from high incidence areas , even in the absence of pulmonary involvement . neurosurgery is uncommonly required during pregnancy , but it requires a multidisciplinary approach with a specific anaesthetic plan , keeping in mind the safety requirements of both the mother and the foetus .

intracranial space occupying lesion [ sol ] during pregnancy presents several challenges to the neurosurgeons , obstetricians and anaesthesiologists in not only establishing the diagnosis , but also in the perioperative management as it requires a careful plan to balance both maternal and foetal well - being . it requires modification of neuroanaesthetic and obstetric practices which often have competing clinical goals to achieve the optimal safety of both mother and foetus . intracranial tuberculoma should be considered in the differential diagnosis of intracranial sol in pregnant women with signs and symptoms of raised intracranial pressure with or without neurological deficits , especially when they are from high incidence areas . we report a 28-week pregnant patient with intracranial sol who underwent craniotomy and excision of the lesion , subsequently diagnosed as cranial tuberculoma .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

mutant mice either in the mixed c57bl/6 129 background or pure 129 background were maintained in specific pathogen the surgical procedure was performed as described ( 15 ) . in brief , mice were anesthetized by avertin ( 0.2 ml of 2.5% solution per 10 g body wt ) , and a 0.5-cm midline incision was made in the peritoneum . the distal two thirds of the cecum was ligated with a silk suture , and the cecum was punctured once with an 18.5-gauge needle , and then gently squeezed to ensure that the holes were completely open . the cecum was returned to the peritoneal cavity , and the body wall was stitched and the incision was closed with 9-mm stainless steel wound clips . sham controls were operated on in the same manner but without ligation and puncture . in some experiments peritoneal lavage was harvested after injection of 3 ml of pbs with 2% fcs and used for further assays ( see below ) . in other experiments , mice were bled through the tail vein at 1.5 , 3 , 6 , and 12 h after clp and sera were used for further assays . statistical analysis for survival was performed using the stata program ( stata corp . , college station , tx ) . polyclonal igm was isolated from normal mouse serum ( sigma chemical co. , st . sera were precipitated by ammonium sulfate ( 50% saturation ) , and the precipitate was dissolved in pbs and dialyzed . recovered solution was filtered through a 0.45-m filter and applied to a 5-ml protein g sepharose column ( sigma chemical co. ) to remove igg . the flow - through was reconstituted with nacl to a final concentration of 0.5 m and applied to a 4-ml anti - igm sepharose column ( zymed laboratories , inc . bound protein was eluted with 0.1 m glycine/0.15 m nacl , ph 2.5 , and neutralized with 1 m tris , ph 8.0 . protein content in each fraction was determined by spectrophotometry , and positive fractions were pooled , dialyzed , and concentrated ( amicon , inc . leonore a. herzenberg ( stanford university , stanford , ca ) , and 5e11 hybridoma secreting pc - specific igm was obtained from dr . j. latham claflin ( university of michigan medical school , ann arbor , mi [ 12 , 16 ] ) . anti - ptc and anti - pc igm was isolated with anti - igm affinity column from ascites produced in recombination activating gene ( rag)-2deficient mice . western blot and elisa were used to assess igg contamination and degradation of igm . all batches of purified igm had no detectable degradation and < 1% igg contamination ( data not shown ) . endotoxin levels in purified igm was < 200 eu / ml as determined by limulus amebocyte lysate assay ( associates of cape cod , woods hole , ma ) . in igm reconstitution experiments , sigm - deficient mice were given 0.5 mg i.v . peritoneal lavage fluid was assayed for escherichia coli , neutrophils , and the levels of endotoxin ( lps ) , tnf- , and il-6 . e. coli colony - forming units ( cfu ) were determined by overnight culture of serial dilutions of peritoneal lavage fluid on luria broth agar plates at 37c . colonies of e. coli were identified in a background of heterogeneous colonies by morphology and confirmed by culture on macconkey ii agar and by assay with the enteric identification system ( organon teknika , durham , nc ) . neutrophils in the peritoneal lavage were analyzed by flow cytometry using pe - conjugated anti - granulocyte antibody and fitc - conjugated anti - cd19 antibody ( pharmingen , san diego , ca ) . 10 live cells were collected for each sample , and neutrophils were identified as grcd19 . tnf- and il-6 were measured by elisa using commercial kits ( endogen , inc . , cambridge , ma ; and intergen co. , purchase , ny ) . concentrations of tnf- and il-6 in individual samples were determined by comparison with a standard curve derived from the cytokine standard supplied with the kits . lps concentrations of individual samples were calculated using a standard curve derived from the lps standard provided with the kit . mutant mice either in the mixed c57bl/6 129 background or pure 129 background were maintained in specific pathogen the surgical procedure was performed as described ( 15 ) . in brief , mice were anesthetized by avertin ( 0.2 ml of 2.5% solution per 10 g body wt ) , and a 0.5-cm midline incision was made in the peritoneum . the distal two thirds of the cecum was ligated with a silk suture , and the cecum was punctured once with an 18.5-gauge needle , and then gently squeezed to ensure that the holes were completely open . the cecum was returned to the peritoneal cavity , and the body wall was stitched and the incision was closed with 9-mm stainless steel wound clips . sham controls were operated on in the same manner but without ligation and puncture . in some experiments peritoneal lavage was harvested after injection of 3 ml of pbs with 2% fcs and used for further assays ( see below ) . in other experiments , mice were bled through the tail vein at 1.5 , 3 , 6 , and 12 h after clp and sera were used for further assays . statistical analysis for survival was performed using the stata program ( stata corp . , college station , tx ) . polyclonal igm was isolated from normal mouse serum ( sigma chemical co. , st . sera were precipitated by ammonium sulfate ( 50% saturation ) , and the precipitate was dissolved in pbs and dialyzed . recovered solution was filtered through a 0.45-m filter and applied to a 5-ml protein g the flow - through was reconstituted with nacl to a final concentration of 0.5 m and applied to a 4-ml anti - igm sepharose column ( zymed laboratories , inc . bound protein was eluted with 0.1 m glycine/0.15 m nacl , ph 2.5 , and neutralized with 1 m tris , ph 8.0 . protein content in each fraction was determined by spectrophotometry , and positive fractions were pooled , dialyzed , and concentrated ( amicon , inc . , beverly , ma ) . leonore a. herzenberg ( stanford university , stanford , ca ) , and 5e11 hybridoma secreting pc - specific igm was obtained from dr . j. latham claflin ( university of michigan medical school , ann arbor , mi [ 12 , 16 ] ) . anti - ptc and anti - pc igm was isolated with anti - igm affinity column from ascites produced in recombination activating gene ( rag)-2deficient mice . western blot and elisa were used to assess igg contamination and degradation of igm . all batches of purified igm had no detectable degradation and < 1% igg contamination ( data not shown ) . endotoxin levels in purified igm was < 200 eu / ml as determined by limulus amebocyte lysate assay ( associates of cape cod , woods hole , ma ) . in igm reconstitution experiments , sigm - deficient mice were given 0.5 mg i.v . peritoneal lavage fluid was assayed for escherichia coli , neutrophils , and the levels of endotoxin ( lps ) , tnf- , and il-6 . e. coli colony - forming units ( cfu ) were determined by overnight culture of serial dilutions of peritoneal lavage fluid on luria broth agar plates at 37c . colonies of e. coli were identified in a background of heterogeneous colonies by morphology and confirmed by culture on macconkey ii agar and by assay with the enteric identification system ( organon teknika , durham , nc ) . neutrophils in the peritoneal lavage were analyzed by flow cytometry using pe - conjugated anti - granulocyte antibody and fitc - conjugated anti - cd19 antibody ( pharmingen , san diego , ca ) . 10 live cells were collected for each sample , and neutrophils were identified as grcd19 . tnf- and il-6 were measured by elisa using commercial kits ( endogen , inc . , cambridge , ma ; and intergen co. , purchase , ny ) . concentrations of tnf- and il-6 in individual samples were determined by comparison with a standard curve derived from the cytokine standard supplied with the kits . lps concentrations of individual samples were calculated using a standard curve derived from the lps standard provided with the kit . to examine the role of natural igm in the immediate response to microbial infection , we determined the susceptibility of sigm - deficient mice to acute septic peritonitis induced by clp that releases endogenous bacteria from the cecum into the peritoneal cavity . 32 h after clp , 70% of sigm - deficient mice died compared with 20% of the wild - type mice ( fig . 1 ) , indicating that the absence of natural igm rendered mutant mice much more susceptible to the acute bacterial infection ( p < 0.0001 ) . the increased susceptibility of mutant mice to clp was due to the absence of sigm , because reconstitution of mutant mice with a single dose of 0.5 mg i.v . of total igm isolated from normal mouse serum 4 h before clp restored their survival to the same level as wild - type mice ( fig . sigm - deficient mice were also more sensitive to challenge by individual species of pathogenic bacteria such as group b streptococcus . the lethal dose to 50% ( ld50 ) animals challenged was 10-fold lower for sigm - deficient mice than for wild - type mice ( our unpublished observations ) . sigm - deficient mice also showed increased incidence of spontaneous bacterial infection by opportunistic bacteria , including pasteurella pneumotropica , in specific pathogen these data show that natural igm is required for the protection against bacterial infection . resistance to clp is dependent on complement , mast cells , and tnf- ( 1720 ) . the susceptibility of sigm - deficient mice to clp is similar to mice deficient in complement component c3 or c4 or mast cells ( 1719 ) . the requirement of natural igm for resistance to clp is probably based on its ability to bind to bacteria and activate complement . to test this possibility , we determined the immediate response by assaying the levels of tnf- , il-6 , and lps , neutrophil infiltration , and bacterial load in the peritoneal lavage 3 h after clp . as in c3-deficient mice , the levels of tnf- and il-6 in mutant mice were approximately half those in wild - type mice ( table 1 ) . without clp , both sigm - deficient and wild - type mice had very few neutrophils in the peritoneum ( < 1% ) and similar numbers of cells in the lavage after clp ( data not shown ) . again , as in c3-deficient mice , the percentage of neutrophils in the peritoneal lavage recovered from sigm - deficient mice was significantly reduced compared with that from wild - type mice ( 55 vs. 82% ; table 1 , and data not shown ) . furthermore , 10 times more e. coli were recovered from the peritoneal lavage of sigm - deficient mice than from wild - type mice ( table 1 ) . associated with the higher bacterial load , approximately twice the amount of endotoxin ( lps ) was detected in sigm - deficient mice as in wild - type mice . reconstitution of sigm - deficient mice with total igm restored the levels of tnf- and neutrophils and reduced e. coli load in the peritoneal lavage ( table 1 ) , consistent with the increased survival . these data show that the effects resulting from the absence of sigm on the induction of tnf- , neutrophil infiltration , and bacterial load in the peritoneum are very similar to those seen in the absence of c3 , indicating that natural igm functions through the complement pathway . a single bound igm molecule is sufficient to activate complement to lyse a red blood cell ( 21 ) . binding of natural igm to bacteria immediately after infection likely results in the activation of complement through the classical pathway . since serum from sigm - deficient mice lysed antibody - opsonized red blood cells just as efficiently as serum from wild - type mice in a hemolytic assay ( data not shown ) , the increased susceptibility of sigm - deficient mice to clp is probably associated with the absence of igm - mediated complement activation . c3- or c4-deficient mice appear to be even more sensitive to clp than sigm - deficient mice as indicated by 100% mortality within 24 h ( 17 ) . this may be because complement can also be activated through the alternative and lectin pathways , and complement is important in the efficient clearance of bacteria . in addition , sigm - deficient mice have relatively normal levels of iggs ( 13 ) , some of which are probably natural antibodies . although the igg proteins may contribute to the survival of sigm - deficient mice , they are clearly not sufficient to compensate fully the absence of natural igm . uncontrolled bacterial infection in the peritoneum leads to a fatal systemic infection and inflammation . to determine the systemic inflammatory response to clp in the absence of natural igm , we collected serum from both sigm - deficient and wild - type mice at 1.5 , 3 , 6 , and 12 h after clp and assayed for the levels of lps , tnf- , and il-6 . we divided the sera into four groups based on the genotype of the mice and whether the mice died or survived at 32 h after clp . as shown in fig . 2 , the levels of serum lps were similar to those of sham controls 3 h after clp but significantly higher at 6 and 12 h after clp . among the four groups of mice , sigm - deficient mice that died had a significantly higher level of lps by 6 h after clp , consistent with previous findings indicating that natural igm is involved in the clearance of lps ( 22 ) . similarly , sigm - deficient mice that died had a significantly elevated level of tnf- 3 h after clp ( fig . although the initial local release of tnf- is beneficial to the containment of bacterial infection , systemic release of tnf- is usually associated with wide - spread inflammation . the level of proinflammatory cytokine il-6 was also elevated in the serum 6 and 12 h after clp in sigm - deficient mice as well as in wild - type mice that died ( fig . il-6 is a major cytokine that induces the production of acute phase proteins , such as c - reactive protein , that are thought to have similar functions as natural antibodies ( 2325 ) . the similar levels of serum il-6 in both sigm - deficient and wild - type mice suggest that acute phase proteins were similarly elevated in both types of mice . thus , natural igm appears to have some unique functions in the immediate response against bacterial infection that can not be replaced by acute phase proteins . in the absence of natural igm reconstitution of sigm - deficient mice with polyclonal igm from normal mouse serum restored their resistance to clp ( fig . 1 ) . therefore , it was of obvious interest to test whether the resistance can be restored by reconstitution with monoclonal igm specific to ptc or pc . ptc is a common membrane component and anti - ptc is the most widely expressed specificity by natural igm ( 11 , 12 ) . pc is chemically related to ptc and is found as a determinant of the pneumococcal cell wall ( 26 ) . anti - pc igm has been shown to confer protection against certain types of streptococcus pneumoniae ( 27 , 28 ) . 3 h after clp , mice were killed and peritoneal lavage was harvested to assay for the levels of tnf- and lps , neutrophil infiltration , and bacterial load . as in reconstitution with polyclonal igm , reconstitution with anti - ptc igm resulted in elevated levels of tnf- and neutrophils and a reduced e. coli load and lps in the peritoneal lavage ( table 1 ) . the difference between the two monoclonal igm preparations was probably due to their differences in specificity , because both were > 98% pure , had minimal endotoxin contamination , and reconstituted to a similar level as assayed by elisa of serum taken 3 h after clp ( data not shown ) . the resistance conferred by anti - ptc igm was clearly evident but it appeared not to be as effective as polyclonal igm , probably because polyclonal igm bind bacteria through many different antigenic determinants . that anti - ptc igm had a clear effect suggests that it is involved in the immediate defense against bacterial infection under physiological conditions . activation of complement is essential for the initial containment of systemic bacterial infection as demonstrated by the susceptibility of complement - deficient humans , guinea pigs , and mice to bacterial infection ( 17 , 2931 ) . c3a and c5a peptides function as chemoattractants to recruit leukocytes to the site of infection . opsonization of bacteria by c3b and ic3b promotes efficient phagocytosis by neutrophils . in addition , complement is required for the efficient activation of mast cells to release tnf- for the initiation of a local inflammatory response ( 17 ) . however , the optimal clearance of bacteria by complement requires the presence of natural antibodies . gram - positive bacteria are generally resistant to complement - mediated lysis because the thick peptidoglycan layer in their cell wall prevents insertion of the membrane - attack complex ( 32 ) . although gram - negative bacteria are effectively lysed by complement , these bacteria contain lps in their cell walls which when released in substantial amount can be fatal within hours ( 33 , 34 ) . furthermore , sialic acids of the pneumococcal capsular polysaccharides can inhibit the activation of the alternative complement pathway ( 35 ) . the capsular polysaccharides also inhibit phagocytosis by macrophages and neutrophils ( 36 ) . effective responses to bacterial infection require antibodies to opsonize bacteria for efficient phagocytosis and to neutralize the released endotoxin ( 22 ) . what is the source of antibodies before the onset of a specific humoral response to a particular infectious agent ? natural igm is ideally suited for these purposes because of its polyreactivities and the exquisite ability to activate complement , and because normally it is already present . the susceptibility of sigm - deficient mice to clp and restoration of resistance by reconstitution with polyclonal igm and to a lesser extent by anti - ptc igm clearly demonstrate a critical role of natural igm in the immediate defense against bacterial infection sigm - deficient ( / ) and wild - type ( + /+ ) mice at 6 - 8 wk of age were subject to clp . igm - reconstituted sigm - deficient mice ( / igm ) were given as a single dose of 0.5 mg i.v . mice were monitored for survival within the first 32 h. analyses of peritoneal lavage 3 h after clp sigm - deficient mice ( / ) , wild - type mice ( + /+ ) , and igm - reconstituted sigm - deficient mice were subject to clp . peritoneal lavage was carried out by injection of 3 ml i.p . of pbs with 2% fcs 3 h after clp . of purified polyclonal igm from normal mouse serum or monoclonal igm specific to ptc or pc 4 h before clp . peritoneal lavages of sigm - deficient and wild - type mice were each pooled from seven mice , and the levels of tnf- , il-6 , and lps , e. coli counts , and neutrophils were assayed ( see materials and methods ) . the levels of tnf- and lps , e. coli counts , and neutrophils were assayed in peritoneal lavage from individual igm - reconstituted mice . comparison of the levels of lps , tnf- , and il-6 in the serum of sigm - deficient and wild - type mice at different time points after clp . sera were collected at 1.5 , 3 , 6 , and 12 h after clp and divided into four groups based on the genotype of the mice and whether the mice died or survived at 32 h after clp . , the numbers of mice used in each category are as follows : / dead , 12 ; / survived , 10 ; + /+ dead , 10 ; and + /+ survived , 16 . numbers of sham controls for tnf- , il-6 , and lps were 2 , 1 , and 1 , respectively .

to evaluate the role of natural immunoglobulin ( ig)m in the immediate response against microbial infection , we tested mutant mice that are deficient in secreted ( s)igm in an acute peritonitis model induced by cecal ligation and puncture ( clp ) . 20% of wild - type mice died within 32 h of clp , whereas 70% of sigm - deficient mice died within the same time period . the increased susceptibility was associated with a reduced level of tumor necrosis factor ( tnf)- , a decreased neutrophil recruitment and an increased bacterial load in the peritoneum , and elevated levels of endotoxin and proinflammatory cytokines in the circulation . resistance to clp by sigm - deficient mice was restored by reconstitution with polyclonal igm from normal mouse serum . reconstitution with a monoclonal igm specific to phosphatidylcholine , a conserved cell membrane component , has a modest effect but a monoclonal igm specific to phosphocholine is not protective . these findings demonstrate a critical role of natural igm in the immediate defense against severe bacterial infection .

Materials and Methods Mice. CLP. IgM Purification. Assays. Results and Discussion Figures and Tables

oxidative stress has been defined as the cellular imbalance of prooxidants versus antioxidants that overwhelms the cell 's capacity to scavenge the oxidative load and contributes to the pathogenesis of various diseases . reactive oxygen species ( ros ) are free radicals derived from molecular oxygen that play a key role in promoting oxidative stress . there are several products of oxygen metabolism , both nonradicals and radicals that form ros such as hydrogen peroxide ( h2o2 ) and superoxide anions ( o2 ) . contributors of ros can modify the intracellular redox status through unfavorable interactions with endogenous regulators of oxidative stress . superoxide radicals can interact with mitochondrial nitric oxide to form peroxynitrite which can alter antioxidant enzymes such as aconitase and the mitochondrial complexes of the electron transport chain . on the other hand , the presence of oxidative stress can alter normal cellular homeostasis by modifying proteins involved in dna repair ; activating signal transduction pathways involved in cell survival and inflammation ; as well as , inducing cellular apoptotic pathways that are detrimental to the cell . for many years , scientists have tried to combat free radical generation and superoxide production through the utilization of the exogenous antioxidant supplementation , such as ascorbate , vitamin e , as well as linoleic acid . however , many of these trials have failed showing no significant decrease in cancer incidence , death , or major cardiovascular events . herein , we will focus on several novel signaling pathways affecting ros generation , such as p53 signaling and the interaction between p53 and manganese superoxide dismutase ( mnsod ) and how to potentially target these pathways for cancer therapy . oxidative stress has been repeatedly shown to contribute to the progression of multiple diseases , such as cancer , diabetes , ulcerative colitis , cardiovascular disease , pulmonary disease as well as neurodegenerative diseases . nevertheless , the biological significance of oxidative stress can be beneficial or detrimental depending on certain parameters such as concentration , duration of action , cell type exposed , the type of free radicals and reactive metabolites involved , and the activities of the associated signal transduction pathways . the mitochondrial electron transport chain remains to be one of the main sources of intracellular oxidative stress . during mitochondrial respiration , electrons flow through four integral membrane protein complexes to finally reduce molecular oxygen to water . however , approximately 1 - 2% of molecular oxygen undergoes incomplete reduction , resulting in the formation of superoxide anions and mitochondria - mediated ros generation . though mainly produced from mitochondrial respiration , superoxide anions can be detoxified via endogenous antioxidant enzymes such as manganese superoxide dismutase ( mnsod ) to hydrogen peroxide , which is further converted to water via the enzymatic actions of various antioxidant enzymes including glutathione reductases , peroxiredoxins , glutathione transferases , as well as catalase which all function in the removal of hydrogen peroxide . nevertheless , it is common for cells in response to stress to enhance ros generation . oxidoreductases are enzymes that are often activated during the cellular stress response and catalyze the transfer of electrons from the electron donor ( reductant ) to the electron acceptor ( oxidant ) with associated formation of superoxide anions and ros as byproducts . there are several enzymes that act as oxidoreductases and contribute to intracellular ros generation , such as cyclooxygenase , lipoxygenase [ 12 , 13 ] , cytochrome p450 enzymes , nitric - oxide synthase , xanthine oxidase , and mitochondrial nadh : ubiquinone oxidoreductase ( complex i ) . napdh oxidases of the nox family are also oxidoreductases that produce superoxide anions as a primary product and one of the key sources of intracellular ros formation . nadph oxidases ( nox ) are endogenous enzymatic heterogenic complexes that reduce molecular oxygen to superoxide , in conjunction with nadph oxidation , which can be converted to various ros . nox can be activated by a myriad of cellular stress stimuli such as heavy metals [ 18 , 19 ] , organic solvents , uv and ionizing irradiation [ 21 , 22 ] . once the cellular stress response is initiated two cytosolic regulatory units of nox , p47 , p67 , and the small g protein rac translocate to the membrane and associate with cytochrome b558 ( consisting of two subunits gp91phox ( nox2 ) and p22phox ) , which acts as a central docking site for complex formation . emerging evidence has linked nox enzymes to oxidative stress that may contribute to disease progression [ 11 , 17 , 24 , 25 ] . the radicals generated from nox activation are capable of modulating various redox - sensitive signaling pathways involved in the activation of mitogen - activated protein kinases ( mapks ) and transcription factors ( nf-b ) [ 2628 ] causing regulation of nox activation to be complex . oxidative stress can be generated endogenously , as well as promoted exogenously by multiple environmental factors . uv is known to damage dna and other intracellular proteins through direct and indirect mechanisms . uv exists in three forms uva ( 400320 nm ) , uvb ( 320290 nm ) , and uvc ( 290100 nm ) . uva and uvb are the most biologically significant , with uvc being most absorbed by ozone . uv is known to directly induce the cross - linking of neighboring pyrimidines to form pyrimidine dimers in dna that result in mutagenic dna lesions [ 3035 ] . however , uv is known to promote ros generation that can damage a large number of intracellular proteins and can indirectly damage dna . high levels of ros are detrimental and can cause damage to various biomolecules , which include the fatty acid side chains of membrane lipids that form reactive organic products such as malondialdehyde and 4-hydroxynonenal , both of which can generate dna adducts and point mutations . lipid peroxidation not only affects dna stability , but can also alter lipid membrane proteins that are involved in signal transduction pathways to promote constitutive activation and downstream cellular proliferation . furthermore , previous studies have shown that products of lipid peroxidation served as intermediates in the activation of signaling pathways that involved phospholipase a2 and the mapk pathway , both associated with uv - induced carcinogenesis [ 3739 ] . although there are various sources of endogenous oxidative stress , mitochondria are the major cellular organelles that contribute to intracellular ros generation . mitochondria consume approximately 8090% of the cell 's oxygen for atp synthesis via oxidative phosphorylation . in the early 1920s otto warburg and colleagues theorized that defective oxidative phosphorylation during cancer progression caused tumor cells to undergo a metabolic shift requiring high rates of glycolysis that promoted lactate production in the presence of oxygen . this phenomenon became known as aerobic glycolysis and later coined the warburg effect . some of the metabolic enzymes that are altered during cancer progression the electron transport chain consists of a constant flow of electrons through mitochondrial intermembrane complexes with molecular oxygen being the ultimate electron acceptor . the process of the electron transport chain is used to pump protons into the mitochondrial inner membrane creating an electrochemical gradient . however , leaking electrons contribute to the incomplete reduction of molecular oxygen , resulting in superoxide anion formation . mitochondria are readily susceptible to oxidative damage for various reasons : ( 1 ) lack of effective base excision repair mechanisms ; ( 2 ) the close proximity of mitochondrial dna to ros generation ; ( 3 ) lack of mitochondrial dna protective histones . therefore , alterations in mitochondrial ros generation and protection via antioxidant expression are key in the detrimental effects of disease progression . however , when this balance is altered in favor of free radical generation , normal physiology is altered and the pathogenesis of disease is promoted . antioxidants are endogenous defense mechanisms utilized by the cell to fight against fluctuations in free radical generation , which include both enzymatic and nonenzymatic contributors . ascorbic acid ( vitamin c ) and -tocopherol ( vitamin e ) are nonenzymatic antioxidants that have been previously shown to effectively scavenge free radicals . on the other hand , antioxidants such as glutathione peroxidase and superoxide dismutase are enzymatic antioxidants that catalyze the neutralization of free radicals into products that are nontoxic to the cell . superoxide dismutase catalyzes the dismutation of superoxide anions leading to the formation of hydrogen peroxide and molecular oxygen . . currently , there are three major superoxide dismutase enzymes within the human cell : manganese superoxide dismutase ( mnsod ) , copper - zinc superoxide dismutase ( cu , znsod ) , and extracellular superoxide dismutase ( ecsod ) . mnsod is localized in the mitochondrial matrix , cu , znsod is found primarily in the cytosol and can be detected in the mitochondrial intermembrane space , and ecsod is a homotetrameric glycosylated form of cu , znsod found in the extracellular space . mnsod is ubiquitously found in both prokaryotes and eukaryotes , and its increased activity is often associated with cytoprotection against oxidants . mnsod can be induced by various mediators of oxidative stress such as tumor necrosis factor , lipopolysaccharide , and interleukin-1 . this antioxidant enzyme is nuclear encoded by a gene localized to chromosome 6q25 , a region often lost in cancers such as melanoma . mnsod is synthesized in the cytosol as a larger precursor with a transit peptide on the n - terminus and imported to the mitochondrial matrix via proteolytic processing to the mature form . most cancer cells and in vitro transformed cell lines have diminished mnsod activity compared to normal counterparts . in addition , deficiencies in mnsod may contribute to oxidative stress generation that promotes neoplastic transformation and/or maintenance of the malignant phenotype . in looking at the correlation between mnsod expression and cancer progression , mutations within the mnsod gene and its regulatory sequence in addition , our laboratory as well as others has shown that overexpression of mnsod reduces tumor multiplicity , incidence , and metastatic ability in various in vitro and in vivo models [ 5457 ] . p53 is a well - characterized transcription factor known to induce its tumor suppressor activity by activating genes known to play a role in cell cycle arrest , such as p21 and gadd45 . these genes , once activated , arrest the cell cycle to allow for adequate dna repair to restore normal cell proliferation . however , if the cell becomes overwhelmed by the stressor or the dna damage can not be repaired , p53 can ultimately induce apoptosis . senescence is characterized by irreversible loss of proliferative potential , acquisition of characteristic morphology , and expression of specific biomarkers such as senescence - associated -galactosidase . nonetheless , emerging evidence suggests ros as a common mediator of senescence with the involvement of superoxide dismutase and p53 . reported that rnai - mediated knockdown of sod1 in primary human fibroblasts induced cellular senescence mediated by p53 . furthermore overexpression of mnsod induced growth arrest in the human colorectal cancer cell line hct116 and increased senescence which required the induction of p53 . on the contrary , p53 can suppress senescence through the inhibition of the mtor pathway via multiple mechanisms [ 6163 ] . nevertheless , this diverse biological spectrum of p53 regulation of cellular function remains complex and is dependent on the source of activation and cell type . there are various sources of p53 activators , which include nucleotide depletion , hypoxia , ultraviolet radiation , ionizing radiation , as well as many chemotherapeutic drugs can act as activators of p53 ( i.e. , doxorubicin ) . in normal cells , p53 remains at a low level and is under strict control by its negative regulator mdm2 . , p53 can bind to the promoter region of the mdm2 gene to promote transcription of mdm2 mrna [ 64 , 65 ] . following proper translation into a functional protein , mdm2 acts as an e3 ligase during p53 activation . mdm2 can polyubiquitinate p53 leading to proteasomal degradation . however , mdm2 can also monoubiquinate p53 leading to intracellular trafficking . the decisive role of p53 to induce cell cycle arrest , senescence , or apoptosis involves intricate posttranslational , as well as , transcription - dependent and transcription - independent mechanisms . the tumor suppressor p53 is a well - characterized transcription factor known to induce the transactivation of proapoptotic genes such as bax , puma , noxa , bid and represses the transcription of anti - apoptotic genes such as bcl-2 , bcl - xl , and survivin [ 68 , 69 ] . many of the transcription - independent mechanisms of p53 were discovered through the use of inhibitors of transcription / translation , as well as p53 truncated mutants with altered subcellular localization , dna binding , and cofactor recruitment . the p53 monomer consists of various multifunctional domains including the n - terminal transactivation domain ( residues 173 ) , a proline - rich region ( residues 6397 ) , the highly conserved dna - binding core domain ( residues 94312 ) , a tetramerization domain located within the c - terminus ( residues 324355 ) , and an unstructured basic domain ( residues 360393 ) ( figure 1 ) . there are multiple polymorphisms that occur within the tp3 gene that may enhance or alter p53 functionality . discovered functional differences in polymorphic variants that enhanced p53-mediated apoptosis independent of its transactivation abilities . a common sequence polymorphism that occurs within the proline - rich domain encoding arginine at position 72 exhibited a fivefold increase in inducing apoptosis compared to the common proline ( pro72 ) variant . these results suggested two mechanisms of arg 72 apoptotic enhancement : ( 1 ) increased mitochondrial localization ; ( 2 ) enhanced binding of the arg 72 variant to the negative p53 regulator e3 ligase , mdm2 . although increased binding to mdm2 did not augment p53 degradation , it was suggested that the altered confirmation of the p53 arg 72 variant enhanced the binding ability and facilitated greater nuclear export . this suggests the importance of understanding the regulation of structure - activity relationships in polymorphic forms of p53 in transcription - independent apoptosis . during p53-mediated apoptosis , a distinct cytoplasmic pool of p53 translocates to the mitochondria . to promote mitochondrial translocation , the e3 ligase , mdm2 monoubiquitinates p53 . since the p53 protein lacks a mitochondrial localization sequence , p53 interacts with bcl-2 family proteins via bcl-2 homology ( bh ) domains . the presence of the bh domain allows proteins to regulate and interact with other bcl-2 members that consist of multiple bh domains . once p53 arrives at the mitochondrial outer membrane , p53 binds to bak inducing a conformational change and bak homo - oligomerization that results in mitochondrial outer membrane permeabilization ( momp ) . momp allows for the release of pro - apoptotic signaling molecules from the outer and inner mitochondrial membranes into the cytosol triggering the intrinsic apoptotic signaling cascade . ros generation has been suggested as an alternative p53 apoptotic target independent of cytochrome c release . . found that ros generation regulated the mitochondrial membrane potential ( ) , which was found to be a key constituent in the induction of p53-mediated apoptosis . interestingly , during ros generation , apoptosis occurred in the absence of bax mitochondrial translocation , bid activation , as well as cytochrome c release . several studies have suggested that the downstream effects of p53-mediated apoptosis are regulated by bax expression . it has been shown that the introduction of recombinant bax protein into isolated mitochondria induced cytochrome c release . the ability of bax to initiate pore formation in synthetic membranes has been shown to regulate cytochrome c release resulting in the induction of apoptosis [ 75 , 76 ] . discrepancies exist with in vivo studies showing bax being localized in the cytosol , rather than within the mitochondrial membrane at physiological conditions . herein , we show how p53 has been shown to play a dual role in early - versus late - stage cancer progression . during the process of carcinogenesis for example , loss of upstream activators of p53 , for example , atm and chk2 , can prevent p53 activation , contributing to unregulated cell cycling and promoting tumorigenesis . in addition , mutations within the p53 protein can alter necessary structure conformational changes and dna binding properties needed for efficient p53 activation . lastly , many of these mutations lead to loss of downstream genes such as bax or noxa which are pro - apoptotic and necessary for regulation of cellular proliferation and death signaling . the process of tumor formation is a multistage process that involves both the activation of protooncogenes , and the inactivation of tumor suppressor genes , such as pten and p53 . the multistage carcinogenesis paradigm consists of three well - characterized stages : initiation , promotion , and progression . during the initiation stage , there is the induction of mutations within critical target genes of stem cells , for example , h - ras ; however in the skin carcinogenesis model , the epidermal layer remains phenotypically normal . during the tumor promotion stage , a noncarcinogenic agent such as a phorbol ester can be used to induce the clonal expansion of the initiated stem cells through epigenetic mechanisms . this stage is often used by investigators to identify potential therapeutic targets due to its reversibility . during the tumor progression stage , malignancy takes place , being characterized by enhanced invasiveness via the activation of proteases , and metastasizes via tumor cells entering into the lymphatics and loss of tumor suppressor activity ( e.g. , p53 ) . the two - stage skin carcinogenesis mouse model has been well characterized and used in numerous studies to screen anti cancer agents . an initiator , such as dimethylbenz[a]anthracene ( dmba ) , is applied to the skin to initiate dna damage within skin cells . following dmba treatment , a tumor promoter such as 12-o - tetradecanoylphorbol-13-acetate ( tpa ) is applied topically to the same area repeatedly for the duration of the study to promote the clonal expansion of mutated cells during the promotion stage . interestingly , during the early stages of dmba / tpa - mediated tumor promotion both oncogenes and tumor suppressor genes are activated , resulting in increased cell proliferation being accompanied by increased cell death ( figure 2 ) . many of the tumor - promoting mechanisms utilized by phorbol esters are directly linked to the involvement of cell surface membranes [ 80 , 81 ] . tpa can mediate its pleiotropic actions through intercalating into the cellular membrane and inducing the activation of the ca - activated phospholipid - dependent protein kinase , protein kinase c ( pkc ) both in vitro and in vivo . tpa can directly activate pkc via molecular mimicry by substituting for diacylglycerol , the endogenous substrate , increasing the affinity of pkc for ca which leads to the activation of numerous downstream signaling pathways involved in a variety of cellular functions including proliferation and neoplastic transformation . in addition , it is known that a direct correlation exists between phorbol ester - mediated tumor promotion and enzymatic activation of pkc [ 82 , 83 ] . pkcs are involved in numerous cellular processes including cell differentiation , tumorigenesis , cell death , aging , and neurodegeneration ; however the induction of the signaling pathway is determined by the intracellular redox status and the isoform that is activated . the pkc family consists of a myriad of isoforms that have been divided into three classes : ( a ) classical or conventional pkcs ( cpkc : , i , ii , and ) ; ( b ) novel pkcs ( npkc : , , , and ) ; ( c ) the atypical pkcs ( apkc : , , and ) which are classified based on sensitivity to ca and diacylglycerol ( dag ) . in various types of cancers pkc has been shown to be upregulated while pkc and pkc are downregulated . furthermore , overexpression of pkc has been shown to enhance the formation of skin carcinomas . moreover , tpa treatment leads to the concomitant activation of the redox - sensitive transcription factor activator protein-1 ( ap-1 ) . there are 3 jun isoforms ( c - jun , jun - b , and jun - d ) and 4 fos family members ( c - fos , fra-1 , fra-2 , and fos - b ) whose activation is modulated by oxidants such as superoxide and hydrogen peroxide , while dna binding activities are modulated by the intracellular redox status [ 8890 ] . kiningham and clair reported a reduction in tumorigenicity and ap-1 dna binding activity following overexpression of mnsod in transfected fibrosarcoma cells . furthermore , the protein expression of bcl - xl , an antiapoptotic ap-1 target gene , was decreased , as well . in addition , pkc activation was reduced in mnsod transgenic mice treated with dmba / tpa compared to their nontransgenic counterparts . these results suggest a mechanistic linkage between mnsod expression , mitogenic activation , and ap-1 binding activity . another activated signaling pathway that has been defined following dmba / tpa treatment is the ras - rac1-nadph oxidase pathway , which leads to p53 mitochondrial translocation and apoptosis . nadph oxidase forms a stable heterodimer with the membrane protein p22 , which serves as a docking site for the sh3 domain - containing regulatory proteins p47 , p67 , and p40 . upon tpa treatment , rac , a small gtpase , mitochondrial p53 has been shown to interact with mnsod , resulting in decreased enzymatic activity and promoting oxidative stress propagation . the primary role of mnsod is to protect mitochondria from oxidative damage . in 2005 , zhao et al . found that tpa treatment , both in vitro and in vivo , can induce p53 mitochondrial translocation . in addition , p53 not only came in contact with the outer mitochondrial membrane but was able to localize to the mitochondrial matrix . interestingly , following p53 mitochondrial translocation and matrix localization , p53 interacted with the mitochondrial antioxidant enzyme mnsod that resulted in a reduction in mnsod activity and propagation of oxidative stress . however , the question remains : does mitochondrial p53 contribute to or suppress tumor promotion during the early stages of skin carcinogenesis ? jb6 cells were originally derived from primary balb / c mouse epidermal cell culture . through nonselective cloning , it was discovered that clonal variants existed within the jb6 cell lineage that were either stably sensitive ( p+ ) or resistant ( p ) to tumor promoter - induced neoplastic transformation [ 9597 ] . in addition , jb6 cells remain the only well - characterized skin keratinocytes for studying tumor promotion and screening anti - cancer agents . in 2010 , we utilized the jb6 p+ and p clonal variants to determine if a relationship existed between tumor promotion and early - stage tpa - induced p53 activation . surprisingly , we found that p53 was only induced in promotion - sensitive p+ cells and not promotion resistant ( p ) cells , therefore suggesting that p53 expression is highly associated with early stage tumor promotion . we then assessed bax protein expression levels , as a marker for p53 transcriptional activity , and found that bax expression is only induced in jb6 p+ cells and not p cells , suggesting that p53 expression , as well as transcriptional activity , is highly associated with early - stage tumor promotion following tpa treatment . mnsod expression was also measured in both jb6 p+ and p cells and was found to be highly expressed in promotion - resistant p cells compared to promotable p+ cells . tpa - mediated ros generation was measured in p+ and p cells ( unpublished data ) , and promotion resistant cells contained significantly lower levels of ros following tpa treatment when compared to their promotable counterparts . it is known that reduced mnsod expression contributes to increased dna damage , cancer incidence , and radical - caused diseases [ 99 , 100 ] . consistent with that , an increase of several markers of oxidative damage such as 4-hne , 8-oxodg , and lipid peroxidation has been seen in both in vitro and in vivo studies following tpa treatment [ 57 , 85 , 101 , 102 ] , suggesting the involvement of oxidative stress in the promotion of tumorigenesis . these results imply the importance of redox regulation in modulating cellular functions during the early stage of tumor promotion . we questioned whether the ros generated from the mnsod - p53 mitochondrial interaction was sufficient to promote tumorigenicity . therefore , we utilized promotion - resistant jb6 p cells that exhibited no p53 protein expression or transactivation following tpa treatment to address this question . interestingly , we found that when jb6 promotion - resistant cells were transfected with wild - type p53 , these cells were able to transform and form colonies in soft agar , in comparison to their control counterparts . these results suggest a dual role of p53-mediated ros generation during the early stages of skin carcinogenesis and how the presence of p53 is necessary for tumor promotion in skin ( figure 3 ) . the contradictory role of p53 in promoting cell survival or death is the result of the ability to regulate the expression of both pro- and antioxidant genes . for example , p53 can promote the generation of ros through the induction of genes involved in mitochondrial injury and cell death which include bax , puma , and p66 and ros - generating enzymes such as quinine oxidoreductase ( nqo1 ) and proline oxidase . however , p53 can upregulate the expression of various antioxidant enzymes to modulate ros levels and promote cell survival such as aldehyde dehydrogenase 4 and mammalian sestrin homologues that encode peroxiredoxins and gpx1 , which are major enzymatic removers of peroxide . previous reports suggest the presence of a p53 binding region at 328 bp and 2032 bp upstream of the transcriptional start site of the mnsod gene [ 104 , 105 ] . others suggest that p53 represses mnsod gene expression by interfering with transcription initiation , inhibiting gene activators at the promoter level by forming an inhibitory complex suppressing gene transcription and protein - protein interactions . p53-mediated mnsod expression is regulated in conjunction with other cell proliferative transcription factors such as nf-b . kiningham and clair demonstrated the presence of an nf-b binding site within the intronic enhancer element of the mnsod gene . it was later shown that mutation of the nf-b site within the enhancer element abrogated p53 induced mnsod gene transcription . in addition , knockdown of p65 via sirna reduced mnsod gene transcription via p53 as well . overall the effects of p53 on mnsod gene expression have been suggested to be concentration dependent , with low concentrations of p53 increasing mnsod expression via corroborative nf-b binding promoting cell survival and high concentrations of p53 suppressing mnsod expression by interfering with important transcriptional binding elements such as sp1 . however , the remaining human tumors contain wild - type p53 with defects in the downstream mediated p53-signaling pathways . this , in turn , provides novel areas of discovery in stabilization and restoration of wild - type p53 activity . currently , many drug companies are focused on utilizing p53 interactions as targets for pharmacological intervention . there are various protein - protein interactions that occur within the cell that positively or negatively regulate p53 expression and function . for example , mdm2 is an e3 ligase of p53 that polyubiquitinates p53 , priming the tumor suppressor for proteasomal degradation . many have found that , by blocking this interaction through peptides or transcriptional inhibitors , longer durations of p53 activation have resulted . some of the therapeutic strategies that are currently being utilized are peptides that increase p53 activation through inhibition of mdm2 function . three - dimensional structural models of the hdm2-p53 interaction along with biochemical data [ 111 , 112 ] have identified three residues that are important to this interaction , phe19 , trp23 , and leu26 [ 111 , 112 ] . from this data , an 8-mer peptide was generated and showed promising results in inducing apoptosis in tumor cells that overexpressed hdm2 . however , these conditions were difficult to optimize with a smaller molecule therefore causing this peptide to be therapeutically inefficient . also nutlins have been utilized to disrupt the mdm2-p53 interaction resulting in reactivation of the p53 response [ 114 , 115 ] . gene replacement therapy is another therapeutic modality that has been explored in treating tumors lacking or containing mutant p53 . this technique utilizes adenoviruses , as well as retroviruses to achieve high expression of p53 in tumor cells . promising results have been seen with retroviral vectors in patients with nonsmall cell lung cancers . on the contrary , although we have seen the enhancement of tumorigenicity in our in vitro p53 transfection studies , we have not tested stably transfected cells in in vivo xenograft mouse models , nor have we tried other tissue types . therefore , the reintroduction of the p53 gene into tumors may have contradictory outcomes depending on the cell type and tissue microenvironment . this concern has echoed through various studies , persuading investigators to opt to combine gene therapy with chemotherapy and radiotherapy [ 118121 ] . for decades in addition , p53-mediated ros generation has been limited to the induction of apoptosis . currently , the ability of wild - type p53 to contribute to tumor promotion has received considerable attention . we have shown that the p53-mnsod interaction contributes to the early stage of tumor promotion . in addition , it has been consistently shown that mnsod activity is altered in human tumors . therefore , designing diagnostic tools to assess mnsod activity , as well as p53 activation , can be used to effectively design individualized treatments for cancer patients . for example , following chemotherapeutic treatment , patients that have higher levels of p53 expression and exhibit lower levels of mnsod can receive an sod mimetic that can upregulate mnsod or synthetic compounds that can downregulate p53 activity to decrease ros - mediated apoptosis and potential relapse within these patients . overexpression of mnsod through gene therapy introducing genetically engineered dna / liposomes containing the human mnsod transgene into preclinical and clinical models has been shown to be protective in normal tissues against ionizing irradiation . the final product ( vlts-582 ) is a dna / liposome formulation that consists of a double - stranded dna bacterial plasmid containing human mnsod cdna in conjunction with two lipids { cholesterol and dotim ( 1-[2-[9-(2)-octadecenoyloxy]]-2-[8-(2)-heptadecenyl]-3-[hydroxyethyl ] imidazolinium chloride ) } . recent studies suggest that this formulation has been successful in murine models and has been administered orally to patients concurrently with a weekly chemotherapy regime exhibiting no dose - limiting toxicities . although proven therapeutically efficacious , more studies are needed to improve ( 1 ) delivery of the transgene to the targeted tissue ; ( 2 ) reducing rapid elimination of the transgene ; ( 3 ) control of the expression of the transgene within targeted tissues . on the other hand , a topical application of an sod mimetic has also been described . the mn ( iii ) porphyrin mn te-2-pyp possesses highly potent sod activity as facilitated by the redox properties of the metal center and the positive charge to the ortho - n - ethylpyridyl nitrogens . mn te-2-pyp has been proven effective in vitro and in various human diseases such as stroke [ 125 , 126 ] , diabetes [ 127 , 128 ] , and cancer and radiation - related treatment [ 129132 ] . in preclinical animal models , topical application of mn te-2-pyp was shown to reduce levels of oxidative damage and reduced cell proliferation without interfering with p53-mediated apoptosis when applied prior to tpa treatment . these data support the concept that overexpression of mnsod when applied in conjunction with standard chemotherapeutics or during the tumor promotion stage is protective in both preclinical and clinical models . nevertheless , both p53 and mnsod have been shown to posses reduced activity and/or mutated in most human diseases including cancer . therefore , more therapeutic quests are needed to detect and restore both mnsod and wild - type p53 activity . however , future therapeutic optimization strategies should have minimal nonspecific drug - related toxicities and be based on the stage of cancer progression which may reveal a therapeutic window for treatment intervention . in summary , reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases . in addition , the tumor suppressor p53 has been shown to be activated during the early stage of skin carcinogenesis and contributed to the propagation of oxidative stress . , p53 physically interacts with mnsod . as a result , this interaction reduces the free radical scavenging abilities of mnsod , promoting enhanced ros generation which has been shown to act as a tumorigenic stimulus during cancer progression . this suggests that wild - type p53 may play a direct role in promoting oxidative stress and contributing to the ros - mediated tumor - proliferative stimuli . in addition , others have shown that mutant p53 can , in fact , translocate to the mitochondria and interact with mnsod . however , lontz et al . observed following doxorubicin treatment of lymphoma cell lines with varying wt or mutant p53 levels , mitochondrial function , as evidenced by complex i / ii activities , was only compromised in lymphoma cells expressing wild - type and not mutant p53 . therefore , the continuation of deciphering mechanistic differences in tumors containing wild - type or mutant p53 can lead to the development of therapeutic p53-mediated interventions and a clearer understanding of chemoresistance in both wild - type and mutant p53 human tumors . several studies have suggested that mnsod may play a primary protective role against tissue injury . mnsod has been found to be depleted in a variety of tumor cells , as well as in vitro transformed cell lines , suggesting that mnsod may act as a novel tumor suppressor , protecting cells from oxidant - induced carcinogenesis . consistent with that , accumulating evidence suggests that a number of antioxidants or drugs with antioxidant properties can reduce mediators of tumor promotion . clair et al . showed that transfecting mouse 10 t 1/2 cells with human mnsod cdna promoted differentiation with 5-azacytidine treatment and protected against neoplastic transformation . in addition , transfecting human mnsod cdna into mcf-7 breast cancer cells and uacc-903melanoma cells suppressed their malignant phenotype and suppressed growth in nude mice [ 54 , 138 ] . we have shown that the cumulative induction of endogenous antioxidant enzymes ( i.e. , catalase , total sod and mnsod ) is efficient in reducing tumor incidence and multiplicity . in addition , the induction of endogenous antioxidant enzymes via dietary administration can suppress p53 mitochondrial translocation . tpa can induce p53 mitochondrial translocation ; however , this phorbol ester also decreases the mitochondrial membrane potential , as well as mitochondrial complex activities and respiration . other studies have shown that mnsod overexpression in mice protects complex i from adriamycin - induced deactivation in cardiac tissue . these results suggest that antioxidant expression protects against fluctuations in mitochondrial functions which suppress p53 mitochondrial translocation , p53-mediated ros , and both downstream apoptotic and cell proliferation signaling pathways . on the contrary , connor et al . suggest that overexpression of mnsod in ht-1080 fibrosarcoma cells and 253j bladder tumor cells enhanced the migatory ability and invasiveness of tumor cells , through the upregulation of matrix metalloproteinases . although some tumors express higher levels of mnsod , the downstream effects of enhanced antioxidant expression are dependent on the tumor type and susceptibility to oxidative damage , underlying oncogenic mutations and the stage of disease progression . therefore the question remains , are the effects of the p53-mnsod interaction protumorigenic or anti - tumorigenic ? to definitively answer this question further investigation of this interaction is needed . however , there are several factors that must be considered in determining the fate of the p53-mnsod interaction , which include the stage of disease progression as well as tumor microenvironment . it has been shown that p53 activation is required in tumor promotion and can mediate ros generation . however , the duration of enhanced ros generation , severity of oxidative damage , and the status of the cellular antioxidant capacity can all contribute to the proliferative / apoptotic switch that occurs during the response to cellular stress . overall , further studies are needed to clearly assess the status of mnsod during the various stages of carcinogenesis to enhance the efficacy of standard treatment regimens currently being used . consistent with that , defining the downstream effects of the p53-mnsod complex formation can expand our knowledge of the molecular mechanisms that contribute to the early stage of tumorigenesis and how they may be altered during cancer progression . with further knowledge , modulators of mnsod , p53 and their associated regulators can be therapeutically useful in the treatment of cancer and various stages of tumor progression .

the formation of reactive oxygen species ( ros ) is a result of incomplete reduction of molecular oxygen during cellular metabolism . although ros has been shown to act as signaling molecules , it is known that these reactive molecules can act as prooxidants causing damage to dna , proteins , and lipids , which over time can lead to disease propagation and ultimately cell death . thus , restoring the protective antioxidant capacity of the cell has become an important target in therapeutic intervention . in addition , a clearer understanding of the disease stage and molecular events that contribute to ros generation during tumor promotion can lead to novel approaches to enhance target specificity in cancer progression . this paper will focus on not only the traditional routes of ros generation , but also on new mechanisms via the tumor suppressor p53 and the interaction between p53 and mnsod , the primary antioxidant enzyme in mitochondria . in addition , the potential consequences of the p53-mnsod interaction have also been discussed . lastly , we have highlighted clinical implications of targeting the p53-mnsod interaction and discussed recent therapeutic mechanisms utilized to modulate both p53 and mnsod as a method of tumor suppression .

1. Introduction 2. Oxidative Stress 3. Manganese Superoxide Dismutase (MnSOD) 4. The Tumor Suppressor p53 5. MnSOD-p53 Mitochondrial Interaction 6. Clinical Implications of the MnSOD-p53 Interaction 7. Concluding Remarks

a severe ocular injury sometimes results in an anterior segment anomaly and a posterior segment disturbance , simultaneously . a variety of corneal injuries can cause an opaque cornea that impedes the visualization of the fundus during vitreoretinal surgery . for this circumstance , an endoscopy - assisted vitrectomy is one solution , and the application of temporary keratoprosthesis ( tkp ) is another solution [ 25 ] . a penetrating keratoplasty ( pkp ) procedure is usually performed after a pars plana vitrectomy ( ppv ) using tkp . however , the long - term outcomes of this triple procedure ( ppv + tkp + pkp ) were not so optimistic for the traumatized eyes [ 25 ] . the main reasons for these unfavorable results were ciliary body malfunction and secondary graft failure . in our center , a different strategy was used in treating patients with coexisting vitreoretinal injury and severe corneal opacification . after a tkp assisted ppv , an allograft corneal transplant was not performed at the same time ; instead , the patient 's trephined corneal button was sutured back . herein , we present the anatomic outcome of this procedure ( tkp + ppv + replacement of the trephined cornea [ rtc ] ) . all subjects in this study were selected from the database of the eye injury vitrectomy study ( eivs ) , which was established in january 1997 . the details and protocols of eivs the type and zone of the injury conform to the recommendations of the united states eye injury registry and the international society of ocular trauma ( birmingham eye trauma terminology ( bett ) ) ; a system for classifying mechanical injuries of the eye . the intraocular area of the injured eye was examined and assessed during vitreoretinal surgery , and the results were recorded by the chief surgeon as baseline information for the register of eye injury form . all of these data were entered into the eivs electronic database using epidata ( the epidata association , odense , denmark ) . for this study , we selected cases from january 2008 to june 2012 in the eivs database . the inclusion criteria include ( 1 ) eyes that suffered from an open globe injury with retinal detachment ; ( 2 ) tkp + ppv + rtc was performed ; ( 3 ) the injured eye was tamponade with silicone oil ; and ( 4 ) the postoperative follow - up was no less than twelve months . the exclusion criteria are patients who have missing records ; those without complete follow - up information ; those who suffered open globe injuries with no retinal detachment ; those who had an allograft transplant during ppv due to corneal infection or severe corneal destruction ; those with gas tamponade eyes ; and those who had a follow - up in less than twelve months . the preoperative examinations include posttraumatic vision , intraocular pressure ( iop ) , and b - scan ultrasonography . most of the relevant intraoperative assessments include type and zone of the injury , causes of corneal opacification , the length of the wound , extension of the severe ciliary body injury ( defined as inability to recognize the structure of ciliary processes in at least two quadrants ) , severe choroidal damage ( defined as a communication between the vitreous cavity and the suprachoroidal cavity caused by choroidal rupture , and which is surgically unrepairable ) , severe loss of the retinal tissue ( defined as retinal loss in at least two quadrants ) , and the retinal reattachment rate during an operation . the follow - up information includes a postoperative vision , iop , and corneal transparency and the anatomical outcome of the injured eyes , which were categorized into four types according to the following flowchart ( figure 1 ) : anatomically restored ( defined as iop no less than 8 mmhg , retina is reattached , and silicone oil is removed ) ; silicone - oil sustained ( defined as iop less than 8 mmhg , silicone oil can not be removed , and the eyes are permanently tamponade with silicone oil ) ; enucleated due to phthisis bulbi ; and recurrent with retinal detachment . under general anesthesia , the pathologic cornea was trephined and the corneal button was protected by a viscoelastic material and kept in a humid container . ocular landers wide field temporary keratoprosthesis ( ocular instruments , usa ) was sutured with a 6 - 0 vicryl suture ( johnson & johnson services , inc . , usa ) . in addition to vitrectomy , surgical procedures may include synechiolysis of the anterior segment , cyclopexy , iridoplasty , lensectomy , membrane peeling , retinotomy and retinectomy , foreign body extraction , subretinal hemorrhage removal , use of perfluorocarbon liquids , and endophotocoagulation . at the end of the surgery , instead of an allograft transplant , 74 eyes of 73 patients met the criteria in this study ( 66 male and 7 female ) . the mean age of the patients was 34.6 15.8 years old ( range , 5~69 ) . for posttraumatic vision , there was no light perception ( nlp ) in 54 eyes ; light perception ( lp ) in 17 eyes ; and hand motion ( hm ) in 3 eyes . the mean posttraumatic iop was 4.7 2.7 mmhg ( range , 2~14 ) . the average interval of injury and vitrectomy ( ioiv ) was 23.8 12.4 days ( range , 5~60 ) . the mean length of the wound was 12.9 6.9 mm ( range , 5~30 ) . the retina of 2 eyes ( 2.9% ) could not be reattached during the surgery . 10 eyes ( 13.5% ) were enucleated due to atrophia bulbi during follow - up . one year after the tkp + ppv + rtc procedure , the vision results were nlp in 28 eyes ( 43.8% ) ; lp in 18 eyes ( 28.1% ) ; hm in 13 eyes ( 20.3% ) ; and finger count in 5 eyes ( 7.8% ) . 16 out of 54 eyes ( 29.6% ) improved from preoperative nlp to postoperative lp ; 29 eyes ( 45.3% ) had improved vision after the tkp + ppv + rtc procedure ; and 35 eyes ( 54.7% ) remained unchanged . the mean postoperative iop during the final visit was 8 3.09 mmhg ( range , 5~16 ) . 10 eyes ( 13.5% ) were enucleated due to atrophia bulbi ; 46 eyes ( 62.2% ) were silicone - oil sustained ; 15 eyes ( 20.3% ) were anatomically restored ; and 3 eyes ( 4.0% ) experienced recurrent rd . when treating patients with coexisting corneal opacification and vitreoretinal disorders , the triple procedure ( ppv + tkp + pkp ) has been commonly used [ 25 ] . however , open globe eye trauma is a unique clinical entity since the injured eye would usually have a complex and multiple intraocular structure disorganization . special attention must be paid to this situation ; and the routine triple procedure ( ppv + tkp + pkp ) may need some modifications for the injured eyes . for instance , in our practice , an allograft was not transplanted during an exploratory vitrectomy at the acute phase of the injury . instead , the patient 's opaque cornea was sutured back . first of all , for severely injured eyes , postoperative persistent hypotony is common ; and a longstanding or permanent silicone - oil tamponade may be needed . if pkp is performed on such eyes , the success rate will dramatically be reduced due to insufficient nourishment of the cornea , which is caused by an inadequate aqueous flow and an unavoidable silicone oil - endothelium contact [ 911 ] . roters et al . reported a series of 34 severely injured eyes that underwent the ppv + tkp + pkp procedure ; 8 eyes were considered phthisical and 10 eyes had a longstanding hypotony . during the follow - up period , the author pointed out that there is a higher risk of graft failure if silicone oil is not removed . in their case series , 21 eyes ( 62% ) had silicone oil - corneal endothelium contact ; and all of those grafts failed . in our case series , 46 eyes ( 62.2% ) experienced persistent hypotony and the silicone oil could not be removed ; and 10 eyes ( 13.5% ) were enucleated due to atrophia bulbi . only 15 eyes ( 20.3% ) were anatomically restored . these figures only demonstrate a small percentage of the injured eyes in our series , which have pkp indications . secondly , hematocornea , which is a common reason for corneal opacification after a severe ocular trauma , is sometimes reversible . brodrick observed that 6 eyes had corneal blood staining due to contusion injuries , which occurs in traumatic hyphema cases . he observed that the cornea became completely clear in 4 out of 6 eyes , although the processes may take a long time . in our case series , hematocornea accounts for 67.6% of corneal opacification . during the one - year follow - up , all eyes with hematocornea showed some degree of clearing ; and in 6 eyes , the cornea became clear enough ; hence , pkp was no longer necessary . for these cases , if allografts were hastily performed , the patients would have suffered a series of unnecessary troubles , such as long - term use of topical steroids and immunosuppressive agents , and a lifetime vigilance for graft rejection . therefore , eyes with corneal blood staining will be observed for at least one year to see if it can resolve by itself . lastly , the injured eyes are usually still congested and inflamed at the time of vitrectomy , which increases the risk of graft failure . . found that a transplant failure was more frequent for eyes that were grafted within 8 weeks of trauma . besides , in areas with donor tissue scarcity , allograft is often not available for the limited time frame of vitrectomy surgery . suturing back the patient 's trephined corneal button allows the pkp to be performed in a relatively quiet eye and provides sufficient time for waiting for the donor . it is undeniable that suturing back the patient 's opaque cornea may cause some inconvenience in observing the fundus after the surgery . however , in many patients , the transparency of the cornea gets better over a period of time after surgery , and the fundus can usually be examined through the area of the transparent cornea . for patients with a completely opaque cornea , there are two major issues we can take into account when determining the visual potential of the eye or making plans for additional surgery : one is the previous surgical record with detailed description of the status of the retina and the other is the level of iop and the sensitivity of light perception and light projection postoperatively . b - scan and endoscopy can be used to help determine the status of the retina . in conclusion , from this large case series with a coexisting corneal and vitreoretinal injury , we found that only 20.3% of the eyes can be anatomically restored and have real value for pkp . there was also a high incidence ( 62.2% ) of persistent hypotony and longstanding silicone - oil tamponade for severely injured eyes . corneal blood staining was the most common reason ( 67.6% ) for using tkp . during follow - up therefore , instead of using the triple procedure ( ppv + tkp + pkp ) as a routine treatment for every patient with coexisting vitreoretinal injury and severe corneal opacification , we advocate suturing back the patient 's trephined cornea during the primary procedure and perform pkp on a later stage , as a secondary procedure for carefully selected cases .

in this case series of 74 patients with coexisting vitreoretinal injury and severe corneal opacification , after temporary keratoprosthesis ( tkp ) assisted pars plana vitrectomy ( ppv ) , an allograft corneal transplant was not performed at the same time ; instead , the patient 's trephined corneal button was sutured back . one year after the surgery , if intraocular pressure of the injured eyes was above 8 mmhg , removing silicone oil was attempted , and penetrating keratoplasty could be performed . finally , 10 eyes ( 13.5% ) were enucleated due to atrophia bulbi ; 46 eyes ( 62.2% ) were silicone - oil sustained ; 15 eyes ( 20.3% ) were anatomically restored ; and 3 eyes ( 4.0% ) experienced recurrent retinal detachment . these figures only demonstrate a small percentage of the injured eyes in our series , which have pkp indications . it is a practical option to suture back the patient 's trephined cornea following a tkp assisted ppv ; keratoplasty was reserved for selected cases .

1. Introduction 2. Patients and Methods 3. Results 4. Discussion

tooth development involves a complex interaction of physiologic growth processes and morphologic stages to achieve tooth 's final form and structure . prevalence of supernumerary teeth ranges from 0.1% to 3.6% of which mesiodens comprise one - third . incidence of mesiodens of 1.6% and distribution of 87% , 12% , and 1% with one , two , and multiple supernumerary teeth respectively has been reported . mesiodens may cause delayed or impaired eruption , displacement , rotation , dilacerations , root resorption , crowding , diastema , cyst formation , infection , or mesiodens pulpitis . the case we present here is about a 15-year - old boy reported with the chief complaint of malaligned upper front teeth causing unesthetic appearance . he had permanent dentition with a full complement of teeth until second molar in both arches and angles class i molar relation both sides . two conical mesiodens were diagnosed one present in the midline and other present palatal to former . right and left central incisor had 180 and 90 torsiversion , respectively [ figure 1 ] . lower right central incisor had gingival recession may be due to occlusal trauma from mesiodens . lateral cephalogram , orthopantomograph and maxillary occlusal radiographs were taken to evaluate the status of the mesiodens along with complete dentition [ figure 1 , table 1 ] . radiographs showed two conical shaped mesidens in the midline with completely formed short roots and no associated root resorption or pathology . model analysis showed 2 mm spacing in the maxillary arch and 2.5 mm of tooth material excess in the mandibular arch and boltons discrepancy with 1 mm excess of tooth material in mandibular anterior region . pre - treatment records cephalmetric analysis after a detailed examination the decision was made to : extract the two mesiodens to relieve the patient of his problems associated with speech and appearancealign upper and lower arches with fixed orthodontic appliancemanagement of boltons discrepancy and lower crowding by proximal stripping of lower anteriors . extract the two mesiodens to relieve the patient of his problems associated with speech and appearance align upper and lower arches with fixed orthodontic appliance management of boltons discrepancy and lower crowding by proximal stripping of lower anteriors . orthodontic treatment was carried out with fixed mechanotherapy using standard edgewise appliance ( 0.022 0.028 ) . two lingual buttons were bonded on each central incisor and derotation was done initially with elastomeric chain [ figure 2a and b ] . the final derotation was done with 0.016 stainless steel multi - loop wire ligated in the brackets bonded on central incisors [ figure 2c and d ] . after complete derotation spacing of 4.5 mm was present in the maxillary arch with 1 mm of overjet . to enable space consolidation bite opening was done with 0.018 stainless steel wire with second order bends and curve of spee and palatal root torquing of incisors was done in 0.019 0.025 stainless steel wire for 4 months followed by elastomeric chain from right first molar to left first molar . proximal stripping was done ( 2.5 mm ) in mandibular anterior region to relieve minimal crowding and eliminate bolton discrepancy followed by finishing for 4 months . finally , composite restoration was done on attrited left maxillary and mandibular central incisors to enhance esthetics . fixed spiral space maintainer was bonded in both arches and case debonded after 1 year 8 months of active treatment [ figure 3 ] . after a detailed examination the decision was made to : extract the two mesiodens to relieve the patient of his problems associated with speech and appearancealign upper and lower arches with fixed orthodontic appliancemanagement of boltons discrepancy and lower crowding by proximal stripping of lower anteriors . extract the two mesiodens to relieve the patient of his problems associated with speech and appearance align upper and lower arches with fixed orthodontic appliance management of boltons discrepancy and lower crowding by proximal stripping of lower anteriors . orthodontic treatment was carried out with fixed mechanotherapy using standard edgewise appliance ( 0.022 0.028 ) . two lingual buttons were bonded on each central incisor and derotation was done initially with elastomeric chain [ figure 2a and b ] . the final derotation was done with 0.016 stainless steel multi - loop wire ligated in the brackets bonded on central incisors [ figure 2c and d ] . after complete derotation spacing of 4.5 mm was present in the maxillary arch with 1 mm of overjet . to enable space consolidation bite opening was done with 0.018 stainless steel wire with second order bends and curve of spee and palatal root torquing of incisors was done in 0.019 0.025 stainless steel wire for 4 months followed by elastomeric chain from right first molar to left first molar . proximal stripping was done ( 2.5 mm ) in mandibular anterior region to relieve minimal crowding and eliminate bolton discrepancy followed by finishing for 4 months . finally , composite restoration was done on attrited left maxillary and mandibular central incisors to enhance esthetics . fixed spiral space maintainer was bonded in both arches and case debonded after 1 year 8 months of active treatment [ figure 3 ] . management of supernumerary teeth depends on the type and position of the tooth requiring analysis of all the clinical and radiographic findings . in the clinical management of mesiodens very often there is confusion whether and when they can be extracted . if teeth are causing no complications and are not likely to interfere with orthodontic tooth movement they can be monitored with yearly radiographic review , but if associated with complications , it is usual to extract such teeth . in this particular case , considering the age of the patient as well as the problems associated with the mesiodens , extraction of the mesiodens was carried out , since both maxillary central incisors had totally erupted showing complete root formation according to delayed approach of extraction of mesiodens . an innovative mechanism of using reciprocal anchorage for correction of torsiversion in central incisors was used . two anchor units were made by the figure of eight ligature and 0.018 sectional stainless steel wire from lateral incisor to molar on both sides . first elastomeric chain was given from distopalatal lingual button on right central incisor to right molar hook . second elastomeric chain was given from mesiobuccal lingual button on left central incisor to left molar hook . a third elastomeric chain was given from mesiobuccal lingual button on right central incisor to distopalatal lingual button on left central incisor [ figure 2a ] . the difficulty encountered during this derotation was a need to reposition distopalatal lingual buttons of both central incisors for effective force application [ figure 2b ] and hindrance from mandibular incisors . glass ionomer cement blocks were given on posterior mandibular teeth to raise the bite and prevent hindrance from mandibular incisors during derotation . the case also had bolton discrepancy of 1 mm which was managed by proximal stripping in mandibular anteriors because of increased mandibular tooth material and incisor mandibular plane angle . therefore , the authors were successful in timely management of a case of twin mesiodens , which was of great esthetic concern to the parents and helped to pervert development of psychological trauma to the child and to eliminate complications due to mesiodens . this paper reports an innovative orthodontic and esthetic management of a patient with erupted twin mesiodens , which had caused torsiversion of maxillary central incisors and attrition due to occlusal trauma .

mesiodens is the most common type of supernumerary teeth found in the premaxilla between the two central incisors . early and proper diagnosis and appropriate treatment plan is critical in eluding the extent of treatment needed . this case report presents the successful orthodontic and esthetic management of an unusual case of indian origin with twin mesiodens in the maxillary arch causing torsiversion and attrition of mandibular incisors due to occlusal trauma .

Introduction Case Report Treatment plan Treatment progress Discussion Conclusions

giant cavernous hemangiomas ( gch ) , measuring more than 6 cm , may harbor unique radiological findings of tumefactive appearance , including mass - effect , midline shift , perilesional edema , and contrast enhancement , with a conspicuous absence of calcifications . such a radiological finding in a child with raised intracranial pressure ( icp ) should be distinguished from intracranial neoplastic lesions . in this report , we describe the clinico - radiological features of a supratentorial gch in a 2-month - old infant and a 14-year - old boy who were admitted with the diagnosis of intra - tumoral bleed . the pathological result made us review the unique radiological findings in retrospect , providing a word of caution while diagnosing such cases . a 2-month - old infant , with no congenital problems , presented to our pediatric emergency following refusal of feeds and vomiting . on examination , the infant had right - sided hemiplegia and anterior fontanelle was bulging . a computed tomography ( ct ) scan showed a circumscribed hyperdense mass lesion involving the left frontal lobe , with chinking of the ipsilateral ventricle and hypodense hemisphere . a magnetic resonance imaging ( mri ) scan revealed a circumscribed mass lesion involving the left frontal lobe , which contained blood of different ages [ figure 1a e ] . the infant underwent left frontal craniotomy and mid - frontal gyrus transcortical approach to mass lesion . postoperatively , the infant was ventilated for 48 hours and extubated a day later . at 6 months of follow - up , ( a ) plain ct scan ( axial section ) showing a hyperdense mass lesion in the left temporal region , with ipsilateral hypodense hemisphere , ( b - d ) mri scan ( t1- , t2-weighted , flair ) showing the left temporal mass lesion with varying densities of blood , indicating cavernoma , and ( e ) ct angiogram showing normal cerebral vasculature a 14-year - old boy presented with a 7-month history of progressive left - sided hemiparesis and bifrontal , throbbing headache . there was rapid worsening of the weakness and associated headache and vomiting over 5 days preceding his first admission at a local hospital . ct of the head revealed a large mass lesion occupying the right parieto - occipital region . as mri facility was not available nearby and he was not in a position to be shifted for mri , he was taken up for tumor excision . intraoperatively , the mass lesion was found to be highly vascular and surgery was abandoned . on arrival at our center , the child was not obeying any commands , had left - sided hemiparesis , and had no vision . radiological evaluation at our center included ct and mri scans , which revealed a well - defined mass in the right parieto - occipital region of size 10 8 8 cm , extending into the atrium of the right lateral ventricle [ figure 2a f ] . on t1 scan , multiple low- to high - signal intensities were observed within the lesion , signaling blood of different ages . upon administration of contrast , minimal enhancement of the lesion was observed . on t2 scans , multiple hypo- to hyperintense signals within the lesion the lesion had herniated out of the previously performed craniectomy defect , thereby easing icp . ( a ) ct scan at presentation , showing status craniectomy and a mass lesion involving the right parieto - occipital region , ( b ) mri scan ( t1-weighted ) showing heterogenous mass lesion , harboring hyperintense signals in a salt and pepper fashion , ( c - d ) contrast - enhanced mri scan showing minimal uptake of contrast by the lesion , ( e ) mri scan ( t2-weighted ) showing hyperintense signals , interspersed with hypointense signals , indicating hemosiderin deposits and blood at various stages , and ( f ) postoperative ct scan showing total excision of gch with a provisional diagnosis of malignant neoplasm , the child underwent re - exploration via previously performed craniectomy . the mass lesion was well defined , encapsulated , multilobulated , and extraparenchymal , and had a reddish brown color . the lesion was excised as a single specimen , as there was a good plane of dissection between the tumor and brain parenchyma . the tumor was firm , rubbery , and compressible , with multiple blood - containing cavities . postoperatively , the patient gradually regained consciousness and the left - sided weakness improved to a near - normal state . the excised specimen showed a relatively circumscribed lesion composed of blood - filled vascular channels of varying sizes . there were thick - walled vascular channels with intervening stroma showing variable fibrosis and moderate lymphomononuclear cell infiltration . elastic fibers within the vessel wall were highlighted as black fibers upon elastic van gieson staining . ( a ) low power showing the relatively circumscribed lesion composed of blood - filled vascular channels of varying sizes ; ( b ) higher magnification ( 200x ) showing thick - walled vascular channels with intervening stroma showing variable fibrosis and moderate lymphomononuclear cell infiltration ; ( c ) many vessels showed fibrin thrombi ; and ( d ) elastic fibers highlighted within the vessel wall as black fibers ( elastic van gieson stain ) a 2-month - old infant , with no congenital problems , presented to our pediatric emergency following refusal of feeds and vomiting . on examination , the infant had right - sided hemiplegia and anterior fontanelle was bulging . a computed tomography ( ct ) scan showed a circumscribed hyperdense mass lesion involving the left frontal lobe , with chinking of the ipsilateral ventricle and hypodense hemisphere . a magnetic resonance imaging ( mri ) scan revealed a circumscribed mass lesion involving the left frontal lobe , which contained blood of different ages [ figure 1a e ] . the infant underwent left frontal craniotomy and mid - frontal gyrus transcortical approach to mass lesion . postoperatively , the infant was ventilated for 48 hours and extubated a day later . at 6 months of follow - up , ( a ) plain ct scan ( axial section ) showing a hyperdense mass lesion in the left temporal region , with ipsilateral hypodense hemisphere , ( b - d ) mri scan ( t1- , t2-weighted , flair ) showing the left temporal mass lesion with varying densities of blood , indicating cavernoma , and ( e ) ct angiogram showing normal cerebral vasculature a 14-year - old boy presented with a 7-month history of progressive left - sided hemiparesis and bifrontal , throbbing headache . there was rapid worsening of the weakness and associated headache and vomiting over 5 days preceding his first admission at a local hospital . on examination , he was drowsy , but obeyed commands infrequently . ct of the head revealed a large mass lesion occupying the right parieto - occipital region . as mri facility was not available nearby and he was not in a position to be shifted for mri , he was taken up for tumor excision . intraoperatively , the mass lesion was found to be highly vascular and surgery was abandoned . on arrival at our center , the child was not obeying any commands , had left - sided hemiparesis , and had no vision . radiological evaluation at our center included ct and mri scans , which revealed a well - defined mass in the right parieto - occipital region of size 10 8 8 cm , extending into the atrium of the right lateral ventricle [ figure 2a f ] . on t1 scan , multiple low- to high - signal intensities were observed within the lesion , signaling blood of different ages . upon administration of contrast , minimal enhancement of the lesion was observed . on t2 scans , multiple hypo- to hyperintense signals within the lesion the lesion had herniated out of the previously performed craniectomy defect , thereby easing icp . ( a ) ct scan at presentation , showing status craniectomy and a mass lesion involving the right parieto - occipital region , ( b ) mri scan ( t1-weighted ) showing heterogenous mass lesion , harboring hyperintense signals in a salt and pepper fashion , ( c - d ) contrast - enhanced mri scan showing minimal uptake of contrast by the lesion , ( e ) mri scan ( t2-weighted ) showing hyperintense signals , interspersed with hypointense signals , indicating hemosiderin deposits and blood at various stages , and ( f ) postoperative ct scan showing total excision of gch with a provisional diagnosis of malignant neoplasm , the child underwent re - exploration via previously performed craniectomy . the mass lesion was well defined , encapsulated , multilobulated , and extraparenchymal , and had a reddish brown color . the lesion was excised as a single specimen , as there was a good plane of dissection between the tumor and brain parenchyma . the tumor was firm , rubbery , and compressible , with multiple blood - containing cavities . postoperatively , the patient gradually regained consciousness and the left - sided weakness improved to a near - normal state . the excised specimen showed a relatively circumscribed lesion composed of blood - filled vascular channels of varying sizes . there were thick - walled vascular channels with intervening stroma showing variable fibrosis and moderate lymphomononuclear cell infiltration . elastic fibers within the vessel wall were highlighted as black fibers upon elastic van gieson staining . ( a ) low power showing the relatively circumscribed lesion composed of blood - filled vascular channels of varying sizes ; ( b ) higher magnification ( 200x ) showing thick - walled vascular channels with intervening stroma showing variable fibrosis and moderate lymphomononuclear cell infiltration ; ( c ) many vessels showed fibrin thrombi ; and ( d ) elastic fibers highlighted within the vessel wall as black fibers ( elastic van gieson stain ) misdiagnosis in such cases is common due to their rarity and variable spectrum of radiological findings . radiological findings range from completely cystic lesions to neoplasm - like masses with striking contrast enhancement and mass effect . furthermore , these heterogenous lesions may have peripheral hemosiderin rim and no significant contrast enhancement and mass effect . these patients often present with seizures due to deposition of epileptogenic iron / hemosiderin pigments in the surrounding regions of the brain . classical cavernous hemangioma has presence of hemosiderin rim and products of blood breakdown within the lesion . however , giant mass lesions , with significant mass effect and no calcifications pose a real diagnostic challenge . the differential diagnoses for these radiological findings include neoplastic lesions such as oligodendroglioma , hemorrhagic and calcified meningioma or primitive neuro - ectodermal tumor , anaplastic astrocytoma , ependymoma , metastatic melanoma , and rare supratentorial atypical teratoid rhabdoid tumor . non - neoplastic differential diagnoses include thrombosed arteriovenous malformations , toxoplasmosis , and cysticercosis . the present case had clinical features of raised icp , radiologically evident contrast - enhancing mass , no calcification , and perilesional edema . even the excised specimen was fleshy , tough , and rubbery , containing blood - filled cavities , suggesting a primitive neuroectodermal tumor with intratumoral hemorrhage . hence , the definite diagnosis rests on the pathological evaluation of the excised specimen , as in the present case . english literature describes nine cases of gcm among children , who had tumor - like presentations . it is supposed that recurrent bleeds occur within the mass lesions , followed by organization of the clot , pseudo - capsule formation , and secondary expansion , thus causing a mass effect . surrounding brain edema is highly unusual , as these lesions are encapsulated and do not invade the surrounding brain . intraoperatively , surgical strategies to prevent excessive bleeding include the use of bipolar cautery so as to shrink the tumor volume and then excising it in toto or coagulating it until its sponge - like tissue becomes fibrotic and then excising it piecemeal .

this study reports two children , including a 2-month - old infant , harboring giant cavernous hemangiomas ( gch ) in the supratentorial compartment , causing raised intracranial pressure and focal deficits . relevant demographic details , clinical presentation , and radiological findings of gch are discussed in light of tumefactive presentation . differential diagnoses of such radiological findings are elaborated .

Introduction Case Report Case 1 Case 2 Discussion

dementia is a growing problem , affecting over 800,000 people in the united kingdom at an annual cost of 23 billion . informal care by friends and family accounts for 55% ( 12bn ) of this cost ; this would have to be covered by health and social services , if carers were unable to cope . the number affected by dementia is expected to rise with the ageing population , placing a heavier burden on both families and health and social services in the future . from an economic perspective , it is important to support informal carers in their role to enable people with dementia to remain living at home as long as possible . in response to the increasing pressure being placed on scarce health and social care resources , the uk government has made a commitment to double spending on dementia research to 66 million per year by 2015 following calls by the alzheimer 's society to increase funding substantially ; therefore , it is timely to consider the appropriateness of current health economics instruments used to measure quality of life . in a survey of the general uk population , respondents were asked to list up to five things that affected quality of life . over 60% stated relationships with family and other people , 43% selected their own health , and 38% selected the health of somebody they were close to . caring has been found to affect mental health more than physical health , with up to 30% of carers of people with dementia experiencing depression . with increased public expectations of what treatments should be made available on the national health service ( nhs ) , choices have to be made about whether or not to fund specific health care interventions . informed funding decisions can only be made after a rigorous economic analysis of the costs and benefits of competing alternatives has taken place . in the uk , the national institute for health and care excellence ( nice ) assesses evidence on the clinical - effectiveness and cost - effectiveness of treatments . the nice guide to technology appraisal states that effectiveness should be reported in quality - adjusted life years ( qalys ) , with the eq-5d their preferred questionnaire for measuring health - related quality of life component of the qaly . guidance on whether the nhs should fund a treatment is based on whether the cost per qaly falls below an arbitrarily chosen funding threshold of 20,00030,000 . however , despite nice favouring the use of the eq-5d , there are arguments for including alternative outcome measures . the eq-5d is dominated by physical health questions , which places a patient 's identity on the carer . carer interventions can cross the health and social care sector ; therefore , instruments that focus on physical functioning underestimate the full effects on quality of life . in this paper , we argue for the routine inclusion of broader quality of life measures alongside the eq-5d in research involving carers of people with dementia . recent developments include three instruments : the icecap - o , the carer experience scale ( ces ) [ 11 , 12 ] , and the adult social care outcomes toolkit ( ascot ) . the icecap - o was developed through prior research into quality of life of members of the general population aged 65 + . it is an appropriate instrument as many carers of people with dementia fall into this age group . the icecap - o has been validated for use with the general older population and in a hospital setting [ 14 , 15 ] . the carer experience scale was developed through qualitative research with carers and validated through a survey of carers in the general population . the ascot was developed to measure social care related quality of life for the care recipient but the domains might be applicable for evaluation of quality of life for carers too . while similar in format to the eq-5d , these instruments contain domains which measure the capability of an individual to achieve a range of outcomes . to explore whether these instruments would be suitable for use with carers of people with dementia , a qualitative approach was adopted . qualitative research allows a deeper exploration of a subject and can be conducted alongside quantitative research to enhance understanding and put results into a meaningful context . while it is not often possible to generalise findings across a whole population , qualitative research is a useful tool for informing the choice of instruments used in quantitative methods . the qualitative research described in this paper was undertaken to explore the question what do family carers of people with dementia perceive as affecting quality of life and how well do capability based instruments capture these aspects of quality of life ? framework analysis was selected as it is systematic and allows transparency in the data analysis . it is the opposite of more inductive approaches , such as grounded theory , as the focus is not on developing a new theory but instead on describing and interpreting participants ' views . the coreq checklist is used to report the qualitative research presented in this paper . carers were recruited through distributing information sheets face - to - face at alzheimer 's cafes in north wales and through the mailing list of the neurodem ( wales dementias and neurodegenerative diseases research network ) research participant register , a register of carers and people with memory problems who have given permission to be contacted about research projects . the information sheet explained the aim of the study , and that information was being collected as part of a phd study examining quality of life measurement in carers of people with dementia . carers were asked to contact the lead author ( carys jones ) , if they were interested in participating . it did not matter whether participants were current or former carers because a lived experience viewpoint was sought . convenience sampling was used and participants were selected opportunistically to ensure as homogeneous a sample as possible . no target sample size was set ; participants were recruited until data saturation occurred . due to the potentially sensitive nature of the topic , semistructured interviews were held at a location convenient to the carer , typically their home , between november 2010 and july 2011 . interviews were planned to be held with only the interviewer and participant present ; however , in two of the interviews the person with dementia was also present . interviews were conducted by the lead author ( carys jones ) , a female phd student . before the interview commenced , carys jones asked participants to read the information sheet again and provide written consent to take part . participants were reminded that they could stop the interview or ask questions at any time . repeat interviews were not conducted . an interview schedule containing open ended questions about the participant 's experiences as a carer was used . questions were designed to encourage participants to talk about both past experiences and concerns about the future . the interview schedule was not tested prior to use ; however , after each interview , the schedule was reviewed to determine whether modifications were needed . interviews lasted between 22 and 54 minutes and were recorded using a digital recorder , with additional notes taken during the interview . interviews were transcribed verbatim by carys jones with identifying information , such as names , changed to protect confidentiality . the lead author familiarised herself with data through repeated listening to the recordings and reading of all of the transcripts . the domains of the eq-5d , icecap - o , ces , and ascot were used as predetermined codes . the icecap - o measures an individual 's capability to achieve an outcome regardless of whether they carry out the functioning ; for example , respondents are asked whether they can have all the love and friendship that they want rather than if they have all the love and friendship that they want . the domains of the icecap - o are love and friendship , thinking about the future , doing things that make you feel valued , enjoyment / pleasure , and independence . there are four possible levels for each domain : i can have all , i can have a lot , i can have a little , and i can not have any . the icecap - o is scored from 0 ( no capability ) to 1 ( full capability ) . its six domains are activities outside caring , support from family and friends , assistance from organisations and the government , fulfilment from caring , control over caring , and getting on with the person you care for . the ces is scored from 0 ( worst caring state ) to 100 ( best caring state ) . domains of the ascot are control over daily life ; personal cleanliness and comfort ; food and drink ; personal safety ; social participation and involvement ; occupation ; accommodation cleanliness and comfort ; and dignity . each domain has four levels and the ascot is scored between 0.17 ( no care needs met ) and 1 ( all care needs met ) . the predetermined codes ( i.e. , the domains of the capability instruments ) were sought in the data using a line by line coding method in nvivo by carys jones . a sample of transcripts was reviewed by coauthors rhiannon tudor edwards and barry hounsome to improve rigour ; however , as carys jones had led the research and conducted the interviews , she was more immersed in the data and was ultimately responsible for coding decisions . similar ideas thought to affect quality of life were grouped into categories , which were then refined into broader themes . the original transcripts were cross - checked to ensure that the themes and their interpretation were grounded in the participant 's descriptions . sections not relevant to the theme have been removed and replaced by ellipses ( ) . repetition and hesitations not thought to add meaning , such as erm , you know , and umm , have been removed without ellipses . in compliance with the terms of the data protection act , contact details for participants were stored securely in a password protected file on a computer that only carys jones had access to . hard copies of consent forms were archived in a locked cabinet in a locked room , the key being held by carys jones . four themes were identified : social network and relationships ; interactions with agencies ; recognition of role ; and time for oneself . this theme encompasses the social support that carers perceive they have and how their relationships with both the person with dementia and others had changed . a change in the ability to communicate with the person being cared for was a source of upset . we always had this very strong relationship and we always used to think the same things once the alzheimer 's started all his personality changed , that all went , as if we were n't on the same wavelength at all . we always had this very strong relationship and we always used to think the same things once the alzheimer 's started all his personality changed , that all went , as if we were n't on the same wavelength at all . ( c1 ; female , bereaved spouse ) . in the case of the child carer , as dementia had progressed , it facilitated a closer relationship than had been experienced before . i had my arm around her and i always try and massage her back or just touch her hand or just try and be quite tactile with her . it 's come to it that this has got to happen for us to actually give her a hug . i had my arm around her and i always try and massage her back or just touch her hand or just try and be quite tactile with her . she was n't a very tactile person at all it 's come to it that this has got to happen for us to actually give her a hug . family and friends were seen as a secondary social support network , both for practical care tasks and emotional support . one does n't do much with his dad but he 'll come and say to me oh tidy yourself up and i 'll take you out for a meal.the other son , we see him nearly every day , and he does what his dad ca n't do anymore . he mends things listening to a lot of people , i think i 'm alright . one does n't do much with his dad but he 'll come and say to me oh tidy yourself up and i 'll take you out for a meal.the other son , we see him nearly every day , and he does what his dad ca n't do anymore . he mends things listening to a lot of people , i think i 'm alright . relationships with friends and family could also become strained , if there was a lack of understanding about dementia . they used to come here and the first questions they used to ask him do you remember how we used to do so and so ? in the end i had to tell them not to remind him , or ask him questions . because you 'd see then that robert would get quite frustrated . it 's family , recently i 've been trying to get through ( to ) them there 's a problem but they 're not interested , as far as they 're concerned you look fine so you are fine . they used to come here and the first questions they used to ask him do you remember how we used to do so and so ? in the end i had to tell them not to remind him , or ask him questions . because you 'd see then that robert would get quite frustrated . it 's family , recently i 've been trying to get through ( to ) them there 's a problem but they 're not interested , as far as they 're concerned you look fine so you are fine . carers were anxious about socialising in a wider circle , if they felt that the behaviour of the person with dementia might cause embarrassment . i 've been to the memory clinic , or memory caf rather , on two or three occasions , but i do n't feel that either brenda or myself have benefitted from that . what it amounts to is that you sit at a table possibly with other people , have a cup of tea and a biscuit , and you might have a talk by the fire brigade or the police , which in the case of brenda really is of no interest and occasionally she makes adverse remarks very loudly during the lecture , which was an embarrassment . i 've been to the memory clinic , or memory caf rather , on two or three occasions , but i do n't feel that either brenda or myself have benefitted from that . what it amounts to is that you sit at a table possibly with other people , have a cup of tea and a biscuit , and you might have a talk by the fire brigade or the police , which in the case of brenda really is of no interest and occasionally she makes adverse remarks very loudly during the lecture , which was an embarrassment . social activities , such as dining out or shopping , were restricted , if it was felt that the person with dementia was not enjoying the experience . he 'd become very agitated if he was somewhere strange , and with strange people . we used to go to the christmas dinner , but he always wanted to come home . he 'd become very agitated if he was somewhere strange , and with strange people . we used to go to the christmas dinner , but he always wanted to come home . this theme refers to the carers ' perceptions about their experiences with medical staff , social services , and organisations such as crossroads and the alzheimer 's society . this was typically caused by the person with dementia not acknowledging that there was a problem and refusing to see a doctor or , once an appointment was made , the doctor not confirming the symptoms as being dementia . the lengthy process of getting a diagnosis caused stress and self - questioning about whether there really was something wrong with the person being cared for . naturally for self preservation [ sic ] reasons , clinicians , doctors are very reluctant to say that the patient has alzheimer 's disease . it took a year to get the diagnosis . which i think is probably actually fairly quick compared to some people . but it was actually almost a bit of a relief to actually know that we were n't sort of imagining that it [ sic ] . ( c3 ; female , spouse ) . naturally for self preservation [ sic ] reasons , clinicians , it took a year to get the diagnosis . which i think is probably actually fairly quick compared to some people . but it was actually almost a bit of a relief to actually know that we were n't sort of imagining that it [ sic ] . carers felt that information received from various agencies was fragmented and not received at an appropriate stage of the illness . some stated that they would have liked more information at the time of diagnosis ; others mentioned that at the start of the transition from spouse to carer they did not want to hear about practical care tasks that might become necessary as the person with dementia deteriorated , such as dealing with incontinence and feeding needs . some carers also found the amount of extra support received immediately after a dementia diagnosis to be overwhelming . two carers mentioned that it was difficult to access health and social care services during the night - time . services unless it was an emergency situation as he did not want to be seen as a burden . occasionally i refused it because i thought i could go my own way , but i was in the wrong . one of the things that was missing in my case , having any support at night the cpn said you can ring me any time which was n't quite true as her mobile would be turned off because she was tending someone else or something else , and at night time i did not like calling because it 's her time off . occasionally i refused it because i thought i could go my own way , but i was in the wrong . one of the things that was missing in my case , having any support at night the cpn said you can ring me any time which was n't quite true as her mobile would be turned off because she was tending someone else or something else , and at night time i did not like calling because it 's her time off . ( c5 ; male , spouse in long - term care ) . where services had not met the carer 's expectations , it got to a stage where it was 2 o'clock in the afternoon and she was still in bed , and i felt pretty desperate about it . i got in touch with the social services that suggested that perhaps if someone came in , she would respond to a figure of authority they used to come in for perhaps ten or fifteen minutes , if she would still not get up they said because they 're not allowed to physically intervene they would come and ask for my help anyway . i felt that was a bit of a fiasco . at the end of the couple of months or whatever i had a bill for 300 , and i did n't really feel that i 'd had very much in the way of assistance . oh , we 're not at liberty to give out specific advice like [ sic ] which homes shall i go to . the cpn , community health care people , they said the same : we 're not allowed to recommend homes . ( c5 ; male , spouse in long - term care ) . it got to a stage where it was 2 o'clock in the afternoon and she was still in bed , and i felt pretty desperate about it . i got in touch with the social services that suggested that perhaps if someone came in , she would respond to a figure of authority they used to come in for perhaps ten or fifteen minutes , if she would still not get up they said because they 're not allowed to physically intervene they would come and ask for my help anyway . at the end of the couple of months or whatever i had a bill for 300 , and i did n't really feel that i 'd had very much in the way of assistance . oh , we 're not at liberty to give out specific advice like [ sic ] which homes shall i go to . the cpn , community health care people , they said the same : we 're not allowed to recommend homes . caring can be associated with increased levels of stress and depression ; however , positive aspects such as fulfilment from caring were identified . in the past charles would have dealt with a lot of the things that i now may be do n't deal with but i help him with he would have dealt with all the financial side of things and the paying of the bills and although i drove , he used to like to do a lot of the driving..he was n't a great diy person or anything like that but it 's all the sort of small things that you do n't really think that he automatically used to do that i now find myself doing . so i 'm probably busier than i have been for quite a few years ( laughing ) . ( c3 ; female , spouse ) . in the past charles would have dealt with a lot of the things that i now may be do n't deal with but i help him with he would have dealt with all the financial side of things and the paying of the bills and although i drove , he used to like to do a lot of the driving..he was n't a great diy person or anything like that but it 's all the sort of small things that you do n't really think that he automatically used to do that i now find myself doing . so i 'm probably busier than i have been for quite a few years ( laughing ) . seven participants mentioned events which they believe triggered or accelerated the onset of dementia in the person they cared for . carers felt guilt or blamed themselves for those events even if they were unavoidable , such as having their own health problems . when i had the operation i was away from her for two weeks cause i had the operation in cambridge and she had to look after the dogs at home . so she did n't see me for two weeks and when i came back she was quite distressed . when i had the operation i was away from her for two weeks cause i had the operation in cambridge and she had to look after the dogs at home . so she did n't see me for two weeks and when i came back she was quite distressed . all of the carers had successfully adapted to the change in role from being a spouse / child to a carer . i 'm very much like right , ok , things happen , i need to work on it . i 'm very much like right , ok , things happen , i need to work on it . carers were keen to help others through recording their own experiences and raising awareness of dementia and its progression over time . and i do try and talk to people that i know are going through it , right at the beginning and try and give as much of my information as i can , the things that we went through . that 's why i 'd quite like to get this little diary that i 've got published as a little book . and i do try and talk to people that i know are going through it , right at the beginning and try and give as much of my information as i can , the things that we went through . that 's why i 'd quite like to get this little diary that i 've got published as a little book . the theme of time for oneself recognises the perceived value of having time away from the person being cared for and hence time away from caring . participants spoke of being able to resume activities they had previously discontinued because the person being cared for had not shared their interest . my wife and i were opposites when we met at the time i thought this might be a good marriage , because we can then each benefit from each other 's experience . but it never really worked out like that ; i tended to abandon all my academic interests i did n't begrudge at the time , but now that she 's off , and i have every other day to myself and doing things which i did in my youth i'm taking up movie making again , and things like this , which i did before we got married . my wife and i were opposites when we met at the time i thought this might be a good marriage , because we can then each benefit from each other 's experience . but it never really worked out like that ; i tended to abandon all my academic interests i did n't begrudge at the time , but now that she 's off , and i have every other day to myself and doing things which i did in my youth i'm taking up movie making again , and things like this , which i did before we got married . a sense of frustration was evident when the carer was not able to spend time away from the person being cared for . television now has become just action ; all he can watch is these action things like schwarzenegger i ca n't stand them but i 've got to sometimes go along with it and try and read we do have two televisions at home but if i go to watch he 'll find ( me ) and say what you watching ? i think i 'll watch that then cause he wants to be with me . television now has become just action ; all he can watch is these action things like schwarzenegger i ca n't stand them but i 've got to sometimes go along with it and try and read we do have two televisions at home but if i go to watch he 'll find ( me ) and say what you watching ? i think i 'll watch that then cause he wants to be with me . as well as having a greater feeling of independence , the carers of people who had moved into long - term care also spoke of the guilt they experienced at having to make the decision . it 's awful , in one way it gives me the freedom but on the other hand i feel awful picking her up for a little while , you know , having a good time and then just dumping her . it 's awful , in one way it gives me the freedom but on the other hand i feel awful picking her up for a little while , you know , having a good time and then just dumping her . interventions involving carers of people with dementia might have multiple objectives , such as improving burden , coping skills , and general quality of life . the need to select appropriate outcome measures for economic evaluations has been recognised [ 23 , 24 ] . by focusing on health - related quality of life measurement , the nice guide to technology appraisal bodies allocating research funding should check that outcome measures listed for proposed research match the objectives of the intervention rather than relying on the use of historically popular measures . the capability instruments that we choose to explore there is scope to improve current research practice by considering these alternative measures to capture quality of life . in the context of family medicine , using instruments which are sensitive enough to detect subtle changes in quality of life will lead to more informed decisions being made when scarce health care resources are being allocated . as coast discussed , there is a role for qualitative work in the traditionally quantitative field of health economics ; however , researchers must be careful to use methods appropriately to produce work which passes the scrutiny of both health economists and qualitative researchers . the aim of this study was to elicit what carers of people with dementia perceived as impacting on their quality of life , and hence whether capability based instruments capture these aspects of quality of life and are appropriate for use in future health services and health economics research . four themes were identified : social network and relationships ; interactions with agencies ; recognition of role ; and time for oneself . spouses felt a sense of loss for the person they used to know and sometimes found it difficult to communicate with the person their spouse had become . the subsequent layer of social support was the wider network of friends and family , reflecting the findings of etters et al . , who in a review of carer burden noted the importance of positive family relationships and support . . also found that the closer the kinship to the person being cared for , the higher the level of burden perceived . carers in this study had experienced a reduction in their extended social support network as a result of avoiding social situations and loss of friends due to normal aging . in a trial of a counselling and support intervention compared to usual care for spouses of people with alzheimer 's disease , it was found that carers who utilised support services were able to keep their partner at home longer than those who did not . the mechanism behind this was thought to be an improvement in response to behavioural problems and increased carer satisfaction with social support , which relates to the theme of social network and relationships found in this study . the social network and relationships theme overlapped with all three capabilities based instruments , with the ces exhibiting the most overlap . only the anxiety / depression domain of the eq-5d was thought to have a clear conceptual overlap with the social network and relationships theme . interactions with agencies were closely tied to the domain of assistance from organisations and the government on the ces . , the diagnosis process was a source of frustration , sometimes caused by the person with dementia refusing to admit to having problems with their memory , and sometimes caused by the medical professional not being supportive . in some cases , the person with dementia appeared to be fine during an appointment so the staff would only assess the symptoms of dementia ( or lack of them ) that they witnessed themselves . previous qualitative work involving practitioners revealed four obstacles that delayed a formal diagnosis ; therapeutic nihilism ; risk avoidance ; concerns about self - competency in managing dementia care ; and availability of resources . in our study the carers perceived the delay in diagnosis to be mainly concerned with risk avoidance and therapeutic nihilism on the part of the professionals . under the carers ( equal opportunities ) act 2004 , carers are entitled to an assessment of their needs by the social services . if there is a delay in diagnosis , it will hold up the process of the carer being assessed and supported . opinions were mixed about whether carers should be given a lot of information at the time of diagnosis or whether knowing the potential outcomes associated with dementia would be distressing . having more knowledge about dementia has previously been linked to family carers displaying an increased preference for moving the person with dementia into a long - term care facility . the authors of the study hypothesized that people who were more aware of the potential decline associated with dementia were able to recognize that they might not be able to cope with caring . in our study , carers praised staff who they thought had given them good practical advice and information about caring . these findings suggest that the level of information offered to carers should be judged on a case by case basis , with further information given freely if requested . the biggest change in role experienced by carers in our study occurred for the daughter who was looking after her mother . she described a reversal of roles , where she now assumed the parent role and her mother had regressed to being like a child . other carers compared the experience to looking after a child , a perception also found in the quinn et al . quinn et al.also found that those with a close emotional relationship with the person being cared for prior to the commencement of caring had lower levels of burden and saw caring as rewarding . as well as describing stress and burden , carers spoke of positive experiences arising from their new role . these ranged from closer relationships between the carer and the person being cared for to a sense of feeling appreciated . one bereaved carer had become a volunteer befriender to continue giving in a carer role . in this study , the carers of people in long - term care had become involved in fundraising and raising awareness of dementia as a way of helping others . recognition of role is reflected in the control over caring domain of the ces , the occupation domain of the ascot and the doing things that make you feel valued domain of the icecap - o . as well as experiencing fulfilment from caring , participants acknowledged a need for time away from caring . this time was used to catch up on chores as well as pursuing leisure activities . the ability to be independent depends on the level of support received from the social network and agencies so this theme is closely linked to the first two . younger carers often have more competing time demands as they juggle caring , working , and looking after their own family , and this was found to be the case for the child carer included in our study . however , a review of caregiver burden and depression suggested that adult child caregivers are more likely to have alternative roles and social activities outside of caregiving , which might moderate the stresses associated with caregiving . the ascot has three domains that cover the time for oneself theme : control over daily life ; accommodation , cleanliness , and comfort ; and personal cleanliness and comfort . the icecap - o domains of independence and thinking about the future were also linked to the theme . two themes of the eq-5d were thought to overlap with the theme : self - care and usual activities . exploring quality of life and how experiences shape the capability of individuals to successfully cope with caring is of great importance in dementia care . the eq-5d , which focuses on physical health , had the least amount of conceptual overlap with the identified themes . two eq-5d domains were not thought to fit in with the quality of life themes identified by this work : pain / discomfort and mobility . as nice prefers cost - effectiveness to be reported as a cost per qaly , calculated with a preference based utility measure , there is a need to continue using the eq-5d in research involving carers of people with dementia . the themes emerging from this exploratory qualitative analysis suggest that capability based instruments are a potential addition to the health economists ' toolkit for measuring quality of life in carers of people with dementia . all domains of the icecap - o and ces were thought to overlap with the identified themes . this is unsurprising as both instruments were designed to measure capability based quality of life in similar populations ; the icecap - o for an older population ; and the ces for carers . the ascot had three domains that did not fit with the themes : dignity , food and drink , and personal safety . however , the ascot was developed primarily for use with the person receiving social care rather than their carer . for this reason it might be that the icecap - o and ces are more suitable for carer research . the icecap - o was designed to capture capability based quality of life of people aged 65 years and over ; so while it will be suitable for a lot of spousal carers , it will not be appropriate for younger people , such as child carers . some people do not self - identify as carers , and as such may feel more comfortable filling out a questionnaire such as the icecap - o which asks more broadly about quality of life , instead of answering the questions specific to caring that appear in the ces . the ces was developed using interviews with carers for a range of illnesses , and results from this study suggest that it would be equally suitable for dementia carers . in research aiming to capture a greater insight into the pragmatic experience of caring for people with dementia , the ces would be preferable to the icecap - o . limitations . qualitative work should be interpreted in its context , which restricts the generalisability of results . carers in this study were recruited through the alzheimer 's society and a research register . as such , participants were engaged with a number of local services already and might not be representative of families who do not yet have a formal dementia diagnosis . carers were offered a choice of interview location ; in two interviews the person with dementia was present , which might have made the carer uncomfortable discussing the negative impact of caring . all participants were white and living in suburban or semirural locations ; it is unclear whether different themes would emerge from the experiences of carers of different ethnicities or living in an urban area with better access to services and this is an area that needs to be explored in future work .

background . in an ageing population , many individuals find themselves becoming a carer for an elderly relative . this qualitative study explores aspects of quality of life affected by caring for a person with dementia , with the aim of identifying whether capability based questionnaires are suitable for measuring carer quality of life . methods . semistructured interviews lasting up to an hour were conducted , november 2010july 2011 , with eight family carers of people with dementia . interviews typically took place at the participants ' homes and were recorded and transcribed verbatim . framework analysis was used to code and analyse data . domains from three capability based questionnaires ( icecap - o , carer experience scale , and ascot ) were used as initial codes . similar codes were grouped into categories , and broader themes were developed from these categories . results . four themes were identified : social network and relationships ; interactions with agencies ; recognition of role ; and time for oneself . conclusions . by identifying what affects carers ' quality of life , an appropriate choice can be made when selecting instruments for future carer research . the themes identified had a high degree of overlap with the capability instruments , suggesting that the capabilities approach would be suitable for future research involving carers of people with dementia .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

they were isolated from a herbaceous perennial plant called peganum harmala family ( zygophillacea)-(syrian rue or harmal ) ( 1).the plant had been used extensively in the traditional medicine of middle east , india , china and africa as emmenagogue , hallucinogen , snuff , abortifacient , antidepressant , anti - inflammatory , antimalarial , anti - leishmania and anti - microbial ( 1 ) . nowadays , many -carbolins have been found to possess antibacterial , antiviral , antiparasitic and anticancer activities ( 1 ) . chloroquine ( cq ) is still the safest , cheapest and the most efficient among other antimalarials , ( 2 ) but unfortunately it started to lose its token as a potent drug due to emergence of chloroquine resistant and tolerant strains of plasmodium falciparum . the former is associated with loss of drug response to higher doses while the latter is accompanied by higher incidence of the disease reoccurrence ( 3 , 4 ) . this issue has urged the scientist to search for other alternatives or agents that can chemosensitize cq and reduce its resistance or tolerance ( 5 ) . in this communication , both the anti - plasmodium and cq resistance reversal potencies of three different -carbolins alkaloids ; harmalin , harmol and harmalol were tested against both 3d7 ( cq sensitive ) and k1 ( cq resistant ) strains of p. falciparum . both cq sensitive and resistant strains of p. falciparum k1 and 3d7 were cultured in o+ red blood cells suspended in a complete malaria culture medium ( cmcm ) containing rpmi-1640 , 25 mm hepes ( ph 7.4 ) , 0.75 mm hypoxanthine , 0.5% albumax , 24 mm sodium bicarbonate , 11 mm glucose and 50 g / l gentamicin . the culture was incubated at 37 c in a micro - aerophilic atmosphere containing 90% n2 , 5% co2 and 5% o2 . furthermore , the medium was changed every 24 h and parasitemia was monitored using giemsa stained thin blood smears ( 6 , 7 ) . prior to drug screening , the parasites were synchronized using sorbitol synchronization technique described by ( 8) . stock solutions of 100 mm of chloroquine and each of the mentioned phytochemicals were prepared in pbs ( ph 7.4 ) and a mixture of [ methanol : dimethylsulphoxide ( dmso ) ( 1:1 ) ] respectively . aliquots of 50 l of prbcs suspension ( synchronized at the ring stage at 2% parasitemia and 2% hct ) were uploaded to a 96 well flat bottomed microtiter plates ; featured serial dilution of cq or each phytochemical ( 1 nm to 1 m ) as well as drug , rbcs and prbcs controls wells , were incubated for 48 h at 37 c with prbcs ( synchronized at the ring stage with 2% parasitemia and 2% hct ) . drug dilution was done so that dmso concentration did not exceed its cytotoxic threshold against p cells and rbcs ( < 0.5% ) . three plates were prepared for each drug and each dilution was done in triplicate . at the end of the incubation period , the plates were freeze - thawed and then 100 l of sybr green - i lysis buffer was added to each well . the mixture was incubated at room temperature for 1 h and finally the fluorescence was measured after 15 seconds of plate agitation ( twice ) in victor plate reader ( perkin elmer , salem , ma ) at an excitation / emission wavelength of 485/535 nm . each of the geometric mean of the first and second pass was used to exclude any measurement error ( 9 ) . both ic50 and ic90 for cq against both p. falciparum k1 & 3d7 were determined using microsoft excel 2007 software according to the recommended protocol of percentage of parasite inhibition versus log [ drug concentration ] . for drug combination assay , working solutions of cq and each of the mentioned -carbolin were prepared from their stocks at concentrations equivalent to 16 times of their ic50 such that their ic50s fall in the fourth two - fold serial dilution . then cq solution was mixed with each test comp at 10:0 , 7:3 , 5:5 , 3:7 and 0:10 ( cq / test comp . in nm then the combinations were uploaded in triplicate and serially diluted for 8 times within a flat - bottomed 96 well plate . after that , an equal volume of prbcs suspension ( synchronized at ring stage at 2% hct and 2 % parasitemia ) was added to the wells that contained ( cq / test comp . ) then the plates were incubated at the standard abovementioned conditions ( section 2 - 1 ) for 48 h and treated in the same way as in the drug sensitivity assay to determine parasite growth and assess both ic50 and ic90 of each combination separately . for each combination ratio , both fic50 and fic 90 ( fractional inhibitory concentration ) were calculated determining the ratio of ic50 or ic90 of each drug within the combination to those values when the drug was incubated alone . at the end , both ic50 and ic90 based isobologram curves were derived through plotting both fic50 and fic90 for each content of the combination ( cq and the test comp . ) on y and x axes respectively . the line that link the two fics , is considered as the line of additivity so that if the plot falls on , the interaction is considered additive ( fig . if it falls above or below the line , then the interaction is considered either antagonism or synergy respectively . some authors dictate that the interaction is considered as complete synergy , if the total fic50 or fic90 ( fic50 or 90 cq+ fic50 or 90 comp ) is equal to or less than 0.5 , antagonism if it is > 2 , additive if it is 0.51 and indifferent if it is in the range of 12 ( 11 ) . both cq sensitive and resistant strains of p. falciparum k1 and 3d7 were cultured in o+ red blood cells suspended in a complete malaria culture medium ( cmcm ) containing rpmi-1640 , 25 mm hepes ( ph 7.4 ) , 0.75 mm hypoxanthine , 0.5% albumax , 24 mm sodium bicarbonate , 11 mm glucose and 50 g / l gentamicin . the culture was incubated at 37 c in a micro - aerophilic atmosphere containing 90% n2 , 5% co2 and 5% o2 . furthermore , the medium was changed every 24 h and parasitemia was monitored using giemsa stained thin blood smears ( 6 , 7 ) . prior to drug screening , the parasites were synchronized using sorbitol synchronization technique described by ( 8) . stock solutions of 100 mm of chloroquine and each of the mentioned phytochemicals were prepared in pbs ( ph 7.4 ) and a mixture of [ methanol : dimethylsulphoxide ( dmso ) ( 1:1 ) ] respectively . aliquots of 50 l of prbcs suspension ( synchronized at the ring stage at 2% parasitemia and 2% hct ) were uploaded to a 96 well flat bottomed microtiter plates ; featured serial dilution of cq or each phytochemical ( 1 nm to 1 m ) as well as drug , rbcs and prbcs controls wells , were incubated for 48 h at 37 c with prbcs ( synchronized at the ring stage with 2% parasitemia and 2% hct ) . drug dilution was done so that dmso concentration did not exceed its cytotoxic threshold against p cells and rbcs ( < 0.5% ) . three plates were prepared for each drug and each dilution was done in triplicate . at the end of the incubation period , the plates were freeze - thawed and then 100 l of sybr green - i lysis buffer was added to each well . the mixture was incubated at room temperature for 1 h and finally the fluorescence was measured after 15 seconds of plate agitation ( twice ) in victor plate reader ( perkin elmer , salem , ma ) at an excitation / emission wavelength of 485/535 nm . each of the geometric mean of the first and second pass was used to exclude any measurement error ( 9 ) . both ic50 and ic90 for cq against both p. falciparum k1 & 3d7 were determined using microsoft excel 2007 software according to the recommended protocol of percentage of parasite inhibition versus log [ drug concentration ] . for drug combination assay , working solutions of cq and each of the mentioned -carbolin were prepared from their stocks at concentrations equivalent to 16 times of their ic50 such that their ic50s fall in the fourth two - fold serial dilution . then cq solution was mixed with each test comp at 10:0 , 7:3 , 5:5 , 3:7 and 0:10 ( cq / test comp . in nm . then the combinations were uploaded in triplicate and serially diluted for 8 times within a flat - bottomed 96 well plate . after that , an equal volume of prbcs suspension ( synchronized at ring stage at 2% hct and 2 % parasitemia ) was added to the wells that contained ( cq / test comp . ) then the plates were incubated at the standard abovementioned conditions ( section 2 - 1 ) for 48 h and treated in the same way as in the drug sensitivity assay to determine parasite growth and assess both ic50 and ic90 of each combination separately . for each combination ratio , both fic50 and fic 90 ( fractional inhibitory concentration ) were calculated determining the ratio of ic50 or ic90 of each drug within the combination to those values when the drug was incubated alone . at the end , both ic50 and ic90 based isobologram curves were derived through plotting both fic50 and fic90 for each content of the combination ( cq and the test comp . ) on y and x axes respectively . the line that link the two fics , is considered as the line of additivity so that if the plot falls on , the interaction is considered additive ( fig . if it falls above or below the line , then the interaction is considered either antagonism or synergy respectively . some authors dictate that the interaction is considered as complete synergy , if the total fic50 or fic90 ( fic50 or 90 cq+ fic50 or 90 comp ) is equal to or less than 0.5 , antagonism if it is > 2 , additive if it is 0.51 and indifferent if it is in the range of 12 ( 11 ) . drug sensitivity assay showed that p. falciparum 3d7 was quietly sensitive to cq ( ic50 = 22.3 0.76 nm ) while k1 was resistant ( ic50= 265 3.3 m ) . meanwhile , both of them were poorly sensitive to each of harmalin , harmalol and harmol ( ic50= 3.993 0.019 , 17.632 0.094 and 14.470.075 m , ic90= 7.14 0.03 , 42.670.12 & 45.060 0.09 m respectively ) . results of isobologram analysis showed that only harmalin could have synergistically reduced cq resistance when it was combined at 7:3 and produced an additive effect at 5:5 ( cq / harmalin ) . the other combinations did not show any interaction except for harmol which showed an additive interaction when it was combined at a ratio of 7:3 ( cq / harmol ) ( fig . 1 , table 1 ) . the ic90 based isobologram shows that harmalin could have synergistically reduced cq tolerance when it was combined with cq at a ratio of 7:3 ( cq / harmalin ) while harmol produced an additive effect at this ratio . overall , according to the results of the two isobolograms , cq / harmalin combination at a ratio of 7:3 represents the most optimum combination to reduce both resistance and tolerance in p. falciparum k1 ( fig . 1 , table 1 ) . medical importance of -carbolin alkaloids has been studied extensively both in vitro and in vivo . they have been used profoundly in the folk chinese , arabic , middle eastern and african medicines to treat various ailments and infectious diseases including leishmaniasis and malaria ( 12 ) . in vitro effect of -carbolins on human cancerous and non - cancerous cells had been studied previously . they were found to have a cytotoxic effect against cancer cell line as possessed genotoxic effect characterized by dna intercalation , induction of chromosomal aberration , induction of dna damage , formation of dna adducts and inhibition of dna excision repair ( 13 , 14 ) . furthermore , they interfere with the intracellular enzymatic system as they were found to be inhibitors of topoisomerase enzyme ( 15 ) , -hydroxylase , monooxygenase , triosphosphate isomerase and cycline dependant kinase . moreover , other studies had proven that they are effective inhibitors of heat shock protein system that protects protein scaffolds against any mis - folding . their effect was more pronounced on cancer cells and they were reported to potentiate other cytotoxic drugs in cancer chemotherapy ( 16 ) . it is noteworthy that different -carbolin induce different extent of cytotoxic action on cancer cell lines ( 16 ) . in our study , we tried to apply the same model on plasmodium cells through combining the three mentioned -carbolin derivatives with cq and screening their anti - plasmodium effect and their potency to reverse cq resistance in p. falciparum k1 . it had been used widely to deduce the type of interaction between two different compounds ( 10 ) . effect of each combination was tested on both ic50 and ic90 of the combined compounds . the former predestines using higher dose to eradicate the parasite whilst the latter requires protracting the treatment period ( 3 , 4 ) . our results showed that p. falciparum was more sensitive to harmalin as compared to harmol and harmalol . furthermore , harmalin was more effective in suppressing both cq resistance and tolerance as seen in the results of the ic50 and ic90 based isobolograms . on the other hand , harmol had succeeded in ameliorating cq tolerance without producing a pronounced effect on its resistance . nevertheless , harmalol did not show any visible interaction when it was combined with cq . the anti - plasmodial effect of the mentioned -carbolins and the ability of some of them to reverse cq resistance in p. falciparum k1 can be attributed to its re - known effects on cellular physiology . they had been found to arrest the cell cycle through dna intercalation and dna adducts formation , compromising dna replication fidelity and dna repair system and inhibition of dna strands break ( 1720 ) . furthermore , they were found to inhibit some vital enzymes involved in cellular replication , viz ; topoisomerase ( 21 ) , cycline dependant kinase ( 22 ) . not only is cellular replication a target for -carbolins action , they were found to affect some other biological targets , such as ; protein kinase c , sodium dependent uptake of the dibasic amino acids , oxygenase , epoxide hydrolase , hydroxylase , epoxide hydrolase and triosephosphate isomerase ( 23 ) . effect of -carbolins on these targets had been studied extensively on cancerous and non - cancerous mammal cells . different derivatives have different intensity of action on each target and indeed , there is a variation in their effect on mammalian and protozoal cells . in spite of the similarity in their action , phytochemicals affect mammalian cells in a way different from their effect on protozoal cells due to the difference in the allosteric site wherein the drug can combine . further studies are recommended to find the interactive effect of the other carbolins and more investigations are required to determine the precise mechanism through which carbolins affect plasmodial targets . plasmodia digest hemoglobin inside the digestive vacuole to use its protein part as a source of amino acids and release heme as a toxic byproduct . hindrance of cq accumulation through inhibition of its influx or triggering its efflux is one of the mechanisms through which plasmodia exhibit cq resistance . agents that inhibit the transporter system that pumps cq outside the vacuole or trigger cq influx can be used to reverse cq resistance ( 4 ) . a recent study has found that cq can affect the dv membrane integrity at concentrations lower than what is required to hinder hemozoin formation . this promotes the dv membrane permeabilization and exodus of some of the dv hydrolytic enzymes especially those whose g.m.wt is low , viz ; cathepsin . cathepsin exodus into the protoplasm induces the cascade of the programmed cell death process of apoptosis . the study denoted to the importance of apoptosis process in progression of cq induced cell death ( 24 ) . the obtained synergism between cq and harmalin is more likely to be attributed to its effect on this apoptotic pathway as the previous studies had pointed out to its powerful effect on arresting the cell cycle but no study had pointed to their effect on membranous drugs transporters . anyway , further investigations are recommended to prove this notion and to justify the interactive effect of this -carbolin with cq . overall , both cq sensitive and resistant strains of p. falciparum 3d7 and k1 show weak to moderate response to harmalin , harmol and harmalol . harmalin could have showed synergy through suppressing cq resistance in k1 , although the degree of suppression was not enough to reverse the resistance and render k1 to be cq susceptible . on the other hand , in spite of their anti plasmodium effect , -carbolins are incapable of producing full eradication of malaria parasite . nevertheless , some of them are effective in reducing cq resistance and tolerance in the resistant strain of p. falciparum . further studies are recommended to monitor their in vivo efficiency and their safety to be used for this purpose .

background : nowadays , scourge of malaria as a fatalistic disease has increased due to emergence of drug resistance and tolerance among different strains of plasmodium falciparum . emergence of chloroquine ( cq ) resistance has worsened the calamity as cq is still considered the most efficient , safe and cost effective drug among other antimalarials . this urged the scientists to search for other alternatives or sensitizers that may be able to augment cq action and reverse its resistance.method:three -carbolin derivatives , namely , harmalin , harmol and harmalol were tested for their anti - plasmodial and cq resistance reversal effects against p. falciparum 3d7 and k1 . sybre green-1 based drug sensitivity assay and isobologram analysis were used to screen the mentioned effects respectively.results:all of them showed moderate anti - plasmodium effect and harmalin was the most effective as compared to the others in reversing cq resistance and tolerance.conclusion:the mentioned phytochemicals are not ideal to be used as conventional antimalarials and only harmalin can be suggested to reverse cq resistance in p. falciparum k1 .

Introduction Materials and Methods Parasite culturing, maintenance and synchronization Stock solution preparation Malaria drug sensitivity assay Estimation of IC Drug combination assay and isobologram analysis Results Discussion Conclusion

in the activation measurements using the nuclear reactor , the neutron capture reaction rates are the product of the neutron flux at the sample position and the neutron capture cross - section of irradiation nuclide . the neutron capture cross - section at particular neutron energy is the probability that a neutron at that energy will be captured by the nuclide . the standard energy for tabulation of thermal neutron cross - section ( 0 ) is that of room temperature ( 293.6k or 20.43 c ) , corresponding to a neutron energy 0.0253 ev or to a neutron velocity of 2200 m / s . however , in many reactors , the temperature ( t ) in the irradiation channels is not 20.43 c , but at higher temperature . westcott developed a method for converting 0 to (t ) , which is the effective cross - section at the actual temperature ( t ) of the irradiation channel , by describing the neutron spectrum as a combination of a maxwellian distribution function , which is characterized by a temperature ( t ) , and a component of epithermal energy neutrons . according to westcott , the reaction rate per target atom is given by:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r = \phi_{0 } \mathop \sigma \limits^{\uplambda } ( t ) = nv_{0 } \sigma_{0 } \left [ { g(t ) + r\sqrt { t / t_{0 } } s_{0 } } \right ] $ $ \end{document}where 0 = nv0 neutron flux defined as the total neutron density times the 2200 m / s neutron velocity ; \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mathop \sigma \limits^{\uplambda } ( t ) $ $ \end{document } effective cross - section ; 0 thermal cross - section for 2200 m / s neutron ; g(t ) parameter which represents the departure of the cross - section from the 1/v law in the thermal region ( g(t ) = 1 if nuclide obeys the 1/v law in this energy region ) and which can be calculated from expression:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g(t ) = { \frac{2}{{\sqrt { \pi e_{0 } } \sigma_{0 } } } } \int\limits_{0}^{\infty } { \sqrt e } \sigma ( e)\sqrt { e / e_{t } } e^ { { - e / e_{t } } } de / e_{t } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{t } = e_{0 } t / t_{0 } $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{0 } = 0.0253 $ $ \end{document } ev , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ t_{0 } = 293.6 $ $ \end{document}k , e neutron energy , and v neutron velocity . c ) to 673.16 k ( 400 c ) for many nuclides were calculated by holden . \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r\sqrt { t / t_{0 } } $ $ \end{document}epithermal index which denotes the strength of the epithermal flux ( it is zero for a pure thermal flux ) and s0parameter which represents the ratio of the resonance integral and thermal cross - section . according to the hogdahl s convention , a method , theoretically less rigorous , but commonly used defines the reaction rate by:3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r = \phi_{th } \phi_{th } + \phi_{epi } i $ $ \end{document}where th , i sub - cadmium cross - section and the epi - cadmium cross - section , respectively ; th neutron flux defined as the thermal neutron density times the 2200 m / s neutron velocity ; epi epithermal flux per unit ln(e ) . from eqs . 1 and 3 it follows that ( see ) : 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{th } = g(t)\sigma_{0 } + { \frac{{\phi_{epi } } } { { \phi_{th } } } } ( 1 - 2\sqrt { e_{0 } - e_{c } } ) g(t)\sigma_{0 } $ $ \end{document}or5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_{th } = g(t)\sigma_{0 } + { \frac{{\phi_{epi } } } { { \phi_{th } } } } [ \updelta i ' - i(1/v ) ] $ $ \end{document}with6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ i(1/v ) = \int\limits_{{e_{c } } } ^{\infty } { g(t)\sigma_{0 } \sqrt { e_{0 } /e } } de / e \cong 2g(t)\sigma_{0 } \sqrt { e_{0 } /e_{c } } $ $ \end{document}where ec cd - cut - off energy ( this cut - off energy is a function of thickness of cd ; ec = 0.55 ev if a small cylindrical cd - filter of 1 mm thickness ) , i(1/v)1/v - part of resonance integral , i and i is the epi - cadmium resonance integral excluding the 1/v part and part , shielded by cd - filter , which depends on the neutron temperature , respectively . clearly , looking at eqs . 1 , 3 , 4 , 5 , and 6 there is one notice that the cross - sections and the resonance integral vary as a function of g(t ) , which are temperature dependence . many authors often assume th = 0 to calculate the resonance integral , it is not completely justified . only the nuclides with cross - section obey the 1/v law up to 12 ev , g(t ) would be unity as mentioned above and s0 = 0 ; then th and the resonance integral are no longer depends on temperature at the irradiation position . thus , in the activation measurements using the nuclear reactor , neutron temperature ( t ) at the irradiation position should be known to preserve the accuracy of the results . in experiment , a well - known method for the determination of the neutron temperature in the irradiation channels is co - irradiation of lu by using lu ( n , ) lu reaction and 1/v - monitors with acceptable accuracy . in this work , we describe a method for the determination of the neutron temperature based on thermal neutron spectrum calculated by mcnp code . as we know , neutron spectrum at an irradiation position can be expressed as a sum of a thermal equilibrium spectrum ; th(e ) ( maxwellian distribution , neutron energy from 0 to 0.55 ev ) and epithermal spectrum in the slowing down region ; epi(e ) ( neutron energy from 0.55 ev to maximal ) . in the paper [ 6 , 7 ] , the neutron spectra were written by semi - empirical functions:7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varphi_{th } \left ( e \right ) = i_{th } \left ( { { \frac{e}{{e_{t}^{2 } } } } } \right)e^ { { - { \frac{e}{{e_{t } } } } } } $ $ \end{document}8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \varphi_{epi } \left ( e \right ) = i_{epi } \left ( { \frac{e}{1ev } } \right)^ { - \alpha } .\frac{1}{e } $ $ \end{document}where ith and iepi are scaling constants for the thermal and epithermal portion ; et is a characteristic energy of maxwellian portion of the spectrum . the neutron temperature is given by:9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e_{t } ( ev ) = 8.617 \times 10^ { - 5 } from the temperature in degrees kelvin , we can convert to temperature in degrees celsius by : t273.15 . thus , if the neutron spectrum at the irradiation position is known , et would be determined by fitting maxwellian portion of the spectrum according to eq . this method was applied to determinate neutron temperature in the irradiation channels 7 - 1 , 1 - 4 , the rotary specimen rack and neutron trap of dalat reactor , vietnam . it is a general purpose monte carlo code , which facilitates independent or coupled neutron , photon and electron transport calculations . the code treats an arbitrary three - dimensional configuration of material and geometric cell and provides a versatile description of the source , the variance reduction techniques , a flexible tally structure and an extensive collection of cross - section data in continuous energy representation . for neutron , all reactions given in a particular cross - section data evaluation are accounted for and cover the energy range from 10 to 20 mev . used mcnp version 4b for the calculation of spectral parameters in typical irradiation channels of the triga reactor , ljubljuna . the comparison between the simulated and experimentally determined parameter f is in good agreement except for channel ic40 . [ 6 , 7 ] also used mcnp for calculation of some characteristic parameters of neutron flux in irradiation channels of dalat reactor . the comparison of results obtained by experimental and monte carlo simulation showed good agreement for all channels . the obtained results in that papers showed that monte carlo calculation of neutron spectra in irradiation channels of reactor provide useful information about the nature of spectra , the neutron flux , as well as reactor s spectral characteristics . the cylindrical reactor is arranged symmetrically by several kinds of material elements . in the center of active region the fuel elements , beryllium and graphite blocks , control rods and irradiation channels are the active region of reactor and form a hexagonal prism lattice in the cylindrical reactor with radius 20.8 cm and height 60.0 cm . outside of the active region is a graphite reflector with thickness of 32.6 cm . . 1.fig . 1configuration of reactor core of dalat reactor configuration of reactor core of dalat reactor the detailed mcnp computation model used for the calculation of neutron spectra in irradiation channels of 7 - 1 , 1 - 4 , the rotary specimen rack and neutron trap of dalat reactor is based on a model thoroughly described by hung et al . kcode ( criticality problem ) and ksrc ( initial spatial distribution of source points ) cards are used for the source description . in this case , mcnp offers several options for the keff values estimate , including the combined average of the absorption , collision and track - length estimator . for all calculations of neutron spectra , the f4 tally was used . f4 is a track - length tally card for calculating average neutron flux over a cell . the s ( , ) thermal treatment was used for h2o , beryllium and graphite . it is a general purpose monte carlo code , which facilitates independent or coupled neutron , photon and electron transport calculations . the code treats an arbitrary three - dimensional configuration of material and geometric cell and provides a versatile description of the source , the variance reduction techniques , a flexible tally structure and an extensive collection of cross - section data in continuous energy representation . for neutron , all reactions given in a particular cross - section data evaluation are accounted for and cover the energy range from 10 to 20 mev . used mcnp version 4b for the calculation of spectral parameters in typical irradiation channels of the triga reactor , ljubljuna . the comparison between the simulated and experimentally determined parameter f is in good agreement except for channel ic40 . [ 6 , 7 ] also used mcnp for calculation of some characteristic parameters of neutron flux in irradiation channels of dalat reactor . the comparison of results obtained by experimental and monte carlo simulation showed good agreement for all channels . the obtained results in that papers showed that monte carlo calculation of neutron spectra in irradiation channels of reactor provide useful information about the nature of spectra , the neutron flux , as well as reactor s spectral characteristics . the cylindrical reactor is arranged symmetrically by several kinds of material elements . in the center of active region the fuel elements , beryllium and graphite blocks , control rods and irradiation channels are the active region of reactor and form a hexagonal prism lattice in the cylindrical reactor with radius 20.8 cm and height 60.0 cm . outside of the active region is a graphite reflector with thickness of 32.6 cm . 1.fig . 1configuration of reactor core of dalat reactor configuration of reactor core of dalat reactor the detailed mcnp computation model used for the calculation of neutron spectra in irradiation channels of 7 - 1 , 1 - 4 , the rotary specimen rack and neutron trap of dalat reactor is based on a model thoroughly described by hung et al . kcode ( criticality problem ) and ksrc ( initial spatial distribution of source points ) cards are used for the source description . in this case , mcnp offers several options for the keff values estimate , including the combined average of the absorption , collision and track - length estimator . for all calculations of neutron spectra , the f4 tally was used . f4 is a track - length tally card for calculating average neutron flux over a cell . the s ( , ) thermal treatment was used for h2o , beryllium and graphite . therefore , this value is quite satisfactory . the energy spectra with 115 groups of energy from 0 ev to 20 mev at 1 - 4 , 7 - 1 channels and rotary specimen rack and neutron trap are calculated . as an example , fig . 2 shows a calculated energy spectrum in 7 - 1 channel and the fitted et - value in the thermal neutron region . the relative error of the calculated spectrum intensity in thermal neutron region is about than 1% and in the epithermal is about 3% for all irradiation channels.fig . 2calculated neutron flux spectrum in 7 - 1 channel of dalat reactor using mcnp4c code ( dark line ) and fitted et - value in the thermal neutron region ( light line ) calculated neutron flux spectrum in 7 - 1 channel of dalat reactor using mcnp4c code ( dark line ) and fitted et - value in the thermal neutron region ( light line ) from the neutron spectra calculated by mcnp code , the et values are determined by fitting the neutron spectra in the thermal neutron region ( neutron energy from 0 to 0.55 ev ) according to eq . the et - fitting values and the temperatures in degrees celsius in the irradiation channels are presented in table 1 . the uncertainty of the neutron temperature values determined from fitting the calculated energy spectra in the thermal neutron region is about 2.5% ; it defends on the uncertainty of the calculated spectrum intensity in thermal neutron region.table 1the fitted et values and the neutron temperatures in irradiation channels of dalat reactor determined from the neutron spectra calculated by mcnp code and experimentirradiation channelsfitted et- values ( 10 ev)calculated temperatures ( c)experimental temperatures ( c)lu au monitorneutron trap2.714 0.02841.8 1.01 - 4 channel2.727 0.02943.3 1.07 - 1 channel2.827 0.03152.0 1.053.0 2.0rotary specimen rack2.698 0.02740.0 1.0 the fitted et values and the neutron temperatures in irradiation channels of dalat reactor determined from the neutron spectra calculated by mcnp code and experiment as a comparison to fitted value , an experiment of the t - determination in 7 - 1 channel was performed . the choice of 7 - 1 channel is suitable for the experiment due to 7 - 1 channel coupled with a pneumatic sample transfer system . the t - determination method was presented in paper . after measuring the irradiated monitors , the westcott factor for lutetium [ g ( t)]lu could be obtained . thus , the neutron temperature also was determined by using the tables [ g ( t)]lu vs. tn in paper . the comparison of the result of the neutron temperature in 7 - 1 channel shows that the value of the neutron temperature agrees well with each other . , it also shows that the neutron temperatures in the irradiation channels of dalat reactor are from 40 c ( in the rotary specimen rack ) to 53 c ( 7 - 1 channel ) in the core of reactor . the neutron temperature in 7 - 1 channel is highest , because it is a dry channel using for neutron activation analysis ( naa ) only . note that , in general , the recommended k0 , au using in the neutron activation analysis in reports is the average k0 , au - value corresponding to an average neutron temperature of 60 c . hence , in k0 naa standardization method , the neutron temperature at the irradiation position is importance for nuclides having ( n, ) reaction which do not exactly obeys the (v ) ~ 1/v law up to 12 ev . the cross - section of ( n, ) reaction of these nuclides depends clearly on the neutron temperature . for this reason , 7 - 1 channel is suitable in naa using k0 standardization method , because there is no need of the correction of neutron temperature . the neutron temperature values in the channels of 7 - 1 , 1 - 4 , the rotary specimen rack and neutron trap of dalat reactor were determined from the neutron spectra in the thermal neutron region , which was calculated by mcnp code . it was compared with the experimental value in 7 - 1 channel and showed that the values are agreement with each other . the results from this study suggest that base on the neutron spectrum calculated by mcnp code , the neutron temperature in the irradiation channels of reactor will be determined by fitting the spectrum in the thermal neutron region according to eq . it is simple , effective and is a good method for the verification of experimental results .

the neutron temperature is a characteristic parameter in irradiation channels of reactor . for nuclides which have resonances in the thermal neutron energy range , their westcott g - factors are different from unity . the values of g - factors and cross - sections of ( n , ) reaction of these nuclides are temperature dependence . the standard energy for tabulation of thermal neutron cross - section ( 0 ) is that of room temperature ( 293.59 k or 20.43 c ) , corresponding to a neutron energy 0.0253 ev or to a neutron velocity of 2200 m / s . however , in the irradiation channels of reactor , the temperature is not exact at 20.43 c . thus , the temperature at the irradiation position must be known to convert 0 to (t ) . a method for determination of the neutron temperature in irradiation channels of dalat reactor is presented by fitting the thermal neutron spectrum obtained from the calculation using mcnp code .

Introduction Base of method MCNP code and configuration of Dalat reactor core MCNP code Configuration of Dalat reactor core Results and discussion Conclusion

this was a prospective observational study of patients attending the department of endocrinology at pusan national university hospital . the study was conducted with the approval of the institutional review board of pusan national university hospital . a total of 264 korean type 2 diabetic patients were consecutively enrolled at the outpatient clinics between may 2008 and december 2009 . all patients fulfilled the following inclusion criteria : age 18 years and estimated gfr ( egfr ) 30 ml / min/1.73 m. we excluded patients with thyroid disorders or who had been medicated within 6 months prior to the study because thyroid function could affect the cystatin c level ( 19 ) . additional exclusion criteria were 1 ) active urinary tract infection , 2 ) renal disease other than diabetic nephropathy , 3 ) neoplastic disorders , 4 ) severe liver dysfunction , 5 ) active or chronic infection or inflammatory disorders , 6 ) pregnancy , and 7 ) a recent ( within 6 months ) history of acute myocardial infarction , stroke , or occlusive peripheral vascular disease . a random spot urine sample and a blood sample were obtained from each patient at the clinic visit . medical histories and anthropometric measurements the egfr level was calculated using the modification of diet in renal disease ( mdrd ) study formula for the korean population : mdrd = 107.904 ( serum creatinine [ mg / dl ] ) age ( 20 ) . a correction factor of 0.667 the serum and urinary cystatin c levels were measured by the latex agglutination test ( modular p800 ; roche diagnostics , mannhein , germany ) . the interassay and intra - assay coefficients of variations of cystatin c in our laboratory were as follows : < 4.2 and < 3.4% , respectively , for serum and < 7.9 and < 10.1% , respectively , for urine . urine specimens with cystatin c levels < 0.01 mg / l were assumed to have a concentration of 0.01 mg / l . the data on urinary cystatin c was also expressed as ratios of urinary cystatin c to urinary creatinine in order to assess different hydration states and renal functions of the patients . the ratio between urine mass concentrations of cystatin c and creatinine in micrograms per millimole was calculated and designated as the urinary cystatin c - to - creatinine ratio ( ccr ) . since we obtained total proteinuria and albuminuria values from each patient at baseline , we were able to estimate the amount of nonalbumin proteinuria through the following calculation : nap - to - creatinine ratio ( napcr ) = protein - to - creatinine ratio ( pcr ) albumin - to - creatinine ratio ( acr ) . the lowest detectable level and the coefficient of variation in our laboratory were as follows : for total proteinuria , 0.7 mg / dl and < 4.8% , respectively ; for albuminuria , 0.2 mg / dl and < 7.4% , respectively . they were managed to give priority to the best treatment according to standard guidelines at each outpatient clinic of two endocrinologists ( i.j.k . and s.s.k . ) . thirteen patients were excluded during follow - up as follows : four patients died of other causes , six patients were hospitalized for acute myocardial infarction and active infections , such as pneumonia , and three patients were diagnosed with additional malignancies during the follow - up period . finally , a total of 237 patients with type 2 diabetes were enrolled for this study . the two tubular damage markers were measured at intervals of 12 1 ( mean sd ) months at the outpatient clinic during the follow - up period . serum creatinine was routinely measured for the estimation of gfr at intervals of 6 1 ( mean sd ) months during the follow - up period using the same methods . the latest egfr calculations were used in the assessment of the annual decline in egfr . chronic kidney disease ( ckd ) stage 3 or greater was defined as having egfr < 60 ml / min/1.73 m in two consecutive measurements from the last follow - up visit . the data are presented as mean sd for normally distributed variables and the medians ( interquartile range ) for nonnormally distributed variables . the distribution of continuous variables was examined for skewness and kurtosis , and logarithm - transformed values of nonnormally distributed variables were used for analysis . we conducted multivariate regression analyses with the annual rates of decline in egfr as dependent variables and urinary ccr and napcr as independent variables , respectively . multivariate logistic analysis , using a backward procedure on the basis of likelihood ratios , was conducted to determine whether both tubular markers would be predictive factors for ckd stage 3 or greater . the odds ratios ( ors ) of urinary levels of cystatin c and nap were calculated with reference to the lowest tertile of each variable . a p value of < 0.05 derived from the two - tailed student t test was considered statistically significant . this was a prospective observational study of patients attending the department of endocrinology at pusan national university hospital . the study was conducted with the approval of the institutional review board of pusan national university hospital . a total of 264 korean type 2 diabetic patients were consecutively enrolled at the outpatient clinics between may 2008 and december 2009 . all patients fulfilled the following inclusion criteria : age 18 years and estimated gfr ( egfr ) 30 ml / min/1.73 m. we excluded patients with thyroid disorders or who had been medicated within 6 months prior to the study because thyroid function could affect the cystatin c level ( 19 ) . additional exclusion criteria were 1 ) active urinary tract infection , 2 ) renal disease other than diabetic nephropathy , 3 ) neoplastic disorders , 4 ) severe liver dysfunction , 5 ) active or chronic infection or inflammatory disorders , 6 ) pregnancy , and 7 ) a recent ( within 6 months ) history of acute myocardial infarction , stroke , or occlusive peripheral vascular disease . a random spot urine sample and a blood sample were obtained from each patient at the clinic visit . medical histories and anthropometric measurements the egfr level was calculated using the modification of diet in renal disease ( mdrd ) study formula for the korean population : mdrd = 107.904 ( serum creatinine [ mg / dl ] ) age ( 20 ) . a correction factor of 0.667 the serum and urinary cystatin c levels were measured by the latex agglutination test ( modular p800 ; roche diagnostics , mannhein , germany ) . the interassay and intra - assay coefficients of variations of cystatin c in our laboratory were as follows : < 4.2 and < 3.4% , respectively , for serum and < 7.9 and < 10.1% , respectively , for urine . urine specimens with cystatin c levels < 0.01 mg / l were assumed to have a concentration of 0.01 mg / l . the data on urinary cystatin c was also expressed as ratios of urinary cystatin c to urinary creatinine in order to assess different hydration states and renal functions of the patients . the ratio between urine mass concentrations of cystatin c and creatinine in micrograms per millimole was calculated and designated as the urinary cystatin c - to - creatinine ratio ( ccr ) . since we obtained total proteinuria and albuminuria values from each patient at baseline , we were able to estimate the amount of nonalbumin proteinuria through the following calculation : nap - to - creatinine ratio ( napcr ) = protein - to - creatinine ratio ( pcr ) albumin - to - creatinine ratio ( acr ) . the lowest detectable level and the coefficient of variation in our laboratory were as follows : for total proteinuria , 0.7 mg / dl and < 4.8% , respectively ; for albuminuria , 0.2 mg / dl and < 7.4% , respectively . they were managed to give priority to the best treatment according to standard guidelines at each outpatient clinic of two endocrinologists ( i.j.k . and s.s.k . ) . thirteen patients were excluded during follow - up as follows : four patients died of other causes , six patients were hospitalized for acute myocardial infarction and active infections , such as pneumonia , and three patients were diagnosed with additional malignancies during the follow - up period . finally , a total of 237 patients with type 2 diabetes were enrolled for this study . the two tubular damage markers were measured at intervals of 12 1 ( mean sd ) months at the outpatient clinic during the follow - up period . serum creatinine was routinely measured for the estimation of gfr at intervals of 6 1 ( mean sd ) months during the follow - up period using the same methods . the latest egfr calculations were used in the assessment of the annual decline in egfr . chronic kidney disease ( ckd ) stage 3 or greater was defined as having egfr < 60 ml / min/1.73 m in two consecutive measurements from the last follow - up visit . the data are presented as mean sd for normally distributed variables and the medians ( interquartile range ) for nonnormally distributed variables . the distribution of continuous variables was examined for skewness and kurtosis , and logarithm - transformed values of nonnormally distributed variables were used for analysis . we conducted multivariate regression analyses with the annual rates of decline in egfr as dependent variables and urinary ccr and napcr as independent variables , respectively . multivariate logistic analysis , using a backward procedure on the basis of likelihood ratios , was conducted to determine whether both tubular markers would be predictive factors for ckd stage 3 or greater . the odds ratios ( ors ) of urinary levels of cystatin c and nap were calculated with reference to the lowest tertile of each variable . a p value of < 0.05 derived from the two - tailed student t test was considered statistically significant . the mean age of the patients was 58.5 11.1 years ( range , 1880 years ) , and there were 115 males and 122 females . the patients were categorized into three groups according to acr : those with acr < 30 mg / g creatinine ( normoalbuminuria group , n = 149 ) , those with acr 30299 mg / g creatinine ( microalbuminuria group , n = 58 ) , and those with acr 300 mg / g creatinine ( macroalbuminuria group , n = 30 ) . age , duration of diabetes , systolic blood pressure ( sbp ) , hba1c , hdl cholesterol , and triglycerides were significantly different between the groups . estimated gfr tended to decrease with increasing degrees of albuminuria ( p value for trend < 0.001 ) . more antihypertensive and lipid - lowering agents were administered in the macroalbuminuria group than in the normo- and microalbuminuria groups . baseline characteristics of metabolic and laboratory parameters in patients with type 2 diabetes the urinary cystatin c level and ccr were significantly higher in the macroalbuminuria group than in the normoalbuminuria and microalbuminuria groups ( both p < 0.001 ) , whereas they were not significantly different between the normoalbuminuria and microalbuminuria groups ( table 1 ) . urinary napcr was significantly higher in the macroalbuminuria group than in the normo- and microalbuminuria groups ( both p < 0.001 ) , and they were also significantly different between the normo- and microalbuminuria groups ( p < 0.001 ) . urinary acr positively correlated with urinary ccr ( r = 0.450 , p < 0.001 ) and urinary napcr ( r = 0.699 , p < 0.001 ) . in addition , at baseline , urinary ccr and napcr , both markers of tubular damage , positively correlated with each other ( r = 0.597 , p < 0.001 ) . the median follow - up period was 29.0 months ( range , 13.044.0 months ) . there was a median annual decline in egfr of 1.31 ml / min/1.73 m / year during the follow - up period . the values of annual egfr decline significantly correlated with both baseline urinary ccr ( r = 0.272 , p < 0.001 ) ( fig . 1b ) in univariate regression analysis ( supplementary table 2 ) . after adjusting for age and significant clinical factors affecting the decline of egfr , both urinary ccr and napcr remained significantly associated with a decline in egfr ( table 2 ) . after additionally adjusting for baseline egfr and serum cystatin c ( only analysis for urinary ccr ) , both urinary ccr and napcr remained significantly associated with the annual decline in egfr in the final model ( r = 0.160 , p = 0.021 ; r = 0.412 , p < 0.001 , respectively ) . single regression analysis of the annual rate of the decline in egfr by using urinary ccr ( a ) and napcr ( b ) . multiple regression analysis of the annual rate of decline in egfr as a dependent variable to test whether urinary ccr and napcr might have prognostic values in early diabetic nephropathy , we analyzed patients with baseline egfr 60 ml / min/1.73 m or with normoalbuminuria . in patients with egfr 60 ml / min/1.73 m , both urinary ccr and napcr showed positive correlations with a decline in egfr in the final model after adjusting for several clinical parameters ( r = 0.144 , p = 0.031 ; r = 0.282 , p = 0.001 , respectively ) . in the normoalbuminuria group , the urinary napcr remained significantly associated with a decline in egfr ( r = 0.261 , p = 0.002 ) in the final model , whereas urinary ccr did not ( r = 0.097 , p = 0.199 ) . in addition , urinary napcr showed a significant association with a decline in egfr ( r = 0.201 , p = 0.018 ) in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria . in univariate logistic regression analysis , the number of patients who progressed to ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) was higher in those in the upper tertiles of both the urinary levels of cystatin c and nap than in those in the lower tertiles ( or 7.26 for urinary cystatin c ; 48.71 for napcr ; p < 0.001 in both ) ( table 3 ) . the increased levels of both urinary ccr and napcr remained significantly associated with progression to ckd stage 3 or greater after adjusting for several clinical factors in each multivariate model . logistic regression of development of ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) at last follow - up the mean age of the patients was 58.5 11.1 years ( range , 1880 years ) , and there were 115 males and 122 females . the patients were categorized into three groups according to acr : those with acr < 30 mg / g creatinine ( normoalbuminuria group , n = 149 ) , those with acr 30299 mg / g creatinine ( microalbuminuria group , n = 58 ) , and those with acr 300 mg / g creatinine ( macroalbuminuria group , n = 30 ) . age , duration of diabetes , systolic blood pressure ( sbp ) , hba1c , hdl cholesterol , and triglycerides were significantly different between the groups . estimated gfr tended to decrease with increasing degrees of albuminuria ( p value for trend < 0.001 ) . more antihypertensive and lipid - lowering agents were administered in the macroalbuminuria group than in the normo- and microalbuminuria groups . baseline characteristics of metabolic and laboratory parameters in patients with type 2 diabetes the urinary cystatin c level and ccr were significantly higher in the macroalbuminuria group than in the normoalbuminuria and microalbuminuria groups ( both p < 0.001 ) , whereas they were not significantly different between the normoalbuminuria and microalbuminuria groups ( table 1 ) . urinary napcr was significantly higher in the macroalbuminuria group than in the normo- and microalbuminuria groups ( both p < 0.001 ) , and they were also significantly different between the normo- and microalbuminuria groups ( p < 0.001 ) . urinary acr positively correlated with urinary ccr ( r = 0.450 , p < 0.001 ) and urinary napcr ( r = 0.699 , p < 0.001 ) . in addition , at baseline , urinary ccr and napcr , both markers of tubular damage , positively correlated with each other ( r = 0.597 , p < 0.001 ) . the median follow - up period was 29.0 months ( range , 13.044.0 months ) . there was a median annual decline in egfr of 1.31 ml / min/1.73 m / year during the follow - up period . the values of annual egfr decline significantly correlated with both baseline urinary ccr ( r = 0.272 , p < 0.001 ) ( fig . 1b ) in univariate regression analysis ( supplementary table 2 ) . after adjusting for age and significant clinical factors affecting the decline of egfr , both urinary ccr and napcr remained significantly associated with a decline in egfr ( table 2 ) . after additionally adjusting for baseline egfr and serum cystatin c ( only analysis for urinary ccr ) , both urinary ccr and napcr remained significantly associated with the annual decline in egfr in the final model ( r = 0.160 , p = 0.021 ; r = 0.412 , p < 0.001 , respectively ) . single regression analysis of the annual rate of the decline in egfr by using urinary ccr ( a ) and napcr ( b ) . multiple regression analysis of the annual rate of decline in egfr as a dependent variable to test whether urinary ccr and napcr might have prognostic values in early diabetic nephropathy , we analyzed patients with baseline egfr 60 ml / min/1.73 m or with normoalbuminuria . in patients with egfr 60 ml / min/1.73 m , both urinary ccr and napcr showed positive correlations with a decline in egfr in the final model after adjusting for several clinical parameters ( r = 0.144 , p = 0.031 ; r = 0.282 , p = 0.001 , respectively ) . in the normoalbuminuria group , the urinary napcr remained significantly associated with a decline in egfr ( r = 0.261 , p = 0.002 ) in the final model , whereas urinary ccr did not ( r = 0.097 , p = 0.199 ) . in addition , urinary napcr showed a significant association with a decline in egfr ( r = 0.201 , p = 0.018 ) in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria . in univariate logistic regression analysis , the number of patients who progressed to ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) was higher in those in the upper tertiles of both the urinary levels of cystatin c and nap than in those in the lower tertiles ( or 7.26 for urinary cystatin c ; 48.71 for napcr ; p < 0.001 in both ) ( table 3 ) . the increased levels of both urinary ccr and napcr remained significantly associated with progression to ckd stage 3 or greater after adjusting for several clinical factors in each multivariate model . logistic regression of development of ckd stage 3 or greater ( egfr < 60 ml / min/1.73 m ) at last follow - up in this study , urinary cystatin c and nap , both clinical tubular damage markers , positively correlated with each other at baseline . both markers were significantly associated with the annual decline in egfr in type 2 diabetic nephropathy . in particular , both tubular damage makers affected a decline in egfr at the early stage of nephropathy in type 2 diabetic patients ( egfr 60 ml / min/1.73 m ) . urinary nap affected egfr decline in patients with both egfr 60 ml / min/1.73 m and normoalbuminuria , although urinary cystatin c did not reach statistical significance . in addition , the increased levels of the two markers were also associated with the progression of ckd stage 3 or greater at the last follow - up . in general , unlike healthy subjects , diabetic patients are continuously exposed to the various metabolic and hemodynamic risks associated with this disease ( 21 ) . recent studies have mainly focused on tubular damage , which is known to correlate with acute kidney injury in patients with diabetic nephropathy ( 410 ) . some cross - sectional studies have reported that several tubular markers increase more in diabetic patients than in healthy controls , and this correlated with the severity of albuminuria ( 48 ) . in our study , nap correlated with the severity of baseline albuminuria , which is consistent with the results of previous studies . however , urinary cystatin c mainly increased in the macroalbuminuria group and was not significantly different between the microalbuminuria and normoalbuminuria groups . microalbuminuria has generally been considered the earliest marker for the development of diabetic nephropathy in clinical settings ( 22 ) . microalbuminuria is diagnosed when significant glomerular damage has occurred ( 23 ) and does not necessarily lead to renal impairment because nephropathy sometimes occurs in the normoalbuminuric patients ( 24,25 ) . in our study , although urinary cystatin c was not associated with a decline in egfr in normoalbuminuric patients , urinary nap was significantly associated with a decline in egfr independent of baseline albuminuria and egfr . it is necessary to assess tubular damage independent of albuminuria in patients with early development and progression of diabetic nephropathy because tubular damage may play a significant role in the normoalbuminuric renal insufficiency . serum cystatin c is the most valid marker to estimate the gfr , rather than serum creatinine , and to predict progression of renal dysfunction ( 26 ) . an increase in urinary cystatin c , independent of serum cystatin c , is suggestive of renal tubular damage ( 13 ) . in our study , urinary cystatin c was a predictor of renal impairment independent of serum cystatin c. thus , urinary cystatin c also plays some role in predicting renal decline independent of serum cystatin c , although serum cystatin c itself , an indicator for the estimation of gfr , is very important for predicting renal decline . naps include -1 microglobulin , -2 macroglobulin , igg , cystatin c , transferrin nephrin , matrix metalloproteinase-9 , and tissue inhibitor of metalloproteinases-1 ( 27 ) . it is well known that each nap is related to renal damage in various chronic renal diseases , including diabetic nephropathy ( 28 ) . because there is little evidence to support the role of nap in diabetic nephropathy , it is worthwhile to investigate this issue . interestingly , in our type 2 diabetic patients , nap has been shown to be a stronger predictor for renal impairment than urinary cystatin c. at normal levels of protein loss , albumin is a minor component of total urinary protein , although albumin becomes the most significant single protein present as protein loss increases ( 29 ) . this supports our result that indicates the clinical value of nap in the prediction of renal impairment in normoalbuminuric patients . a recent study on tubular damage markers in acute kidney injury has demonstrated that unnormalized values are also useful for predicting ongoing injury ( 30 ) . the unnormalized values had similar patterns as the normalized values in baseline characteristics and univariate analysis results ( table 1 and supplementary table 2 ) . the statistical significance of unnormalized values was lost in multivariate analysis , whereas that of normalized values was not ( data not shown ) . interestingly , serum uric acid was found to be associated with renal decline in the most analyzed model in this study and has recently been shown to be related to diabetic nephropathy ( 31,32 ) . uric acid might induce renal microvascular disease independent of blood pressure through stimulation of the renin - angiotensin system ( ras ) and inhibition of endothelial nitric oxide ( 33 ) . in addition , lowering therapy in a diabetic animal model significantly reduced albuminuria and ameliorated tubulointerstitial inflammation , suggesting a role for uric acid in diabetic nephropathy ( 34 ) . first , the follow - up period of this study was relatively short , even though the development and progression of diabetic nephropathy require a longer time frame . however , it is considered that the occurrence of tubular damage , as expressed by biomarkers , could be a significant predictor for relatively rapid decline in renal function during a short period of time . second , we measured the urinary levels of cystatin c , albumin , and protein with single random spot urine samples , although urine samples were collected at the outpatient clinic from patients without illness or renal diseases other than diabetic nephropathy . despite these limitations , it is noteworthy that the two aforementioned tubular damage markers can easily be checked and used to assess the development and progression of diabetic nephropathy in clinical settings . third , although there have been a few studies reporting that urinary cystatin c and nap mainly increase due to tubular rather than glomerular damage , it is unclear whether urinary cystatin c and nap originate from tubular damage alone in clinical conditions involving both tubular and glomerular damage , such as diabetic nephropathy . further studies are needed to investigate whether both urinary cystatin c and nap are biomarkers of tubular damage in clinical conditions with a massive glomerular protein load , such as diabetic nephropathy . in conclusion , it is suggested that urinary cystatin c and nap , along with albuminuria , may be sensitive and specific markers for predicting renal impairment in type 2 diabetic patients and may help to further elucidate the role of tubular damage in the pathophysiological mechanisms of the development and progression of type 2 diabetic nephropathy .

objectivethe aim of this study was to evaluate the association of urinary cystatin c , a tubular damage marker , with the progression of type 2 diabetic nephropathy.reserch design and methodsthe baseline values of serum and urinary cystatin c were measured as primary parameters and those of urinary nonalbumin protein ( nap ) were measured as secondary parameters . in this prospective observational study , a total of 237 type 2 diabetic patients were followed up for 29 months ( 1344 months).resultsboth the urinary cystatin c - to - creatinine ratio ( ccr ) and nap - to - creatinine ratio ( napcr ) were significantly different according to the degree of albuminuria . both markers had strongly positive correlations at baseline . after adjusting for several clinical factors , both urinary ccr and napcr had significant associations with the decline of the estimated glomerular filtration rate ( egfr ) ( r = 0.160 , p = 0.021 ; r = 0.412 , p < 0.001 , respectively ) . urinary ccr had positive correlations with the decline of egfr in the subpopulation of patients with egfr 60 ml / min/1.73 m2 . in patients with egfr 60 ml / min/1.73 m2 and normoalbuminuria , only urinary napcr showed a significant association with the decline of egfr ; urinary ccr did not . in multivariate regression analysis , the number of patients who progressed to chronic kidney disease stage 3 or greater was higher in those in the upper tertiles of both the urinary levels of cystatin c and nap than in those in the lower tertiles.conclusionsthe results of this study suggest that urinary cystatin c and nap may be predictors of the progression of type 2 diabetic nephropathy .

RESEARCH DESIGN AND METHODS Patients Statistical analysis RESULTS Baseline patient characteristics Associations of the annual eGFR decline with urinary CCR and NAPCR Urinary CCR and NAPCR as predictive factors for the development of CKD stage 3 or greater CONCLUSIONS Supplementary Material

primary effusion lymphoma ( pel ) is a rare form of non - hodgkin 's lymphoma characterized by malignant fluid accumulation in the absence of lymphadenopathy . typical sites of accumulation include pleural , pericardial , and ascites fluid . pel is associated with immunodeficiency states such as acquired immunodeficiency syndrome ( aids ) and infectious etiologies such as human herpes virus 8 ( hhv8 ) . the world health organization ( who ) uses the pel term for only hhv - related pel . in our case , the term hhv8-unrelated pel - like lymphoma has been used in the literature to describe such cases . we present the case of a 71-year - old female with no significant past medical history who originally presented to the clinic with shortness of breath , abdominal bloating , and lower extremity edema . post - pericardiocentesis the patient reported only residual orthopnea and night cough when lying down . analysis of the aspirated pericardial fluid revealed monoclonal b - cell lymphocytes compatible with diffuse large b - cell lymphoma , which prompted staging positron emission tomography / computed tomography ( pet / ct ) . of note , immunologic blood work was negative for hepatitis a , hepatitis b , and hepatitis c infection . serum polymerase chain reaction ( pcr ) was negative for cytomegalovirus ( cmv ) and epstein barr virus ( ebv ) . the 18-flourodeoxyglucose pet / ct ( 18-f fdg pet / ct ) was performed using 19.6 mci of intravenously administered radiotracer on a ge discovery ste pet / ct scanner ( ge healthcare , waukesha , wi , usa ) . the patient was imaged 1 and 2 h postinjection . the pet / ct demonstrated uniformly increased radiotracer uptake in a moderate pericardial effusion ( standardized uptake value ( suv ) maximum 3.2 ) , which increased in metabolic activity on delayed imaging ( suv maximum 3.6 ) . of note , the activity was within the fluid density space of the pericardial effusion and not limited to solely the pericardium itself [ figure 1 ] . additionally , there were moderate bilateral pleural effusions and abdominal ascites , which demonstrated similar persistent and uniform radiotracer uptake [ figures 2 and 3 ] [ table 1 ] . these findings are compatible with malignant involvement , particularly given this patient 's history of prior pathology - proven malignant pericardial effusion . axial pet / ct images demonstrate 18-f fdg radiotracer uptake in moderate pericardial effusion ( arrows ) . pet = positron emission tomography , ct = computed tomography , 18-f fdg = 18-flourodeoxyglucose whole body mip . coronal whole body mip image obtained 1 h after the intravenous injection of 19.6 mci 18-f fdg . mip = maximum intensity projection , 18-f fdg = 18-flourodeoxyglucose ( a and b ) the 18f - fdg accumulation over time . metabolically active bilateral pleural effusions ( crosshairs ) at ( a ) 1-h and ( b ) 2-h postinjection . 18-f fdg = 18-flourodeoxyglucose standardized uptake value maximum over time of note , the pet / ct also demonstrated no significant hypermetabolic cervical , thoracic , abdominal , or pelvic lymphadenopathy despite the above - described fdg - avid fluid collections [ figure 4 ] . axial pet / ct images demonstrate 18-f fdg radiotracer uptake ( crosshairs ) in ( a ) abdominal and ( b ) pelvic ascites . pet = positron emission tomography , ct = computed tomography , 18-f fdg = 18-flourodeoxyglucose the patient received chemotherapy and follow - up restaging pet / ct demonstrated interval resolution of the hypermetabolic pericardial , pleural , and peritoneal effusions [ figure 5 ] . pel is a rare subtype of non - hodgkin 's lymphoma characterized by malignant effusions in the body cavities such as the pericardial , pleural , and peritoneal cavities in the absence of lymphadenopathy or organomegaly . pel was first described in 1989 in a patient with hhv8 and human immunodeficiency virus ( hiv ) infection . when pel occurs in the absence of hhv8 , the term hhv8-unrelated pel - like lymphoma has been used in the literature . the described pet / ct findings in our case of hhv8-unrelated pel - like lymphoma are similar to the previously reported pet / ct findings in traditional pel . therefore , the distinction between pel and hhv8-unrelated pel - like lymphoma requires analysis of the fluid and testing for the presence of hhv8 . the distinction between these two closely related clinical entities is important because hhv8-unrelated pel - like lymphoma has a more favorable prognosis than traditional pel . the b - cell markers obtained from flow cytometric analysis have also been found to be helpful in the differentiation between pel and hhv8-unrelated pel - like lymphoma . the cells in pel are usually negative for b - cell markers such as cluster of differentiation ( cd)19 , cd20 , and cd79a . in our patient , we describe the pet / ct findings of hhv8-unrelated pel - like lymphoma , which has only once been previously reported . the presence of malignant fluid accumulation in the absence of lymphadenopathy should raise suspicion of pel . the imaging features of hhv8-unrelated pel - like lymphoma appear to be similar to previously reported pel pet / ct findings . therefore , analysis of the fluid with particular attention to the presence of hhv8 is necessary to make this distinction , which is clinically significant because hhv8-unrelated pel - like lymphoma portends a more favorable prognosis compared with traditional pel .

we present a 71-year - old female with human herpes virus 8 ( hhv8)-unrelated primary effusion lymphoma ( pel)-like lymphoma . dyspnea and pericardial effusion led to pericardiocentesis , diagnosing diffuse large b - cell lymphoma . she underwent positron emission tomography / computed tomography ( pet / ct ) , which demonstrated hypermetabolic pericardial , pleural , and ascites fluid without lymphadenopathy elsewhere . malignant fluid in the absence of lymphadenopathy is a hallmark of pel . pel is associated with immunodeficiency states such as acquired immunodeficiency syndrome ( aids ) and infectious agents such as hhv8 . our patient had no such history and had not received immunosuppressive chemotherapy . we present the pet / ct findings of this rare case of hhv8-unrelated pel - like lymphoma .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

autism spectrum disorders ( asd ) are lifelong conditions severely impairing social skills and autonomy . according to the fifth edition of the diagnostic and statistical manual of mental disorders ( dsm-5 ) , asd core features are : persisting deficits of social communication and interaction ; restricted and repetitive behaviors , interests , activities . compared to the previous edition ( dsm - iv - text revision ) , dsm-5 introduced significant changes . in the diagnostic criteria , language abilities not employed in social communication have been de - emphasized . further , the diagnostic subcategories , that is , autistic disorder , asperger disorder , rett disorder , childhood disintegrative disorder , and pervasive developmental disorder ( pdd ) not otherwise specified , have been abolished . dsm-5 describes three levels of increasing severity of asd , from ( 1 ) ( requiring support ) to ( 2 ) ( requiring substantial support ) , finally to ( 3 ) ( requiring very substantial support ) . we report two explanatory cases affected by asd with the same severity level , according to dsm-5 , but with remarkable differences regarding the neuropsychological and neurobehavioral profile . the maternal grandfather was a very skilled electronics technician , but he lacked empathy and had difficulties relating to others ; as a child , he self - taught himself four languages . the boy was born at full term ; pregnancy and delivery were uncomplicated , psychomotor development was normal . since 18 months of age he appeared isolated , wanted things to be placed in a certain order , played along with water and looked at books for hours . he showed great ability to design projects , e.g. , a new home or railway line and was frustrated by not being able to get the highest grade in maths . applied behavior analysis ( aba ) intervention wechsler intelligence scale for children - iii edition ( wisc - iii ) showed a full intelligence quotient ( iq ) = 102 , with a significant discrepancy ( p < 0.01 ) between verbal iq ( = 115 ) and performance iq ( = 89 ) . comparing the 10 wisc - iii subtests among them [ table 1 ] , the score in vocabulary , involving lexical knowledge , prevailed significantly ( p < 0.01 ) , while the scores in block design ( visuoperceptual abilities , visuomotor coordination ) and in object assembly ( visual analysis , construction of objects ) were significantly lower ( p < 0.01 ) . copy of rey osterrieth complex figure , involving visuoconstructional and visuospatial abilities , was unrecognizable . autism diagnostic observation schedule ( ados ) ( module 3 ) showed a score above the cut - off for autism spectrum in language and communication and above the cut - off for autism in reciprocal social interaction ; the overall result was above the cut - off for autism . childhood autism rating scale second edition - high functioning ( cars2-hf ) showed the presence of mild to moderate asd symptoms . krug asperger 's disorder index ( kadi ) appeared compatible with a highly probable asperger 's disorder diagnosis ; among the findings from this questionnaire the following should be noted : demanding a lot from himself and from others ; too serious appearance ; fluent language and adequate vocabulary before 5 years of age ; tendency to say embarrassing things to others ; unusual way to enact or talk about fantasies or thoughts . the first five from the left are the verbal subtests , the other five are the performance subtests according to dsm-5 criteria , this case had an asd diagnosis with severity level 1 . family history was positive for : learning disorder in the older sister , asd in the younger brother . during pregnancy , psychomotor development was normal except for language as the child began to speak sentences with a slight delay . social - emotional reciprocity was lacking : he often followed the course of his thoughts and conversational exchanges remained limited . comprehension of verbal messages tended to be literal , leading to difficulties in understanding the meaning of metaphors , jokes , and slang expressions . first speech therapy and psychomotricity were conducted , then aba intervention . at school , he had the special needs teacher . wisc - iii showed a full iq = 95 , with a verbal iq = 88 and a performance iq = 104 : this difference was significant at 0.05 level , very near to 0.01 level . comparing the 10 wisc - iii subtests among them [ table 1 ] , the score in comprehension , involving social and practical judgment , was significantly lower ( p < 0.05 ) , while the score in picture completion , involving visual analysis and attention to detail , was almost significantly higher ( p = 0.05 ) . ados ( module 3 ) showed a result above the cut - off for autism both in language and communication , and in reciprocal social interaction ; consequently also the overall result was above the cut - off for autism . kadi was compatible with an extremely low probability of asperger 's disorder diagnosis . according to dsm-5 criteria , the maternal grandfather was a very skilled electronics technician , but he lacked empathy and had difficulties relating to others ; as a child , he self - taught himself four languages . the boy was born at full term ; pregnancy and delivery were uncomplicated , psychomotor development was normal . since 18 months of age he appeared isolated , wanted things to be placed in a certain order , played along with water and looked at books for hours . he showed great ability to design projects , e.g. , a new home or railway line and was frustrated by not being able to get the highest grade in maths . applied behavior analysis ( aba ) intervention wechsler intelligence scale for children - iii edition ( wisc - iii ) showed a full intelligence quotient ( iq ) = 102 , with a significant discrepancy ( p < 0.01 ) between verbal iq ( = 115 ) and performance iq ( = 89 ) . comparing the 10 wisc - iii subtests among them [ table 1 ] , the score in vocabulary , involving lexical knowledge , prevailed significantly ( p < 0.01 ) , while the scores in block design ( visuoperceptual abilities , visuomotor coordination ) and in object assembly ( visual analysis , construction of objects ) were significantly lower ( p < 0.01 ) . copy of rey osterrieth complex figure , involving visuoconstructional and visuospatial abilities , was unrecognizable . bvn 511 ( italian battery of neuropsychological assessment for children and adolescents ) . autism diagnostic observation schedule ( ados ) ( module 3 ) showed a score above the cut - off for autism spectrum in language and communication and above the cut - off for autism in reciprocal social interaction ; the overall result was above the cut - off for autism . childhood autism rating scale second edition - high functioning ( cars2-hf ) showed the presence of mild to moderate asd symptoms . krug asperger 's disorder index ( kadi ) appeared compatible with a highly probable asperger 's disorder diagnosis ; among the findings from this questionnaire the following should be noted : demanding a lot from himself and from others ; too serious appearance ; fluent language and adequate vocabulary before 5 years of age ; tendency to say embarrassing things to others ; unusual way to enact or talk about fantasies or thoughts . the first five from the left are the verbal subtests , the other five are the performance subtests according to dsm-5 criteria , this case had an asd diagnosis with severity level 1 . family history was positive for : learning disorder in the older sister , asd in the younger brother . during pregnancy , psychomotor development was normal except for language as the child began to speak sentences with a slight delay . social - emotional reciprocity was lacking : he often followed the course of his thoughts and conversational exchanges remained limited . comprehension of verbal messages tended to be literal , leading to difficulties in understanding the meaning of metaphors , jokes , and slang expressions . first speech therapy and psychomotricity were conducted , then aba intervention . at school , he had the special needs teacher . wisc - iii showed a full iq = 95 , with a verbal iq = 88 and a performance iq = 104 : this difference was significant at 0.05 level , very near to 0.01 level . comparing the 10 wisc - iii subtests among them [ table 1 ] , the score in comprehension , involving social and practical judgment , was significantly lower ( p < 0.05 ) , while the score in picture completion , involving visual analysis and attention to detail , was almost significantly higher ( p = 0.05 ) . ados ( module 3 ) showed a result above the cut - off for autism both in language and communication , and in reciprocal social interaction ; consequently also the overall result was above the cut - off for autism . kadi was compatible with an extremely low probability of asperger 's disorder diagnosis . according to dsm-5 criteria , the description of these two explanatory cases underlines some critical points of the current setting of dsm-5 classification for asd . according to dsm-5 criteria , both cases fall under asd , due to a significant impairment in social communication and interaction , and to restricted interests and activities . at the same time , both cases ( verbal boys , aged about 7 and a half years , without intellectual disability ) , based on dsm-5 description , fall into the lowest severity level of asd ( level 1 ) , which by definition requires support . however , their respective neuropsychological and neurobehavioral profiles were notably different . while the first boy showed a prevalent impairment of visuoconstructional and visuoperceptual abilities , with language functions even overdeveloped in some respects , the second boy presented an almost specular profile characterized by a predominant involvement of verbal functions , with qualitative impairments in communication , and a preservation of nonverbal functions , some of which ( particularly visual analysis ) were the strength of his development . admittedly , a detailed neuropsychological and neurobehavioral profile represents a basic prerequisite for the treatment of patients with asd . in the past years , when the dsm - iv - text revision ( tr ) was being used , among the pdds , asperger disorder , together with autistic disorder , has been the most studied from the neuropsychological and neurobehavioral perspective . basically , according to dsm - iv - tr criteria , the main distinguishing features that characterized asperger disorder compared with other pdds , were the absence of intellectual disability and the presence of structured language . further , we could observe a cognitive profile characterized by strengths in verbal skills ( e.g. , vocabulary ) and weakness in nonverbal skills ( e.g. , visuospatial and visuomotor abilities ) . , several papers highlighted the differences between neuropsychological profiles of asperger disorder and high - functioning autistic disorder , but over the years the opinion of the authors that devalue these differences has prevailed . according to dsm - iv - tr criteria , both cases fall into pdds ; nevertheless , while the first boy would receive a diagnosis of asperger disorder , the second one , due to his qualitative impairments in communication , would rather receive a diagnosis of autistic disorder , to be considered high - functioning . one of dsm-5 assumptions is the lack of data supporting the distinction between these two entities , but , unfortunately , the current dsm approach ( three severity levels ) does not allow the division of patients with autism into sufficiently homogeneous groups . our report is not meant to suggest a return to the past , but rather to give a cue to improve the current dsm classification . we believe that a further step forward in asd definition and classification is necessary in order to meet the needs of affected individuals from the rehabilitation perspective . in our view it is important to take into account not only the intensity of symptoms , but also their quality , in order to formulate a reliable prognosis , plan an individualized treatment , and monitor the clinical course over time . in addition , description of detailed behavioral phenotypes , allowing the creation of homogeneous subgroups of patients with asd , may be useful also for genetic research .

the fifth edition of the diagnostic and statistical manual of mental disorders ( dsm-5 ) introduced significant changes in the classification of autism spectrum disorders ( asd ) , including the abolition of the diagnostic subcategories proposed by dsm - iv - text revision . dsm-5 describes three levels of increasing severity of asd . the authors report two explanatory cases with asd ( verbal boys , aged about 7 and a half years , without intellectual disability ) . according to dsm-5 , both cases fall into the lowest severity level of asd . however , their neuropsychological and neurobehavioral profile varies significantly . while the first boy showed a prevalent impairment of visuoconstructional and visuoperceptual abilities , the second one presented a predominant involvement of verbal functions , with qualitative impairments in communication . a further step forward in the definition and classification of asd , taking into account both intensity and quality of symptoms , is recommended in order to formulate a reliable prognosis , plan an individualized treatment and monitor the clinical course over time .

Introduction Case Reports Case 1 Neuropsychological and neurobehavioral assessment Case 2 Neuropsychological and neurobehavioral assessment Discussion

eating disorders were described in the early descriptions of patients with asperger syndrome.1 asperger syndrome is a serious and chronic neurodevelopmental disorder , which is presently defined by social deficits , restricted interests , and relative preservation of language and cognitive ability.2 in diagnostic and statistical manual of mental disorders ( dsm)-iv , the syndrome was considered to be separate , but it fell under the broader category of pervasive developmental disorders . in dsm - v , asd is a disorder with persistent deficits in social interaction and communication skills , accompanied by restricted , repetitive patterns of behavior , interests , or activities and by atypical sensory reactivity.3 we know nowadays that eating disorders take various forms and are often presented in asd , complicating both diagnosis and therapy . rastam4 offered a summary of these disorders stating that abnormal eating behaviors are overrepresented in asd , including food refusal , pica , rumination , and selective eating . she considers the connection between anorexia nervosa ( an ) and asd as interesting and states that asperger syndrome is sometimes not recognized in female teenagers with eating disorders . there is a risk that autistic traits in girls with an are overlooked , which may lead to a simplification in the diagnostic consideration and therapeutic procedures . a contemporary review publication has explained that asds are overrepresented in individuals who develop an and also that asds are common in chronic cases of an.5 this comorbidity has been associated with a poorer prognosis.6 the research by baron - cohen et al7 confirms that girls with an have elevated autistic traits . the authors point out that clinicians should consider whether a focus on autistic traits might be helpful in the assessment and treatment of anorexia . early - onset an ( in children under the age of 12 years ) represents approximately 5% of all cases . it is a serious disorder jeopardizing the development of children in the somatic and psychosocial areas , and it seems that its incidence is growing.8 in individual research groups where early - onset is indicated , there is not always agreement as to the precise age definition of early - onset ; some authors describe premenarcheal girls , others refer to the age between 8 and 14 years.9 both an and early - onset eating disorders include syndromes of food avoidance emotional disorder and selective eating . specific psychopathology of early - onset an is very similar to the disorder onset in adolescence.10 an extensive study by halmi et al11 suggests that the predominant feature that precedes all an subtypes is global childhood rigidity , which is a trait that leads to resistance to changes . pooni et al12 found a higher incidence of autistic traits in individuals with early - onset eating disorders ( 816 years ) compared with typically developing peers , namely , repetitive and stereotyped behaviors , and also trends toward higher levels of autistic social impairment . coombs et al13 looked into the relationship between eating disorder psychopathology and autistic symptomatology in a non - clinical sample of school children aged 1114 years with no recorded psychiatric diagnoses , and found a significant relationship between the level of eating disorder symptomatology and asd symptomatology . according to karlsson et al14 eating disorders are common in asd but are often being overlooked . they developed a psychometrically and statistically valid swedish eating assessment for autism spectrum disorders questionnaire detecting eating disorders , which has been designed for individuals with asd aged 1525 years with normal intelligence ; in younger patients , clinical assessment has to suffice at the moment . the question of relationship , similarity , and connection between asd and an has diagnostic and therapeutic importance in clinical practice . apart from the diagnosis of clinical an syndrome , it is necessary to assess the development of cognitive and psychosocial traits of a child , including the possibility of identifying asd or prominent autistic traits . equally , in asd patients it is important to consider the occurrence of eating disorders associated with cognitive and psychosocial peculiarities . the therapeutic basis must respect these development peculiarities and make them part of the therapeutic program . the results imply that young people with an would benefit from a treatment approach tailored to the needs of individuals on the autism spectrum.15 kerbeshian and burd16 have shown an inspiring approach on the case history of a 12-year - old girl with high - functional autism and partial an . they have demonstrated that the treatment approaches used with individuals with neuropsychiatric developmental disorders might be effective in higher functioning individuals with eating disorders . therapeutic implications emphasize the need to improve cognitive and social functions,17 deficits in the field of mentalization,18 and the importance of focusing on working with the family.19 a girl aged 10 years and 9 months was admitted to a children s psychiatric clinic with an eating disorder and an underlying diagnosis of asperger syndrome . the patient s parents had degrees from technical universities and were healthy . the patient s sister , a grammar school student , was 2 years older and had asperger syndrome . she began to form individual words at 8 months and sentences from 24 months , but did not speak much until the age of 4 years . she began to speak fluently at the age of 4 years . at the age of 4.5 years she managed to fit into a small group of children in kindergarten . in the third year of school , she joined a partly new group in a language class and got a new class teacher . she began to dislike going to school ; she had mood swings , sometimes there were suicidal proclamations ; and she withdrew from her peers . at the end of the school year , she did not manage a school trip lasting several days , she ended up disorientated , and she ran away from the teachers several times . she said that she had been confused because of the change in the daily routine that she was used to at school . the diagnosis of asperger syndrome was considered for the first time at this point , yet the adhd diagnosis was erroneously established , and the girl was medicated with atomoxetine , 40 mg per day . when on the medication , she lost appetite and began to reduce the food intake . she had never been a great eater , but until the age of 9 years the parents never noticed any eating problems . she ate small portions of food at precisely the same times daily . in the year leading up to the children s psychiatric clinic admission , she grew 12 cm and her weight reduced by 5 kg . on admission to the children s psychiatric clinic , most of the time , the patient used a pseudo - adult language in conversation and spoke in a high - pitched voice with unnatural intonation . she said on several occasions that she wanted to be the skinniest girl in the world . at other times , she expressed her wish to be a model , fashion designer , and world - famous painter . the eating regimen at the ward was first accompanied by high tension , even affective seizures ; she refused food , and behaved in a bizarre way ( concealed food in clothes , escaped , cried loudly , proclaimed suicide ) . after several days of adapting to the regimen , she began to accept food , and the diet . the asperger syndrome diagnosis was confirmed using the autism diagnostic observation schedule ( ados)20 testing method and following an interpretation of the psychological examination . the girl had a prominently impaired perception of her own body , little interest in social contact , egocentric perception , and infantile expression . she would escape in an imaginary world in which she was a famous and respected artist . her introspection was minimal ; affective seizures grew in number with her growing weight . during the hospitalization , her weight increased by 8 kg following a plan , and she kept her eating regimen even during visits home . the parents requested consultation because of some peculiarities in their daughter s behavior and habits , emphasizing on eating problems . for about 6 months , she expressed her opinion that being fat meant being ugly and mean , and sometimes made tactless remarks about people around her . she insisted on specific odd arrangements when eating ; she had to sit at her own place , have her own dishes , and minimized the food and drink she took . she said repeatedly that she did not want to be fat and old , and she kept asking her parents strange questions on this topic again and again . in the patient s history , there was suspicion of asperger syndrome . according to the parents , she did not have any capability of empathy , never asked personal questions , considered mainly her own self , and was a great egoist . in social contacts , she was aloof and passive in relation to peers and adults ; she only critically commented on what was happening around her without becoming much involved . there were great problems in adapting to changes ( changes in routes , clothes , daily routine ) accompanied by negative reactions or even affective seizures with verbal and brachial aggression . her playing had elements of stereotypes ; there were finger mannerisms . in conversation , she showed signs of impaired communication and social interaction . using the ados testing method , concerning her eating disorder ( minimizing food intake , rigid eating habits , specific arrangements when eating ) , the parents were recommended to approach this as a symptom of the asd diagnosis . the occurrence of an in asperger syndrome has been described in literature.5 good understanding of the connection between these two disorders is crucial both for diagnosis and treatment . the therapeutic points of departure must respect the individual composition of symptoms and their mutual links . the picture of an is always critically influenced by the presence of a pervasive developmental disorder . the clinical guidance of patients with such comorbidities is always more demanding and requires experience with both diagnoses . the patients case histories illustrate the issues and make it possible to share the clinical experience . important issues are raised in the understanding of the comorbidity of these disorders and the implications for treatment . in 1985 , gillberg21 was the first to describe cases where a relationship between children s autism and an was established ( four cases of autistic boys whose close relatives suffered from an ) . the first clear clinical case report was submitted by rothery and garden22 who described the case of a 16-year - old girl with an who was previously diagnosed as having infantile autism . this case illustrates that an does occur in adolescents with autism and that it is important it is diagnosed , so that appropriate treatment can be given . similarly , fisman et al23 described the development of an in a high - functioning autistic adolescent 13-year - old girl . autism was diagnosed when the patient was 4 years old , and a change in eating habits started approximately a year prior to the admission . hospitalization was suggested because of continued weight loss accompanied by increasing refusal to eat and the failure to disengage parents from the patient s eating and weight preoccupation . this case study illustrates , besides a clear comorbidity of both diagnoses , an example where a combined psychotherapeutic and pharmacological strategy resulted in good improvement . kerbeshian and burd16 presented a case report of multiple comorbidities in a 12-year - old girl with high - functioning autistic disorder who developed tourette syndrome , obsessive compulsive disorder , and an . our cases document the difficulty in diagnosing and treating patients with concurrent eating and pervasive developmental disorders . in our first case history of a nearly 11-year - old girl , there was a clear comorbidity of early - onset an and asperger syndrome ; the diagnostic criteria for both the disorders were fulfilled . compared with patients of similar age , the signs of an ( such as the disorder of the body schema , minimizing food intake , lack of introspection ) were more persistent and difficult to influence with the therapeutic procedures commonly applied in cases of eating disorders . the persistence and rigidity so typical of asperger syndrome hindered the work with the patient both in the therapeutic regimen and in the individual , group , and family therapy . the girl needed longer than the usual to adapt to the therapeutic regimen and to accept it , then she rigidly insisted on keeping it , both for herself and for her fellow patients with eating disorders . the success of the therapy depended on the selection of a suitable motivating approach to the patient taking into consideration her traits stemming from the underlying asperger syndrome diagnosis . in our second case history , the eating problem was rather more part of the core symptoms of asperger syndrome , even though the eating disorder was the original reason for the psychiatric assessment . the manipulation around food could be interpreted as a communication means in a child with impaired social and communication abilities . only if this therapy fails would we recommend a more targeted approach to the eating disorder . it is important that we notice these anorectic traits in patients , especially at a young age , which is not typical for the incidence of an . it follows from literature and our clinical experience that the comorbidity of eating disorders and asd is not unusual . our statement is based on the detailed description of two clinical cases of girls with asperger syndrome and symptoms of an . it is necessary to distinguish which symptoms are part of the underlying diagnosis and which are distinctive comorbid symptoms . both diagnosis and therapy should be performed by experts experienced in working with patients with both the diagnoses . we believe that the most efficient in infancy and adolescence is the combined therapeutic strategy , which involves a structured behavioral approach as well as psychotherapy , pharmacotherapy , and family therapy .

eating disorders frequently occur in conjunction with autism spectrum disorders , posing diagnostic and therapeutic difficulties . the comorbidity of anorexia nervosa and asperger syndrome is a significant clinical complication and has been associated with a poorer prognosis . the authors are presenting the cases of an eleven - year - old girl and a five - and - a - half - year - old girl with comorbid eating disorders and asperger syndrome .

Introduction Clinical implication Case history 1 Case history 2 Discussion Conclusion

blunt trauma includes road traffic crash ( rtc ) , altercation , industrial / occupational accidents , sports , and falls . penetrating injuries are results of gunshots , missiles , stabbing , and explosions . pathological diseases such as tumors , osteomyelitis , cysts , osteoradionecrosis may also contribute to facial fracture . rtcs have been reported as the most frequent reason for facial fractures in nigeria , rural and developing world ; while altercations remains the leading causes in urban and developed countries . however , recent report on the war in afghanistan by breeze and associates have identified increasing facial fractures among british troops despite to protective armor worn and advances in on - field resuscitation and critical care that have increased survival in the battle field . falls are common in the very young and elderly . pattern of facial fracture is predicated on etiology , population density , socioeconomic , cultural , race , and time . facial fractures and other maxillofacial injuries have high clinical significant because the anatomical specificity of face provides protection to important vital organs such as the brain and eyes and others like the digestive and respiratory systems . the facial skeleton is one of the most complex arrangements of curving bony structures in the body and consists of bones of the mandible , maxilla , zygoma , bony walls of the nasal cavities , paranasal sinuses , and orbit . injuries to this region can result in serious dysfunctions of sight , smell , breathing ; eating and talking which impact negatively on the victim 's quality of life . moreover , owing to high premium placed on facial appearance in many societies esthetic disturbance could results in adverse psychological consequence . unfortunately , limited specialized manpower needed to treat this injuries and the considerable treatment cost imposes huge burden and demand on the ever dwindling healthcare recourses of developing nation like ours . owing to the forgoing , it is necessary to explore the etiology and pattern of fractures of facial skeleton . such periodic verification of the etiology of maxillofacial injuries will facilitates the assessment of proficiency of road safety measures such as speed limit , drunk driving , crash helmets , and seat belt laws . it will also help to identified the behavioral patterns of commuters in the locality and helps to recommend ways in which injuries to the face can be averted . it will also guide the future funding of public health programs geared towards prevention , provision of facilities , training of manpower ; and aid the healthcare provider to optimize treatment of maxillofacial injuries . hence , this study is aimed at reporting the etiology , pattern , and treatment of facial fractures at our oral and maxillofacial surgery department in usmanu danfodiyo university teaching hospital , sokoto . case records of 40 patients who presented with facial fracture at the dental and maxillofacial surgery department of usmanu danfodiyo university teaching hospital , sokoto between january , 2011 and december , 2011 were retrieved by the first author and the following variables etiology , age , gender , site , and treatment were obtained . this hospital is the only tertiary and referral center in the state and it is strategically located in the state capital , sokoto . sokoto is a city located in the extreme northwest of nigeria , near to the confluence of the sokoto river and the rima river . sokoto is the modern day capital of sokoto state ( and its predecessor , the northwestern state ) . it is situated in northwestern of the country between latitudes 4n to 14n and longitudes 2e and 15e . kebbi state border to the southwest , katsina state to the east , zamfara state to the southeast , and republic of niger to the northwest with a land mass of approximately 25,973 km . sokoto state has a projected population of 3.7 million people based on 2006 census made up of two ethnic groups namely , hausa and fulani . sokoto town , the capital of sokoto state , has a population of approximately 2.5 million . the population is largely rural with farming , cattle rearing , and fishing as the predominant occupations ( > 80% ) . the diagnosis of fracture was based on clinical history , signs and symptoms , visual findings , manual examination , and correct interpretation of plain radiographs . the pattern of facial fracture is determined according to the fractures of mandible , midface , and alveolar bone . fractures of the middle third of the facial skeleton were classified according to the le fort classification . fractures including the base of the skull and frontal bone were not included in the present study . closed reduction and dental wiring with arch bars , direct wires , and eyelet wires combine with mandibulomaxillary fixation ( mmf ) were routine mode of treatment for mandibular fractures . open reduction and internal fixation ( orif ) with intraosseous wire of mandibular fractures were employed when indicated . fractures of the maxillae / le fort fractures were reduced and fixed by eyelets / arch bars combined with mmf and with / without suspension wires . stable zygomatic complex fractures were reduced ( elevated ) intraorally , and unstable ones were supported by antral packs . all patients were placed on oral or intravenous antibiotics for 5 - 7 days except those with established infections who had their antibiotics regimen appropriately extended . data analyses for age , sex , etiology , site of fracture , and treatment given were performed using analyse - it for microsoft excel 2012 . pearson 's chi - square test was used to compare the frequency distribution and statistical significance was set at p 0.05 . there was an overwhelming male dominance in all age groups ( male : female ( m : f ) = 19:1 , odds ratio = 380 ) [ table 1 ] . the most susceptible age group was 21 - 30 years ( 47.5% ) and the least were 0 - 10 years and 51 - 60 years ( 2.5% ) [ figure 1 ] . the most common cause of fracture was rtcs ( 87.5% ) of which 58.3% were motorcycles related and mostly involved riders ( 80.9% ) [ table 2 ] . maxillofacial trauma was highest in the months of october ( 20% ) and june / july ( 15% ) [ figure 2 ] . sex and age distribution age distribution of facial fracture patients etiology and person distribution seasonal variation of facial fractures isolated mandibular fractures were most frequent ( 60% ) and least fractured bone was maxilla ( 7.5% ) [ figure 3 ] . among mandibular fractures , the body was more regularly involved ( 36.5% ) followed by parasymphyseal ( 21.2% ) [ table 3 ] . for midfacial fractures , zygomatic bone and arch there were associated soft tissue injuries with the lower lip mostly affected ( 75% ) especially in rtcs ( p < 0.05 ) . in 14 facial fracture distribution according to involved bone.1 mandible ; 2 maxilla ; 3 zygoma summary data of facial fracture patients closed reduction and dental wiring with arch bars , direct wires , and eyelet wires combined with mmf were the most common form of treatment for mandibular fractures [ figure 4 ] . fractures of the maxillae / le fort fractures were reduced and fixed by eyelets / arch bars combined with suspension wires and mmf . zygomatic complex fractures were treated either conservatively or by either closed or open reduction with gillies temporal or buccal approaches . facial fracture has become a public health issue in many parts of nigeria and globally owing to the attendant mortality , morbidity , and huge socioeconomic consequences . hence , this study was primarily undertaken to look at the pattern of facial fracture and treatment modalities at our relative young center offering maxillofacial trauma services . we hoped that this will help to provide baseline information to guide in prevention and proper planning of maxillofacial trauma care in our hospital and in the region . in this present study , more males were affected than females with a very high odd ratio ; this is the highest reported figure from nigeria and worldwide . our finding , is however , not in concordance with most studies from nigeria , western europe , and japan that showed much narrower gender ratio . fasola et al . , and tanaka et al . , found a 3:1 male - to - female ratio . others include more male exposure to certain risky situations , for example , there are more male drivers on the roads , especially on highways ; men are more likely to practice contact sports ; and men attend bars more often and consequently are more likely to use alcohol and other drugs prior to driving . in addition , culturally in many traditional african and islamic societies , male are often the bread winner of the family and women predominantly take care of the home and only occasionally involved in other activities outside . in works with narrower or equal gender ratio , the female lived a more outdoor life and share similar jobs with their male counterpart , thereby being exposed to similar risk factors . maxillofacial fracture incidence in the present study was highest in october followed by june and july . kontio et al . , in his study found an increase in incidents in june and august , while ravindra and ravindra nair reported significant high in april and may . ogundare et al . , and paes et al . , suggested that increased maxillofacial trauma during summer holiday is due to availability of more leisure time and high alcohol consumption . however , our own data indicates that the months of june and july are peak of raining season , while october is the harvesting time . hence , road travel might be compromised especially with increased density of people plying these roads . the modal age group of 21 - 30 years seen in this present cohort is consistent with the literature . paes et al . , pointed out that this age group reflects people at the prime of their life . they have zest , increased physical energy , and adventurousness , but lack experience and often impetuous ; hence , are likely to disregard traffic regulations . in the present study , the reason in this cohort is unknown ; however , we speculate that owing to preponderance of rtc and its severity , elderly patients might not have survived . the etiology of maxillofacial injuries varies from one country to another and even within the same country depending on the prevailing socioeconomic , cultural , and environmental factors . recent works had established rtc as the leading agent of facial fractures in most developing countries of africa , middle east , asia , and some parts of europe . in contrast to zimbabwe , south africa ; northeastern nigeria and economically advance nations of europe , australia , and united state of america ; interpersonal violence in the form of physical assaults and gunshots has replaced rtc as the predominant cause of facial fracture . according to taher and olasoji et al . , fractures caused by firearm bullets and assault appear to be the most usual cause of facial fracture in iran and northeastern nigeria . rtcs are among the major factor of maxillofacial injuries and according to the results of previous studies the reason of 14.8 - 90.2% of all the skeletal and soft tissues injuries of the face . fasola et al . , noted that rtcs were major cause of death and injury , especially in low and middle - income countries . it is estimated that road traffic injuries will have risen from ninth to third in world disease burden rankings by 2020 , accounting for 2.3 million deaths globally . in developed nations , progress in technology has resulted in more personal and vehicle protective measures being implemented coupled with access to proper medical care , good road and transport facilities ; and regular enforcement of traffic rules and regulations . the reverse scenario exists in developing countries especially of sub - saharan africa which has led to an increase in mortality and morbidity associated with rtcs . the other significant etiologic factors of injuries to the maxillofacial region are assaults / interpersonal violence , falls , sport , industrial , gunshot / warfare , animal , iatrogenic , and suicide / self - inflicted . the current study finding of 87.5% of road traffic collision is similar to that of kadkhodaie in a recent iranian study that found 91% of their facial fracture attributed to rtc . this result also comparable with many nigerian and middle eastern studies that demonstrated rtcs as the most frequent cause of facial fractures . our finding of over 50% of rtc involved head - to - head collision of motorcycles and is reflected by the high frequency of riders involved than pillion passengers which confirmed the works of oginni et al . , and ramli et al . in sokoto metropolis and state as a whole , there is no established public transport system . transport within the city ( when not by foot ) is mainly by motorcycles which operate as one - person taxis . this is quite lucrative and required low start - up capital and has subsequently , attracted many ; particularly unskilled , illiterate , and unemployed youth who flock the city from many rural areas . increase in the use of motorbikes has led to a greater number of accidents and , consequently , multiple facial fractures . other nigerian authors have raised alarm on the increasing menace of commercial motorcyclists on nigerian roads . saheeb and etetafia and earlier nigerian studies repeatedly cautioned that many commercial motorcyclists and motorists ignore the crash helmets / seatbelt regulations despite nigerian traffic code imposition of stiff penalties against noncompliance with seat belts and crash helmet use . additional problems mentioned are the overloading of passengers / pillions and goods , high speed , and imprudence among commercial motorcyclists ( okada ) and motorists . a recent brazilian study attributed their high frequency of rtcs to the fact that there population are largely rural and are farmers . therefore , they travel great distance to transport their produce to the city most often in vehicles overloaded and not road worthy . other reasons postulated for the high frequency of rtcs include lack of street lights , inadequate road safety awareness , unsuitable road conditions without expansion of the motorway network , violation of speed limits , and old vehicles without safety features such as anti - burst locks and energy absorbing materials . furthermore , entry into opposite traffic lane without regard , violation of the right of the way , violation of the highway code , use of alcohol or other intoxicating agents , behavioral disorders , and poor socioeconomical conditions of some drivers . street lighting has been suggested as a relatively low cost intervention with the potential to prevent traffic crashes . in this study , for diagnosis of facial fracture we relied on clinical examination and conventional plain radiography . adeyinka et al . , noted that midfacial skeleton is much more difficult to assess using plain films than is the mandible . the presence of thin bones , fluid - filled spaces ( e.g. , congested sinuses ) , and soft tissues ( e.g. , orbital contents ) make accurate assessment difficult with images that do not offer a high degree of contrast . our low utilization of advance imaging for maxillofacial fractures ( e.g. , computed tomography ( ct ) scan ) is due to poor socioeconomic condition of the study population as health is paid for out of pocket . an average ct scan in sokoto costs 25,000 naira ( about $ 160 ) which is quite exorbitant for most categories of our patients who are predominantly low socioeconomic class . reports in the literature noted that the pattern , incidence , and severity of facial fractures due to rtc often depend on mechanism of injury and mode of transportation predominant in that region . over half of the proportion of facial fractures in this study were mandibular fractures followed by fractures of the zygomatic complex and maxillary bones . this is in agreement with most studies , but contradicts that of ravindra and ravindra nair and others from economically advanced countries where midfacial fractures were more frequently diagnosed . it is mentioned in the maxillofacial literature that the mandible is more vulnerable to fracture owing to its mobility , open arch , atrophy after dental loss and conspicuousness in the lower part of the face . hence , it takes part in the mechanism of hyperextension and hyperflexion of the head in traffic crashes . the present series showed that the body is the most fractured site in the mandible followed by parasymphysis . this is in accordance with recent nigerian studies , but at variance with that of ogundare and elsewhere that demonstrated angle and parasymphyseal fractures as the most common jaw fracture . analysis of our finding showed that the zygoma was the more commonly fractured midfacial bone which is comparable to the literature . however , it contradicts the works of gassner et al . , that reported le fort and orbital fractures as the most frequently affected midfacial bone . it should be highlighted that our result might be affected by the limitations of plain film in assessing midfacial fracture due to low contrast owing to presence of thin bones , fluid - filled spaces ( e.g. , congested sinuses ) , and soft tissues ( e.g. , orbital contents ) . we found in this study high multiple facial injuries and concomitant head injury which is similar to the study of posnic et al . , but contrary to that of singh et al . our study revealed that the mandibular fracture treatment is by closed reduction with mmf and on a few occasion open reduction with transosseous wiring was employed . stable zygomatic fractures were reduced by elevation , and unstable ones supported by antral packs . this treatment modality was chosen as in previous nigerian reports based on affordability , availability , simplicity , and safety ; and plethora of skill and armamentarium . titanium plate osteosynthesis for facial fracture treatment has gained popularity worldwide and is the gold standard . in resource challenged environment however , adopting this trend has been quite difficult owing to the low socioeconomic status of our patients , high cost of acquisition , specialized training and skilled needed , and limited theater space for treatment under general anesthesia especially with other competing surgical specialties . however , a recent review by akadiri and omitola demonstrated that despite the drawbacks of closed reduction results obtained are satisfactory and quite comparable with titanium plate osteosynthesis of orif . we had included in our study only those patients who were treated by the oral and maxillofacial surgery unit . isolated nasal fractures are treated by the ear , nose , and throat ( ent ) surgeons in our hospital which resulted in is none featuring in this article . other parameters pertaining to maxillofacial injuries such as malocclusion , infections , and pseudoarthrosis were missing from our records ; hence , making their analysis impossible . we had included in our study only those patients who were treated by the oral and maxillofacial surgery unit . isolated nasal fractures are treated by the ear , nose , and throat ( ent ) surgeons in our hospital which resulted in is none featuring in this article . other parameters pertaining to maxillofacial injuries such as malocclusion , infections , and pseudoarthrosis were missing from our records ; hence , making their analysis impossible . in summary , rtc was the most frequent cause of facial fracture in our study cohort . a preponderance of male aged 21 - 30 years is in agreement with the literature . furthermore , this current study highlights mandibular fractures as the most common in our environment and low utilization of orif in facial fracture treatment . for a developing economy like ours these include road safety and traffic education to create awareness among our road users with the desire to achieve paradigm behavioral shift that will reduce accidents and carnage on our highways . therefore , a multisectoral strategy that will involve the media , road safety and traffic managers , engineers , town planners , politicians , policy makers , road and motorcycle workers unions , private companies , nongovernmental organizations , and civil associations will facilitate these preventive measures and help to educate road users to be thoughtful and conscious in traffic and to have a responsible and civil attitude to avoid accidents . this study recommends the following : establishment of functional and affordable intra- and intercity public transport systemincorporation of teaching of traffic rules and regulations in our school curriculum at all level of our educational systemeducation and training of road users on adoption of road safety measures such as seatbelt , crash helmet , drunk / drug driving , overspeeding ; and strict reenforcement of road traffic regulationsmaking computer tomography scan more affordable to the generality of the public and extending health insurance coverage both in scope and personsincreasing facilities and provision of theater spaces for facial fractures management . making titanium plates and screws affordable ; thereby , facilitating the adoption of orif in the treatment of facial fractures . establishment of functional and affordable intra- and intercity public transport system incorporation of teaching of traffic rules and regulations in our school curriculum at all level of our educational system education and training of road users on adoption of road safety measures such as seatbelt , crash helmet , drunk / drug driving , overspeeding ; and strict reenforcement of road traffic regulations making computer tomography scan more affordable to the generality of the public and extending health insurance coverage both in scope and persons increasing facilities and provision of theater spaces for facial fractures management . making titanium plates and screws affordable this study recommends the following : establishment of functional and affordable intra- and intercity public transport systemincorporation of teaching of traffic rules and regulations in our school curriculum at all level of our educational systemeducation and training of road users on adoption of road safety measures such as seatbelt , crash helmet , drunk / drug driving , overspeeding ; and strict reenforcement of road traffic regulationsmaking computer tomography scan more affordable to the generality of the public and extending health insurance coverage both in scope and personsincreasing facilities and provision of theater spaces for facial fractures management . making titanium plates and screws affordable ; thereby , facilitating the adoption of orif in the treatment of facial fractures . establishment of functional and affordable intra- and intercity public transport system incorporation of teaching of traffic rules and regulations in our school curriculum at all level of our educational system education and training of road users on adoption of road safety measures such as seatbelt , crash helmet , drunk / drug driving , overspeeding ; and strict reenforcement of road traffic regulations making computer tomography scan more affordable to the generality of the public and extending health insurance coverage both in scope and persons increasing facilities and provision of theater spaces for facial fractures management . making titanium plates and screws affordable

background : facial fracture is gradually become a public health problem in our community due to the attendant morbidity and mortality . hence , the aim of this study was to determine the pattern of facial fracture in dental and maxillofacial surgery department of usmanu danfodiyo university teaching hospital . this cross - sectional study was undertaken to provide information regarding gender , age , etiology , and diagnosis of patients with maxillofacial fractures.materials and methods : a 1-year review of patients diagnosed and treated for facial fractures in usmanu danfodiyo university teaching hospital between january 2011 and december 2011 . the diagnosis was based on radiographic data and clinical examination . the main analysis outcome measures were etiology , age , gender , site , and treatment . data were organized and presented by means of descriptive statistics and pearson 's chi - square test . the level of significance adopted was 5%.results : a total of 40 patients were treated in this period . over 95% were male , 81% were caused by road traffic crash ( rtc ) and 86.4% were in the 21 - 30 years group . most patients ( 52% ) had mandibular fractures , and the most common site was the body . most patients with midfacial fractures had fractures of the zygomaticomaxillary region ( 36% ) , while fractures of the parasymphyseal region were more common in the mandible 156 ( 31% ) . the most common treatment for jaw fractures was mandibulomaxillary fixation ( mmf ) . stable zygomatic complex fractures were reduced ( elevated ) intraorally , and unstable ones were supported by antral packs.conclusions:this study highlights facial fractures secondary to rtc as a serious public health problem in our environment . preventive strategies remain the cheapest way to reduce direct and indirect costs of the sequelae of rtc . it also bring to the fore the necessity to shift to open reduction and internal fixation ( orif ) of fractures .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Limitations of the study CONCLUSION Recommendations

iniencephaly is a rare birth defect having retroflexion of the head combined with severe distortion of the spine . based on the presence or absence of encephalocele , lewis has classified iniencephaly into two groups - iniencephaly apertus and iniencephaly clausus . cardiovascular disorders , diaphragmatic hernias , and gastrointestinal malformations are other additional defects seen in these cases . most of iniencephalic babies are still born or die soon after birth ; however the milder forms of iniencephaly cases are not fatal . here a 25-year - old primi visited obstetric out - patient department with complaints of abdomen and back pain . she had neither antenatal check - up nor had taken folic acid supplementation previously . on abdominal ultrasound , a single - live fetus with hyperextension of cervical spine was found . she spontaneously delivered a female baby at 29-week gestation who died 10 minutes after delivery . on autopsy fetus weighed 900 g and showed severe retroflexion of enlarged head ( 28 cm in circumference , normal is 24.96 cm at 28 weeks and 28.59 cm at 32 weeks of gestation ) , short spine and neck with mandibular skin touching the chest , micrognathia , low set ears , and bilateral clubfoot [ figure 1a ] . radiograph of the necropsy was done which revealed hyperextension of the neck with occiput touching the cervicodorsal vertebrae with soft tissue continuation on the dorsal aspect of neck . cervical and upper thoracic vertebrae showed block vertebrae suggestive of iniencephaly [ figure 1b ] . necropsy of the fetus showed hypoplastic lungs weighing 18 g , small posterior fossa with hypoplastic cerebellum ( weight < 10 g ) , widened foramen magnum with anteroposterior ( ap ) diameter of 3.5 cm , and fusion of cervical and upper four thoracic vertebral bodies anteriorly with crowding of the arches and spinous processes posteriorly [ figures 2a and 2b ] . ( a ) external appearance of fetus showing the retroflexed head and bilateral club foot . ( b ) radiograph of necropsy showing block vertebrae and occipital bone defect . inset shows malformed cervical and upper thoracic vertebrae ( a ) sagital section of fetus showing widened foramen magnum ( red dotted line ) , small posterior fossa ( black arrow ) , and malformed cervical and upper thoracic vertebrae in comparison with normal vertebrae ( white arrow ) ( b ) anterior view of malformed and irregularly fused vertebrae ( yellow arrow ) and normal vertebrae ( white arrow ) here inion ( posterior most part of occipital bone ) fuses with the back leading to the absence of the neck and retroflexion of head . the exact etiology and pathogenesis is not known , both genetic and environmental causes have been implicated . chromosomal abnormalities including trisomy 18 , trisomy 13 , and monosomy x have been associated with this disorder . environmental causes like poor socioeconomic conditions , low parity , and lack of folic acid supplementation , obesity and drugs including sulphonamide , tetracycline , antihistamines , and antitumor agents are shown to have increased risk . in the present case poor nutrition and lack of folic acid supplementation during pregnancy may be the cause . the fetus will show typical star gazing appearance on usg and detailed cns and spine abnormalities may be known by mri or ct . the important features that help us to diagnose a case of iniencephaly are as follows : occipital bone deficit leading to enlarged foramen magnum.irregular fusion of malformed vertebrae.incomplete closure of vertebral arches and bodies.retroflexion of the cervical spine.upward turned face with chin continuous with chest because of the absence of neck . congenital retroflexion of spine is mainly seen in two groups of anomalies , one is anencephaly and other is iniencephaly . iniencephaly clausus should be differentiated from klippel fiel syndrome ( kfs ) and cervical meningomyelocele . anencephaly shows a total or partial absence of neurocranium and retroflexed head is not covered with skin . cervical vertebrae are abnormal in iniencephaly and they are almost normal in anencephaly . in the present case kfs is caused by a failure of segmentation of the cervical vertebrae during early fetal development . although fusion of cervical vertebrae and malformation may be present in both , retroflexion of head is usually not seen in kfs and the presence of retroflexed head should raise suspicion of iniencephaly . at times it is difficult or impossible to differentiate these two ; however it is important to separate the two because kfs is not lethal and can be corrected surgically . other entities that come in differential diagnosis are nuchal tumors such as teratoma , goiter , lymphangioma and jarcho levin syndrome . other findings in our case are hypoplastic lungs , cerebellar hypoplasia , bilateral club foot , and single - umbilical artery . chen in his study has found pulmonary defects and single - umbilical artery in 14.3% of cases . iniencephaly carries a bad prognosis ; there are only six cases of long - term survival of this anomaly in the literature . in summary , iniencephaly is one of the uncommon ntd which causes obstructed labor and necessitates proper obstetric management . it also carries the risk of recurrence in next pregnancies ( 15% ) ; hence the patient should be counseled and encouraged to take folic acid supplementation at least 3 months prior to conception . we present this case for its rarity and isolated iniencephaly without any other major malformations are not always fatal , and should be diagnosed antenatally because proper postnatal surgical therapy can be planned .

iniencephaly is a rare neural tube defect characterized by extreme retroflexion of the head with the absence of neck due to spinal deformities . the important features that help us to diagnose a case of iniencephaly are occipital bone deficit leading to enlarged foramen magnum , fusion of malformed cervical and thoracic vertebrae , and upward turned face with chin continuous with chest because of the absence of neck . the differential diagnoses include anencephaly with spinal retroflexion , klippel fiel syndrome , nuchal tumors such as teratoma , goiter , and lymphangioma and jarcho levin syndrome . previously many case reports on radiological features of iniencephaly are published , but there are very few articles on necropsy findings and differential diagnosis . in the present case we have discussed in detail the necropsy findings of iniencephaly clausus with special reference to differential diagnosis .

Introduction Case Report Discussion

if mitochondrial deficiency causes insulin resistance it must occur before the onset of the insulin resistance . it does not seem possible to answer the question of whether or not mitochondrial deficiency precedes insulin resistance in humans , because t2 dm patients and obese insulin resistant individuals are insulin resistant for many years before they are diagnosed therefore , available evidence comes from studies on laboratory rodents , which develop muscle insulin resistance within a few weeks after being started on a high - fat diet . if the high - fat diet is continued , the rodents become obese and develop the rodent equivalent of the visceral obesity / metabolic syndrome and/or t2 dm ( 17,18 ) . in a number of early studies , high - fat diets were found to induce an increase in the levels of mitochondrial marker enzymes , such as betahydroxybutyrate dehydrogenase and citrate synthase in muscle ( 19,20 ) . more recently , it was found that feeding rats high - fat diets ( 21,22 ) or intermittently increasing plasma fatty acids to high levels ( 23 ) induces an increase in mitochondrial biogenesis in skeletal muscle . this is evidenced by increases in mitochondrial enzyme proteins , in the capacity to oxidize fatty acids , and in mitochondrial dna copy number . the increase in mitochondria appears to be an early , transient event that is lost as the insulin resistance and obesity progress ( 24 ) . the fatty acid - induced increase in muscle mitochondria appears to be mediated by the nuclear receptor peroxisome proliferator activated receptor- ( ppar- ; also referred to as ppar- ) . ppar- is a transcription factor for the genes encoding the enzymes of the mitochondrial fatty acid oxidation pathway . previous studies had shown that overexpression or activation of ppar- in muscle induces an increase in mitochondrial biogenesis ( 25,26 ) . the investigators who performed these studies concluded that the increase in mitochondrial biogenesis was mediated directly by ppar- , because overexpression of ppar- in muscle did not result in an increase in peroxisome proliferator activated receptor coactivator-1 ( pgc-1 ) mrna . however , this did not seem possible , because ppar- regulates expression of only a subset of mitochondrial proteins and , therefore , can not mediate mitochondrial biogenesis , which requires the coordinated activation of numerous transcription factors , a process that is mediated by the transcription coactivator pgc-1 ( 27 ) . it seemed probable , therefore , that an increase in ppar- induces an increase in mitochondria by mediating a posttranscriptional increase in pgc-1 protein . this turned out to be the case , as both overexpression of ppar- or activation of ppar- by raising plasma fatty acids resulted in an increase in pgc-1 ( 22 ) . in light of these findings , it seems clear that mitochondrial deficiency is not necessary for the development of insulin resistance and that , at least in the fat - fed rat model , insulin resistance develops despite an increase in muscle mitochondria . that insulin resistance is also not due to mitochondrial deficiency in humans is evidenced by the finding that insulin - resistant asian indians with t2 dm have a muscle mitochondrial capacity for oxidative metabolism similar to that of nondiabetic indians and higher than that of healthy north americans of northern european ancestry ( 28 ) . various transgenic models have been used to test the hypothesis that a deficiency of mitochondria causes muscle insulin resistance . ( 29 ) studied transgenic mice in which mitochondrial transcription factor a ( tfam ) was knocked out in skeletal muscle . tfam is a transcription factor that mediates transcription of genes encoded in the mitochondrial genome , which includes a number of key mitochondrial respiratory chain proteins . there was a progressive , severe deterioration of respiratory chain function in the muscles of the tfam knockout mice . however , rather than causing insulin resistance , the marked decrease in mitochondrial oxidative capacity resulted in increases in insulin action and glucose tolerance , as well as enhanced basal and insulin - stimulated 2-deoxyglucose uptake in isolated muscles . in another study , pospisilik et al . ( 30 ) examined the effect of deletion of mitochondrial apoptosis - inducing factor , which is required for maintenance of a functional mitochondrial respiratory chain . skeletal muscle apoptosis - inducing factor knockout mice had reduced levels of mitochondrial respiratory chain protein complexes i and iv with an associated decrease in the capacity for substrate oxidation . this reduction in mitochondrial respiratory capacity resulted in improved insulin sensitivity and glucose tolerance , and enhanced insulin - stimulated muscle glucose transport activity . in a third study , kelly and colleagues ( 31 ) generated mice that had combined deficiency of pgc-1 and pgc-1 in skeletal muscle . these totally pgc-1deficient mice had markedly reduced levels of mitochondrial enzymes and 90% decrease in the capacity of muscle to oxidize fat ( 31 ) . this severe mitochondrial deficiency resulted in an improvement in glucose tolerance . in an earlier study by our group , we generated transgenic mice that expressed uncoupling protein-1 ( ucp-1 ) in their skeletal muscles ( 32 ) . the original purpose of this study was to determine the effect of a modest reduction of steady - state atp concentration on mitochondrial biogenesis . however , the amount of ucp-1 expressed in the muscles of the transgenic mice was too high and resulted in disruption of mitochondrial structure ( fig . 1 ) , and caused large decreases in the levels of key mitochondrial enzyme proteins . the mechanism by which a high content of ucp-1 damages muscle mitochondria is not known . despite the severe reduction in mitochondrial function , and the associated accumulation of large amounts of intramuscular lipid ( fig . 1 ) , there were large increases in basal and insulin - stimulated muscle glucose transport activity ( 32 ) . electron microscopic images of muscle from wild - type mice ( a and c ) and from muscles of mice with severely disrupted mitochondria caused by high overexpression of uncoupling protein 1 ( ucp - h ) ( b , low magnification ; d , high magnification ) . * large lipid droplets surrounding abnormal mitochondria . reprinted with permission from han et al . , kelley and colleagues ( 33 ) published an article in which they concluded that insulin resistance in t2 dm is mediated by a deficiency of the electron transport chain ( etc ) enzymes with no deficiency of the citrate cycle or fatty acid oxidation pathway enzymes . they postulated that this results in an imbalance between the etc and the citrate cycle and fatty acid oxidation pathways , and that this imbalance causes insulin resistance . this claim is puzzling , both because their initial reports that t2 dm patients have mitochondrial dysfunction were largely based on the finding of decreases in citrate synthase ( 1,3 ) , and carnitine palmityl transferase ( 2 ) , and because the studies by pospisilik et al . ( 29 ) had shown that severe etc enzyme deficiency results in improved insulin action . to further test the hypothesis that a selective decrease in components of the mitochondrial etc , that results in an imbalance between the etc and the fatty acid oxidation pathway and citric acid cycle is responsible for insulin resistance , rats were made severely iron deficient by means of an iron - deficient diet ( 34 ) . iron deficiency results in decreases in the iron - containing mitochondrial respiratory chain proteins without affecting the noniron - containing enzymes of the fatty acid oxidation pathway and citrate cycle ( 35 ) . some of the iron - deficient rats were fed a high - fat diet . iron deficiency resulted in large decreases in iron - containing mitochondrial respiratory chain proteins in muscle ( 34 ) . citrate synthase and long - chain fatty acyl - coa dehydrogenase , used as markers for the citrate cycle and fatty acid oxidation pathways , were unaffected by the iron deficiency . oleate oxidation by muscle homogenates was increased by high - fat feeding and markedly decreased by iron deficiency despite a high - fat diet . the high - fat diet also increased long - chain acyl - coa dehydrogenase expression . as a result , there was a fivefold increase in the ratio of long - chain acyl - coa dehydrogenase to cytochrome c , indicating a large imbalance between the fatty acid oxidation pathway and respiratory chain . the iron deficiency completely protected against the high - fat diet induced insulin resistance , and also resulted in a higher insulin - stimulated muscle glucose transport activity compared with animals in the low - fat diet , normal iron group ( 34 ) . these studies on rodents showing that mitochondrial deficiency / dysfunction causes improvements in insulin action and glucose tolerance raise the question , why are t2 dm patients insulin resistant despite a reduction in muscle mitochondria ? the hypothesis that was tested in the studies on rodents reviewed above was , does mitochondrial deficiency / dysfunction severe enough to limit fat oxidation in resting muscle cause insulin resistance ? to test this hypothesis , an extremely severe reduction in functional mitochondria is necessary . the rate of substrate oxidation is determined by the demand for energy and is regulated by the rate of atp breakdown / adp production . the energy requirement of resting muscle is determined by housekeeping functions such as maintenance of electrochemical gradients and protein synthesis and is very low relative to the maximum capacity for substrate oxidation . therefore , a very severe reduction in functional mitochondria is necessary in order to result in limitation of resting substrate oxidation in resting muscle . in the various animal models in which mitochondrial deficiency / dysfunction resulted in improved insulin action and glucose tolerance , substrate oxidative capacity was so low that it resulted in a decrease in steady - state atp concentration and an increase in amp ( fig . the increase in amp resulted in activation of amp kinase , which directly stimulates glucose transport and also induces an increase in the glut4 ( fig . the increase in amp kinase activity explains the improvement in insulin action , glucose tolerance , and muscle glucose uptake in rodent muscle with severe mitochondrial deficiency / dysfunction . atp ( a ) , amp ( b ) , amp kinase activity ( c ) , glut4 protein ( d ) , and basal and insulin - stimulated 2-deoxyglucose transport rates ( e ) in muscle of control mice and mice with high overexpression of uncoupling protein 1 ( ucp - h ) and severe disruption of mitochondria . * in contrast to the rodent models of mitochondrial deficiency / dysfunction and improved insulin action in which the decrease in muscle respiratory capacity is so severe they ca nt exercise , t2 dm patients , despite a low vo2max of 26 ml o2 kg min ( 6 ) , are able to exercise sufficiently vigorously to increase their whole body rates of o2 uptake / substrate oxidation 89-fold . since the muscle mass involved in the exercise makes up 20% of body weight , this means muscles of t2 dm patients have a sufficiently high respiratory capacity to increase substrate oxidation 40-fold . in light of this huge reserve capacity , it is clear that a 30% lower than normal content of mitochondria is not sufficient to limit resting substrate oxidation in muscles of t2 dm patients or , therefore , to cause a decrease in atp or an increase in amp and amp kinase activity . it is clear from the many studies showing that mitochondrial deficiency / dysfunction severe enough to limit fat oxidation in resting muscle increases insulin responsiveness , that mitochondrial deficiency plays no role in mediating insulin resistance . studies by kelley and colleagues have supported the concept that fat oxidation is reduced in insulin - resistant individuals . in one of these studies , fat oxidation was decreased and glucose oxidation was increased in the resting , postabsorptive state in 11 t2 dm patients compared with 9 nondiabetic control subjects ( 36 ) . in another , fat oxidation by muscle was lower in 40 obese men than in lean control subjects ( 2 ) . however , in a subsequent study on obese men , kelley and colleagues in collaboration with wolfe ( 37 ) found that obese men derive a greater proportion of energy from fat oxidation than lean men ( 43 vs. 31% ) during mild exercise . in another study , kelley and colleagues found that fat oxidation was not reduced in obese insulin - resistant individuals or t2 dm patients , with fat providing 37% of total energy in the lean control subjects , 47% of energy in the obese insulin - resistant individuals , and 43% in the t2 dm patients ( 38 ) . there have also been reports that plasma fatty acid oxidation was reduced in insulin - resistant individuals , giving the impression that fat oxidation was impaired ( 39,40 ) . however , in these studies , nonplasma fatty acid oxidation ( probably from intramuscular fat ) was increased , so that total fat oxidation was not significantly different between the control and insulin - resistant groups . ( 40 ) , total fat oxidation averaged 0.92 0.08 mol kg min in the control subjects and 1.07 0.18 mol kg min in the obese group . most of the studies by other investigators comparing obese insulin - resistant individuals and/or patients with t2 dm have , however , found that insulin resistant individuals have increased fat oxidation ( 7,37,4147 ) . a hypothesis that used to be rather widely accepted is that insulin resistance is mediated by increased fat oxidation . ( 48 ) that fat oxidation inhibits glucose uptake and metabolism and is , therefore , referred to as the evidence for the randle effect in insulin resistant humans was provided by defronzo and colleagues ( 41 ) , who found that fat oxidation is increased in patients with t2 dm and in obese insulin - resistant individuals compared with normal control subjects . most of the other studies comparing fat oxidation in normal controls with t2 dm patients and/or obese insulin - resistant individuals have also shown that fat oxidation is increased in insulin - resistant obese individuals and patients with t2 dm ( 7,37,4147 ) . in conclusion , the answer to the three questions addressed in this counterpoint article is clearly no . rather than a decrease in mitochondria , an increase in mitochondrial biogenesis occurs in skeletal muscle concomitantly with the development of muscle insulin resistance ( 21,22 ) . rather than causing insulin resistance , a decrease in mitochondria or disruption of mitochondrial function sufficiently severe to limit fat oxidation in resting muscle results in increases in basal and insulin stimulated glucose transport into skeletal muscle and an improvement glucose tolerance ( 2932,34 ) . rather than a reduction in fat oxidation in muscle , the majority of studies show that fat oxidation is increased in obese , insulin resistant individuals and patients with t2 dm ( 7,37,4147 ) .

based on evidence that patients with type 2 diabetes ( t2 dm ) , obese insulin - resistant individuals , and lean insulin - resistant offspring of parents with t2 dm have 30% less mitochondria in their muscles than lean control subjects , it appears to be widely accepted that mitochondrial deficiency is responsible for insulin resistance . the proposed mechanism for this effect is an impaired ability to oxidize fat , resulting in lipid accumulation in muscle . the purpose of this counterpoint article is to review the evidence against the mitochondrial deficiency concept . this evidence includes the findings that 1 ) development of insulin resistance in laboratory rodents fed high - fat diets occurs despite a concomitant increase in muscle mitochondria ; 2 ) mitochondrial deficiency severe enough to impair fat oxidation in resting muscle causes an increase , not a decrease , in insulin action ; and 3 ) most of the studies comparing fat oxidation in insulin - sensitive and insulin - resistant individuals have shown that fat oxidation is higher in t2 dm patients and obese insulin - resistant individuals than in insulin - sensitive control subjects . in conclusion , it seems clear , based on this evidence , that the 30% reduction in muscle content of mitochondria in patients with t2 dm is not responsible for insulin resistance .

DOES A REDUCTION IN MITOCHONDRIA PRECEDE THE DEVELOPMENT OF INSULIN RESISTANCE? DOES A REDUCTION IN MUSCLE MITOCHONDRIA CAUSE INSULIN RESISTANCE? DOES MITOCHONDRIAL DEFICIENCY DECREASE FAT OXIDATION IN T2DM PATIENTS AND INSULIN-RESISTANT OBESE INDIVIDUALS?

for survival and reproduction , the body has developed an intricately balanced system to efficiently control energy homeostasis at multiple levels . in short , the brain continually monitors the systemic metabolic state and adjusts behavior , as well as humoral and neuronal outputs to peripheral effector organs , to ensure an appropriate energy supply . if the central nervous system ( cns ) senses a caloric shortage or surplus , the brain orchestrates responses that alter food intake , nutrient partitioning and physiological functions such as hepatic glucose production , adiposity and thermogenesis . efficient maintenance of the delicate homeostatic balance of energy , glucose and lipid metabolism largely depends on system - wide synchronicity of metabolic processes that can only be achieved by central regulatory influences and master circuits in the brain . disruption of such synchronicity , or failure of any of the key components of this system , are common pathophysiological causes of metabolic disorders such as obesity and diabetes . the most well - studied metabolic sensing region in the forebrain is the hypothalamus , where a number of nuclei such as the arcuate nucleus ( arc ) and ventromedial hypothalamus ( vmh ) express high levels of receptors that bind adipokines ( cell signaling molecules secreted by adipose tissue , such as leptin ) and gut hormones . central leptin resistance , one of the major causes of obesity , is caused by defective leptin sensing in these brain regions ( gautron and elmquist , 2011 ) . in the hindbrain , the nucleus tractus solitarius ( nts)dorsal motor nucleus of the vagus ( dmv ) complex is the best - studied brain area with respect to detection of metabolic feedback , especially from the gastrointestinal system via vagal afferents or the circulation ( berthoud et al . , 2006 ) . in this article and the accompanying poster , we provide an overview of current knowledge regarding coordinated system - wide connections that maintain energy homeostasis , highlighting how disruptions in metabolic sensing and integration between the brain , white adipose tissue ( wat ) and the gastrointestinal system can contribute to metabolic disorders . adipose - tissue communication pathways , it is important to acknowledge that other important cns - peripheral communications ( such as those involving muscle and brown adipose tissue ) also contribute to energy homeostasis . during embryogenesis , the parasympathetic ganglia of both the gut and the brain originate from the neural crest . this common origin could explain why many gut - secreted hormones and peptides and their receptors are also found in the brain ; indeed , many hormones produced by the gastrointestinal tract are referred to as brain - gut hormones . energy homeostasis can be well maintained only when the brain - gut circuit is synchronized . this synchronization is achieved by neuronal and humoral interaction between the brain and gut . in the gastrointestinal tract , nutrient digestion and absorption trigger the secretion of gut satiety signals such as cholecystokinin ( cck ) , peptide yy ( pyy ) and glucagon like peptide-1 ( glp-1 ) for stimulation of vagal sensory nerves to provide feedback to the brain ( berthoud , 2008 ) . vagal sensory terminals also express receptors for other gut hormones and peptides that are involved in metabolic regulation . the nts in the hindbrain is one of the major processors of vagal afferent signals , as evidenced by the finding that upper intestinal lipids inhibit hepatic glucose production through a vagal - nts circuit ( wang et al , 2008 ) . additionally , gut hormones and nutrients can influence several specific brain regions and neurons via the circulation . these factors reach these regions and neurons via transporters across either the blood - brain barrier ( bbb ) and/or less bbb - protected circumventricular organs where neurons can sense blood - borne signals more directly ( cottrell and ferguson , 2004 ; gross , 1992 ) . among these regions , the nts area postrema ( ap ) complex in the hindbrain and the arc in the forebrain are two of the major targets of gut hormones ; insulin , produced in the pancreas , and ghrelin , produced in the stomach , are among the best studied gut hormones acting in these brain areas ( kojima et al . , 1999 ; obici et al . , 2002 ; pocai et al . , 2005 ; tschp et al . , 2000 ) . in response to gut hormones and nutrient signals , these brain areas relay the information to other key regulatory areas , such as the paraventricular nucleus ( pvh ) of the hypothalamus ( to maintain systemic metabolic homeostasis by modulating neuroendocrine and autonomic outputs ) and the suprachiasmatic nuclei [ scn ; where the central biological clock is located ( yi et al . , 2006 ) ] ( to synchronize the behavior and physiology of the whole body and balance the sympathetic and parasympathetic autonomic outflow ) . defective or weakened signal feedback by the brain in response to satiety and nutrients from the gut could cause over - feeding and dis - inhibition of liver glucose production , and thereby promote metabolic diseases ( berthoud , 2002 ) . the autonomic system uses sympathetic and parasympathetic outflow to balance peripheral energy production and expenditure . pre - autonomic neurons located in the pvh project to the lower autonomic centers in the hindbrain and spinal cord and mediate autonomic outflow to different parts of the gut system . good examples of these autonomic - center gut connections are the parasympathetic outflow conveying the central insulin effect on liver glucose production ( obici et al . , 2002 ; pocai et al . , 2005 ) , and the central glucose - sensing effect on liver lipoprotein secretion ( lam et al . , 2007 ) . in contrast , activation of the sympathetic nervous system normally antagonizes peripheral insulin sensitivity . in wat , this antagonism triggers free fatty acid ( ffa ) release ; in liver , it stimulates glucose production via gluconeogenesis and glycogenolysis ; in muscle , it increases ffa oxidation , decreases glucose oxidation and glycogen synthesis , and results in reduced glucose uptake . abnormal elevation of sympathetic outflow could be caused by overactivation of orexin neurons under conditions of sleep deprivation ( yi et al . , 2009 ) , lack of inhibition of neuropeptide y neurons in the arc in the mediobasal hypothalamus ( van den hoek et al . , 2008 ) , or dysregulation of the melanocortin receptor in diverse populations of cells in the forebrain and hindbrain ( skibicka and grill , 2009 ) . feedback from gut hormones to the hypothalamic autonomic control center in the pvh can also influence neuroendocrine output . most of the neuroendocrine - derived hormones ( e.g. epinephrine , glucocorticoids , thyroid hormones and growth hormone ) also counteract insulin s action in the liver and other tissues ( yi et al . , 2010 ) . adipose tissue not only dynamically accumulates and releases lipids , but also acts as an endocrine organ . adipose tissue produces adipokines that inform the brain about whole - body long - term energy - storage status , and the tissue drives the brain s control of energy balance and the long - term regulation of body weight . in terms of adipose tissue crosstalk with the cns , the best studied adipokine is leptin ( halaas et al . , 1995 ; maffei et al . , 1995 ) , although adiponectin , resistin and apelin are also well studied with respect to their specific signaling pathways and functionalities in the cns ( banerjee et al . , 2004 ; , 2012 ; rajala et al . , 2004 ; singhal et al . , 2007 ; it is not yet clear whether other adipokines , including those recently identified [ such as retinol binding protein 4 ( rbp4 ) , visfatin and omentin ] , are involved in adipose - tissue brain communication . in addition to adipokines , wat also communicates with the brain via neuronal inputs ( bartness et al . , 2010b ) . the inability to sense leptin signals ( leptin resistance ) in key areas in the hypothalamus and hindbrain is thought to be one of the underlying mechanisms in the development of obesity . generally , leptin resistance can cause the brain to misunderstand long - term energy stores and , as a result , initiate extra energy production i.e. overfeeding ( hyperphagia ) and glucose production ( hyperglycemia ) , which are typical symptoms of metabolic disorders . the dominant autonomic - wat connections are neuronal sympathetic outputs that initiate lipid mobilization in wat . this sympathetic outflow can be generated from the higher autonomic center in the pvh or the lower centers in the hindbrain and spinal cord . hardwired sympathetic outflow ( mainly by releasing norepinephrine ) is greater than that stimulated by epinephrine released by the adrenal medulla . loss of autonomic control from the pvh [ for instance by lesion of the nuclei ( foster et al . , 2010 ) ] can increase the accumulation of lipids in wat without influencing lipid mobilization . this indicates that obesity could be caused by excess energy deposition owing to malfunction of the sympathetic autonomic nervous system . although the cns as a whole has been well studied with respect to its role in detecting and integrating metabolic signals , the roles of specific cell types , such as astrocytes , have not . astrocytes are a major class of macroglial cells that occupy up to 50% of the total brain volume . beyond their classical roles in neuronal proliferation and differentiation , modulation of synaptic efficacy , and maintenance of ionic and neurotransmitter homeostasis , astrocytes also express adipokine receptors , including leptin receptors ( pan et al . , 2008 ) . thus , a defect in adipokine signals to the brain would affect both neurons and astrocytes . for example , in response to a high - fat diet , astrocytes display increased expression of leptin receptors ( hsuchou et al . , 2009 ) . in addition , a high - fat diet induces astrocytosis , causing an increase in astrocytic components between the blood vessels and the anorexigenic proopiomelanocortin ( pomc ) neurons in the arc of the mediobasal hypothalamus ( horvath et al . , 2010 ) . notably , leptin s role in directing the inhibition of food intake occurs in part through signaling in pomc neurons ( seeley et al . , 1997 ) . in obesity , although leptin concentration is increased in the circulation ( hyperleptinemia ) , the reactive astrocytes with their enlarged ensheathments hamper the pomc neurons ability to sense leptin in blood vessels , which might contribute to leptin resistance . in addition to astrocytes , another important non - neuronal cell type involved in metabolic sensing is the tanycyte , which resides along the wall of the third ventricle . owing to their expression of glucose transporter 2 ( garca et al . , 2003 ) , tanycytes are thought to be involved in glucosensing from the cerebral spinal fluid in the brain ventricle system . for accurate and timely monitoring of the peripheral metabolic state , certain areas are developed by the brain to more directly sense the metabolic feedback from the energy input site ( the gastrointestinal system ) and the energy storing site ( wat ) . these short - term and long - term inputs are integrated at different levels within the cns , and are incorporated into neuroendocrine and autonomic outputs to balance energy intake and expenditure . a malfunction at any level in this input - output circuit could contribute to a homeostasic imbalance and contribute to metabolic disorders . recent progress has been made in the treatment of metabolic syndrome through a combinational strategy , integrating different gut hormones and/or adipokines , while eliminating their potential side effects ( day et al . , 2009 ) . however , there are still many unknowns that make therapeutic strategies problematic . for example , before we completely understand the mechanism of leptin resistance ( for which cell specificity and intracellular signaling have yet to be clarified ) , leptin can not be used to treat obesity . another important unanswered question is how we can target the autonomic system to modulate energy homeostasis . in the past , efforts have been made to determine whether there are specific circuits between autonomic neurons and peripheral organs and tissues . for instance , are the liver and pancreas controlled by the same or different groups of pre - sympathetic neurons ( kreier et al . , 2006 ) ? are there neurochemical characteristics specific to each subtype of pre - autonomic neuron that enable their connections to peripheral organs or tissues ( bartness et al . is it possible to target specific subtypes of pre - autonomic neurons to manipulate their output to specific peripheral organs or tissues ? improved neuroanatomical and neurophysiological methods , combined with genetic tools , will be helpful in addressing these questions and will improve the outlook for anti - obesity therapy .

one of the side effects of our modern lifestyle is a range of metabolic diseases : the incidence of obesity , type 2 diabetes and associated cardiovascular diseases has grown to pandemic proportions . this increase , which shows no sign of reversing course , has occurred despite education and new treatment options , and is largely due to a lack of knowledge about the precise pathology and etiology of metabolic disorders . accumulating evidence suggests that the communication pathways linking the brain , gut and adipose tissue might be promising intervention points for metabolic disorders . to maintain energy homeostasis , the brain must tightly monitor the peripheral energy state . this monitoring is also extremely important for the brain s survival , because the brain does not store energy but depends solely on a continuous supply of nutrients from the general circulation . two major groups of metabolic inputs inform the brain about the peripheral energy state : short - term signals produced by the gut system and long - term signals produced by adipose tissue . after central integration of these inputs , the brain generates neuronal and hormonal outputs to balance energy intake with expenditure.miscommunication between the gut , brain and adipose tissue , or the degradation of input signals once inside the brain , lead to the brain misunderstanding the peripheral energy state . under certain circumstances , the brain responds to this miscommunication by increasing energy intake and production , eventually causing metabolic disorders . this poster article overviews current knowledge about communication pathways between the brain , gut and adipose tissue , and discusses potential research directions that might lead to a better understanding of the mechanisms underlying metabolic disorders .

Introduction Signals to the brain from the gastrointestinal system Autonomic and neuroendocrine outputs to the gastrointestinal system Signals to the brain from white adipose tissue Autonomic and neuroendocrine outputs to white adipose tissue Astrocytes in the ARC are involved in metabolic hormonal sensing Perspectives

construction , expression , and purification of recombinant prion proteins construction of rprp n - terminal deletion mutants rprp5190 and rprp32121 , heterologous expression in escherichia coli , and further purification of the constructs and full - length rprp by high affinity column refolding followed previously described protocols ( 8 , 34 , 35 ) . rprp23231 , rprp31121 , and rprp5190 labeling with fluorescein isothiocyanate all of the rprps were labeled with amino - reactive fluorescein isothiocyanate ( fitc ) for fluorescence anisotropy assays . for the labeling reaction , rprps were dissolved in 0.1 m sodium bicarbonate buffer at ph 8.0 to a final concentration of 510 mg / ml and incubated with fitc ( final concentration , 0.51.0 mg / ml ) for 1 h at room temperature . after incubation , the fitc - rprp23231 conjugate ( fitc - rprp ) was separated from the free dye by gel filtration using a pd-10 desalting column ( amersham biosciences ) equilibrated with tris buffer ( 50 mm , ph 7.4 ) containing 100 mm nacl . after elution , concentration of the labeled protein and the efficiency of labeling were determined based on molar extinction coefficients of rprp23231 ( 63,495 cm m at 280 nm ) , rprp32121 ( 33,015 cm m at 280 nm ) , rprp5190 ( 41,495 cm m at 280 nm ) , and fitc ( 68,000 cm m at 494 nm ) . ribonucleic acid samples total rna from cultured neuroblastoma ( n2a ) cells ( n2arna ) ( 36 ) was extracted using the rneasy midikit ( qiagen ) and the rnaspin mini isolation kit ( ge healthcare , milwaukee , wi ) . high pressure liquid chromatography - purified synthetic single - stranded rna fragment 4359 of saf93 aptamer ( saf93 ) ( 22 ) and oprna , a random 18-mer sequence , derived from a translational operator of ms2 bacteriophage ( 37 ) were obtained from integrated dna technologies , inc . nucleotide sequences were : saf93 , 5-gga ugc aau cuc cau ccc-3 ; and oprna , 5-aaa cau ggg uuc cca ugu-3. synthetic rna samples were maintained lyophilized at -20 c and used in rnase - free water . protein concentration was 1.0 m ( 0.023 mg / ml for rprp23231 , 0.018 mg / ml for rprp5190 , and 0.014 mg / ml for rprp32121 ) for light scattering ( ls ) and fluorescence measurements , 30 m ( 0.69 mg / ml for rprp23231 , 0.57 mg / ml for rprp5190 , and 0.42 mg / ml for rprp32121 ) for cd assays , and 50 nm ( 1.15 g / ml for rprp23231 , 0.9 g / ml for rprp5190 , and 0.7 g / ml for rprp32121 ) for fluorescence anisotropy assays . all of the experiments were performed in 50 mm tris buffer containing 100 mm nacl , ph 7.4 . all of the figures presented in this work are representative of at least three experiments . spectroscopic measurements ls , fluorescence anisotropy , and fluorescence emission were recorded on an isspc1 fluorometer ( iss , champaign , il ) . ls at 90 was measured illuminating the samples at 320 nm and collecting ls from 300 to 340 nm . tryptophan fluorescence of rprp23231 , rprp5190 , and rprp32121 was measured by exciting the samples at 280 nm and collecting the emission from 300 to 420 nm . for anisotropy measurements , the samples were excited at 490 nm , and the emission was observed through a 369 filter . cd spectra were recorded in a jasco j-715 spectropolarimeter ( jasco corporation , tokyo , japan ) at 25 c with circular 0.10-mm - pathlength cells . buffer spectra were subtracted from each sample spectrum , and traces were collected with four accumulations each . nmr spectra were collected at 298 k with a bruker avance 800-mhz spectrometer equipped with gradient triple resonance probes . the spectra were processed using topspin 2.1 ( bruker ) and analyzed with cara 1.8 ( 38 ) . two - dimensional [ n , h]hsqcs were collected with 2048 200 points and 864 scans for the different samples . one - dimensional h - nmr spectra were collected with 2048 points and 128 scans for all samples . for hsqc measurements , we used 0.2 mm uniformly labeled [ n]rprp23231 in 20 mm sodium phosphate buffer , ph 7.4 , 100 mm nacl , and a 10% d2o , 90% h2o mixture in the presence ( at 1:1 molar ratio ) or absence of saf93 . transmission electron microscopy samples were adhered to a carbon - coated grid , blotted to remove excess material , and stained for 10 s with a 2% solution of uranyl acetate prepared in water . the images were digitally collected with a zeiss em 900 electron microscope ( carl zeiss inc . ) . n2a cells were cultured in dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum and 2% antibiotic ( penicillin , erythromycin , and gentamicin ) in a 5% co2 atmosphere for 3 days and then transferred to a 96-well plate ( 5,000 cells / well ) . after transfer , the cells were exposed to samples containing rprp23231 , n2arna , or rprp - rna complex for 3 days . mtt enters the cells by endocytosis and is reduced to formazan by nadh reductase and other enzymes in a reaction that can be measured spectro - photometrically . the amount of formazan reflects the reductive potential of the cytoplasm and the cell viability and generally shows good correlation with other viability tests ( 30 , 39 , 40 ) . live / dead assays were performed following the protocol provided by the kit ( live / dead viability / cytotoxicity kit for mammalian cells ; invitrogen ) . live cells were distinguished by the presence of ubiquitous intracellular esterase activity , which was determined by the enzymatic conversion of the virtually non - fluorescent cell - permeant calcein am to the intensely fluorescent calcein . the polyanionic dye calcein is well retained within live cells , producing an intense uniform green fluorescence in live cells ( ex / em = 495 nm/515 nm ) . ethd-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids , thereby producing a bright red fluorescence in dead cells ( ex / em = 495 nm/635 nm ) . live / dead images were generated in the observer.z1 microscope ( sn : 3834000373 ; carl zeiss inc . ) . western blotting all of the protease - digested ( + pk ) samples were incubated with 20 g / ml proteinase k ( sigma - aldrich ) for 1 h at 37 c . sds - page was performed on 1.5-mm 15% polyacrylamide gels . the blocked membrane was incubated overnight at 4 c with anti - prp monoclonal antibody ( saf84 ; cayman chemical , mi ) diluted 1:500 . following this incubation , the membrane was washed and incubated for 1 h at room temperature with secondary antibody , igg - horseradish peroxidase - conjugated goat anti mouse ( sc-2005 ; santa cruz biotechnology , santa cruz , ca ) diluted 1:200 . the blot was stained with diaminobenzidine tetrahydrochloride and revealed with hydrogen peroxide ( both from sigma - aldrich ) . we first investigated the effects of rna on the full - length recombinant prp ( rprp23231 ) in vitro . it has been shown that prp present in hamster brain homogenates interacts with rna in vitro , acquiring protease resistance , and that rnase treatment can inhibit this conversion without changing the prp content ( 17 ) . questions remain unanswered , however , regarding the size and structure of the rna that are necessary for interaction with prp and for catalysis of this conversion . in light of these results , we investigated the interaction of prokaryotic and eukaryotic rna extracts and of two synthetic rnas ( saf93 and 18-mer oprna ) ( fig . 1 and supplemental table s1 ) with rprp23231 and with two n - terminal rprp deletion mutants ( rprp5190 and rprp32121 ) . the saf93 sequence was chosen based on previous work ( 22 ) that described the isolation of 2-fluoropyrimidine - substituted rna aptamers that bind selectively to disease - associated , -sheet - rich isoforms of prp . ( 23 ) as a high affinity prp ligand ; therefore , we selected the main binding domain ( nucleotides 4359 ) to perform our studies . the oprna random sequence was selected because it has been suggested that prp - nucleic acid interaction is guided by na structure and not by its specific nucleotide sequence ( 22 , 23 ) . the local secondary structures of the synthetic rnas used in this work were predicted with the program genebee , which indicated that both sequences adopt a hairpin - like structure ( not shown ) . thus we report the prp binding behavior of two different base sequences with similar secondary structures . we verified that incubation of the rna extracts with recombinant prp led to an increase in light scattering values for full - length rprp , indicating that the protein aggregates upon rna addition ( table 1 and supplemental table s1 ) . this result was obtained for incubation with both prokaryotic ( from e. coli ) and eukaryotic ( from mammals , n2arna and vrna ; from mosquito cells , c6rna ; and from yeast , scrna ) rna extracts ( supplemental table s1 ) . interestingly , incubation of the rprp5190 n - terminal deletion mutant with all of the rnas investigated did not alter the ls values , revealing an important role for the prp n - terminal region in the rna interaction . we further selected the neuroblastoma cell - derived rna ( n2arna ) to continue our studies because this rna originates from the same tissue as our prion protein constructs ( from mouse ) ( figs . table 1summary of n2arna effects on rprp23231 and rprp5190spectroscopic measurements rprp23231 rna digestion by rnase a rprp5190 rna+rnabefore rprp incubation with rnaafter rprp incubation with rna 6.85 6.63 ls / ls0 1 21.18 2.09 5.96 1 0.86 fi / fi0 1 1.03 1.00 0.82 1 0.88 cm ( cm ) 0 227 35 90 0 6 araw ellipticity values at 222 nm obtained from cd measurementsbnormalized values obtained by dividing each recorded value ( in the presence of n2arna ) by the initial value ( ls0 , initial light scattering ; fi0 , initial fluorescence intensity ) for free protein in solutioncchanges in center of spectral mass values from the tryptophan emission spectra . n2arna digestion with rnase a was performed at room temperature for 1 h at ph 8.0 with a n2arna : rnase ratio of 100:1 summary of n2arna effects on rprp23231 and rprp5190 raw ellipticity values at 222 nm obtained from cd measurements normalized values obtained by dividing each recorded value ( in the presence of n2arna ) by the initial value ( ls0 , initial light scattering ; fi0 , initial fluorescence intensity ) for free protein in solution changes in center of spectral mass values from the tryptophan emission spectra . n2arna digestion with rnase a was performed at room temperature for 1 h at ph 8.0 with a n2arna : rnase ratio of 100:1 figure 1.rna binding to rprp and its effects on rprp secondary structure . a , oprna was titrated into 50 nm fitc - labeled rprp23231 ( black ) , rprp5190 ( dark gray ) , or rprp32121 ( light gray ) . anisotropy values were collected through a 369 filter ( excitation was set at 490 nm ) . b , cd spectra of free rprp23231 ( solid black line ) and in the presence of oprna ( solid blue line ) or saf93 ( solid red line ) and free rprp5190 ( dashed black line ) and in the presence of oprna ( dark green line ) or saf93 ( dark gray line ) . the inset represents the cd spectra of oprna ( dashed black line ) and saf93 ( dotted black line ) free in solution . c , cd spectra of free rprp23231 ( solid black line ) and in the presence of n2arna at 0.69 mg / ml ( dashed black line ) ; free rprp5190 at 0.57 mg / ml ( solid red line ) ; rprp5190 in the presence of n2arna at 0.57 mg / ml ( dashed red line ) ; rprp32121 at 0.42 mg / ml ( solid blue line ) and rprp32121 in the presence of n2arna at 0.42 mg / ml ( dashed blue line ) . the inset represents the cd spectrum of free n2arna at 0.69 mg / ml . for b and c , protein and a , oprna was titrated into 50 nm fitc - labeled rprp23231 ( black ) , rprp5190 ( dark gray ) , or rprp32121 ( light gray ) . anisotropy values were collected through a 369 filter ( excitation was set at 490 nm ) . b , cd spectra of free rprp23231 ( solid black line ) and in the presence of oprna ( solid blue line ) or saf93 ( solid red line ) and free rprp5190 ( dashed black line ) and in the presence of oprna ( dark green line ) or saf93 ( dark gray line ) . the inset represents the cd spectra of oprna ( dashed black line ) and saf93 ( dotted black line ) free in solution . c , cd spectra of free rprp23231 ( solid black line ) and in the presence of n2arna at 0.69 mg / ml ( dashed black line ) ; free rprp5190 at 0.57 mg / ml ( solid red line ) ; rprp5190 in the presence of n2arna at 0.57 mg / ml ( dashed red line ) ; rprp32121 at 0.42 mg / ml ( solid blue line ) and rprp32121 in the presence of n2arna at 0.42 mg / ml ( dashed blue line ) . the inset represents the cd spectrum of free n2arna at 0.69 mg / ml . for b and c , protein and the rprp n - terminal region is necessary for rna - induced aggregation we monitored the interaction of fitc - labeled rprp23231 , rprp5190 , and rprp32121 with oprna by fluorescence anisotropy to further investigate the role of the n - terminal region in rna binding to rprp . increasing concentrations of oprna led to an increase in the anisotropy values only for rna titration in rprp23231 solution , confirming the high affinity interaction between full - length prp and rna ( fig . 1a , black symbols ) . binding of rprp23231 to n2arna was also observed , with a half - maximal effect at 5.95 g / ml ( fig . consistent with measurements taken when rprp5190 is incubated with different rna extracts ( supplemental table s1 ) , anisotropy data confirm the absence of interaction between the n - terminal deletion rprp mutants and rna in the investigated concentration range ( fig . 1a , gray symbols ) . prp - rna interaction leads to secondary and tertiary structural changes only for rprp23231the changes in secondary structure of the prion protein constructs upon interaction with the different rnas were analyzed by cd . to determine whether free rna interferes with the cd spectra , they all showed one negative peak ( 208 nm ) and one positive peak ( 265 nm ) ( fig . 1 , b and c , insets ) . these results show a significant secondary structure signal from the nucleic acids that were investigated . because the rna structure may change upon rprp binding , the rna spectra were not subtracted from the spectra of the complex ( prp - rna ) . instead , the spectrum of the prp - rna complex is displayed together with the sum of each rna spectrum and that of rprp alone ( supplemental fig . , it is possible to infer whether the changes seen are due to modifications in the secondary structure of prp or due only to the addition of the free rna and rprp spectra . interestingly , changes in secondary structure caused by the synthetic rna sequences were different from the effects observed for n2arna . both saf93 and oprna induced an increase in ls ( supplemental table s1 ) , but this increase was much less marked than the one induced by n2arna ( table 1 and supplemental table s1 ) . this result indicates that , for the synthetic rna sequences , an oligomeric species of limited size is populated . unlike n2arna , in the presence of saf93 and oprna , rprp23231 fluorescence emission intensity was reduced and shifts of the spectra ( increase in center of spectral mass values ) to more energetic wavelengths were also observed ( supplemental table s1 ) . because the n2arna is a pool of several rnas with distinct sequences , sizes , and structures , differences between synthetic rnas and the rna pool are to be expected . n2arna was titrated into a 1.15 g / ml ( 50 nm ) fitc - rprp23231 solution , and the anisotropy values were collected through a 369 filter ( excitation was set at 490 nm ) . n2arna was titrated into a 1.15 g / ml ( 50 nm ) fitc - rprp23231 solution , and the anisotropy values were collected through a 369 filter ( excitation was set at 490 nm ) . we observed that n2arna addition led to visible protein aggregation and that the cd spectrum of the complex showed immediate loss of -helical content ( at 222 nm ) in the presence of n2arna ( fig . 1c and supplemental fig . this process is irreversible , because no significant gain in the secondary structure content was observed even after 7 days of incubation ( not shown ) . the aggregation of rprp23231 was confirmed by the increase in ls upon rna addition ( table 1 and supplemental table s1 ) . the fluorescence emission spectrum was displaced toward more energetic wavelengths , as seen from the increase in cm values after n2arna incubation with rprp23231 ( table 1 and supplemental table s1 ) but not when this rna extract was incubated with rprp5190 ( table 1 ) . this result indicates protection of the tryptophan residues , which are mainly exposed to the solvent when rprp23231 is free in solution ( 35 ) . total fluorescence intensity did not change significantly in the presence of n2arna ( table 1 and supplemental table s1 ) . there were no changes in secondary structure ( -helical content ) in either of the rprp n - terminal deletion mutants after incubation with oprna , saf93 , or n2arna , as shown in fig furthermore , neither ls nor fluorescence emission changed significantly in either case upon rna addition , indicating no changes in tertiary structure ( table 1 , last column , and supplemental table s1 for rprp5190 ) . these data agree with prior observations that suggest the importance of the n - terminal region to prp - na interactions ( 24 , 25 , 41 , 42 ) . the samples were treated with pk at 37 c for 1 h ( ratio prp : pk = 100:1 ) , except where indicated ( - ) . lanes 1 and 2 , rprp23231 at 5 m ; lanes 3 and 4 , rprp23231 at 5 m + n2arna at 0.057 mg / ml . molecular mass markers the samples were treated with pk at 37 c for 1 h ( ratio prp : pk = 100:1 ) , except where indicated ( - ) . lanes 1 and 2 , rprp23231 at 5 m ; lanes 3 and 4 , rprp23231 at 5 m + n2arna at 0.057 mg / ml . treatment of rprp23231-n2arna complex with proteinase k prp and prp differ substantially in their conformations . unlike prp , prp is a multimeric assembly characterized by an increase in the amount of -sheet structure and enhanced resistance to proteinase k ( pk ) digestion ( 3 , 4 ) . after observing rprp23231 changes in secondary structure and aggregation upon n2arna binding , we treated the aggregates with pk for 1 h at 37 c ( ratio prp : pk = 100:1 ) . 3 revealed that pk treatment completely digested free rprp23231 , but the rprp23231-n2arna complex was partially resistant to digestion . the same result was observed when pk resistance of the rprp23121-n2arna complex was monitored by sds - page and stained with silver ( supplemental fig . larger aggregates are also formed upon incubation with n2arna and present high resistance to pk digestion ( supplemental fig . treatment of other rprp23231-rna complexes formed by other rna extracts and synthetic sequences saf93 and oprna , however , resulted in total digestion by pk ( not shown ) . we performed the same assay for rprp5190 and verified that this rprp construction does not acquire protease resistance upon incubation with n2arna ( supplemental fig . s3 ) , further indicating that the n - terminal mutants do not interact with the rnas investigated here . superposition of the two - dimensional [ n , h]hsqc spectra of rprp(23231 ) ( black contours ) and rprp23231 72 h after the addition of saf93 ( red contours ) . the inset presents a region of one - dimensional h - nmr spectra of rprp23231 ( black line ) , rprp23231 immediately after saf93 addition ( blue line ) , and 72 h after rna addition ( red line ) . superposition of the two - dimensional [ n , h]hsqc spectra of rprp(23231 ) ( black contours ) and rprp23231 72 h after the addition of saf93 ( red contours ) . the inset presents a region of one - dimensional h - nmr spectra of rprp23231 ( black line ) , rprp23231 immediately after saf93 addition ( blue line ) , and 72 h after rna addition ( red line ) . treatment of n2arna with rnase blocks the prp - rna interaction effects prior treatment of n2arna with rnase a abolished the effect of the nucleic acid on rprp23231 aggregation , as shown in table 1 . ls and fluorescence emission also returned to values close to that of the soluble , nonaggregated rprp23231 . this indicates that the effects seen are specific for rna interaction and not caused by interaction with free ribonucleotides in solution . in contrast , treatment with rnase a after rna incubation with rprp23231 did not completely reverse aggregation and tertiary structure changes ( table 1 ) , suggesting either that the rprp aggregate formed can no longer be solubilized after rna removal or that the rna bound to prp is protected from cleavage . to confirm the latter assumption we applied rnase - treated rprp23231-n2arna samples on agarose gel in the presence of ethidium bromide ( supplemental fig . it is possible to visualize that larger aggregates are formed , and even after rnase treatment there is still rna associated to the protein ( supplemental fig . 4 , we present the nmr data obtained for rprp23231 in the presence or absence of rna ( saf93 ) . we selected this rna sequence because it is homogeneous and can be synthesized in sufficient quantities to perform the nmr measurements . a small region of one - dimensional h nmr spectra taken for free rprp23231 , rprp23231 immediately after rna addition , and 3 days after rna addition is shown in the inset of fig . 4 . these spectra were recorded and processed with the same spectral parameters so that the methyl signal shown could serve as a relative indication of soluble protein concentration . according to the protein sequence , 288 hn cross - peaks are expected in the two dimensional [ n , h]hsqc for rprp23231 . we found 225 hn cross - peaks in the [ n , h]hsqc of the sample prior to rna supplementation . immediately after rna addition it is worth mentioning that the precipitation can be visually detected during the addition of rna . after a few days , the precipitation is no longer visible , and 3 days after rna addition the sample presents 211 hn cross - peaks . as we noticed from the one - dimensional spectra , the amount of signal of this sample accounts for 8% of the signal of the initial sample without rna . the spectra of prp23231 and prp23231 plus rna are similar in regard to the chemical shift of several cross - peaks , indicating that both samples present the same three - dimensional structure . there are , however , many evident chemical shift differences between the spectra of these samples , in particular within the range of 7.88.6 ppm of h chemical shift . furthermore , the area within 7.68.4 ppm of h and 124129 ppm of n chemical shift has many additional cross - peaks . this h spectral range is populated by many amide cross - peaks of unfolded polypeptide backbone . taken together , these nmr data indicate that immediately after the addition of rna , rprp23231 undergoes severe aggregation , which turns slowly into a soluble form of rprp23231 . this is a new form of rprp , which closely resembles the globular region of the free protein but with some modification in the unfolded n - terminal region of rprp23231 . it is worth mentioning that the n terminus ( residues 23 to 120 ) accounts for most of the prp flexibly disordered behavior , whereas the c terminus ( residues 121231 ) encompasses a globular fold ( 35 ) . ultrastructural analysis of rprp - rna complexes to characterize possible morphological differences between the aggregates obtained after incubation with the rna extract , n2arna , and with the small oligonucleotide sequence , saf93 , we collected transmission electron microscopy images of the samples . recombinant prp23231 was incubated with the aforementioned rnas , and the transmission electron microscopy grids were prepared immediately . fig . 5 displays both complexes , and we observed that rprp forms amorphous aggregates upon incubation with rna that are not present in soluble , rna - free rprp samples . both aggregates appear very similar , but the one generated by the saf sequence seems to adopt a more organized structure . these slight differences may account for the different structural changes elicited by the rnas investigated . d , rprp23231 at 2 m ( 46 g / ml ) in the presence of n2arna at 23 g / ml . g , rprp5190 at 2 m ( 38 g / ml ) in the presence of n2arna at 19 g / ml . scale bars , 0.2 m . d , rprp23231 at 2 m ( 46 g / ml ) in the presence of n2arna at 23 g / ml . g , rprp5190 at 2 m ( 38 g / ml ) in the presence of n2arna at 19 g / ml . scale bars , 0.2 m . prp - rna complex alters cultured cell viability a series of aggregates , oligomers , and fibers generated in vitro have been shown to be toxic to cultured cells , leading to death by apoptosis ( 32 ) , but there is no report of toxic prp generated in vitro using total recombinant prp purified from a heterologous system . after structural characterization of the rprp23231-rna interaction , we investigated whether the aggregates formed upon n2arna or saf93 interaction with rprp23231 were toxic to cultured n2a cells using mtt reduction ( 30 ) and viability assays to measure cellular survival . 6 , only the rprp23231-n2arna complex was able to alter the mtt reduction pattern in cultured neuroblastoma cells , leading to a 60% decrease in mtt reduction compared with rprp23231 alone . the complex formed with the synthetic sequence saf93 did not affect the cultures ( fig . 6 ) , and neither did the samples containing only rna molecules , which suggests that the prp changes induced by rna cause formation of a toxic prp species . mtt reduction results were corroborated by viability assays in the presence of saf93 , oprna , and n2arna . the rprp - n2arna complex was the most toxic to cells in culture ( fig . the free oprna sequence was already toxic to cells , but the addition of rprp23231 did not increase the observed cytotoxicity ( not shown ) . samples containing rprp23231 , rna molecules , or protein - rna complexes were added to the culture medium 3 days before mtt addition . the following concentrations were used : rprp23231 at 0.115 mg / ml ( 5 m ) , n2arna at 0.057 mg / ml , rprp - n2arna ( 0.115 mg / ml:0.057 mg / ml ) complex , saf93 at 5 m , and rprp23231:saf93 ( 5 m:5 m ) complex . for simplicity , the results are expressed as percentages of living cells relative to the value for control cells ( cells in culture medium only ) . the odblank was established from the average of the wells containing only dulbecco 's modified eagle 's medium with no cells . samples containing rprp23231 , rna molecules , or protein - rna complexes were added to the culture medium 3 days before mtt addition . the following concentrations were used : rprp23231 at 0.115 mg / ml ( 5 m ) , n2arna at 0.057 mg / ml , rprp - n2arna ( 0.115 mg / ml:0.057 mg / ml ) complex , saf93 at 5 m , and rprp23231:saf93 ( 5 m:5 m ) complex . for simplicity , the results are expressed as percentages of living cells relative to the value for control cells ( cells in culture medium only ) . the odblank was established from the average of the wells containing only dulbecco 's modified eagle 's medium with no cells . all of these results show that prion protein interacts with rna molecules and that this interaction leads to protein aggregation . there is still no single sequence identified that would specifically bind to rprp , but we have shown that the n - terminal prion protein domain is responsible for rna recognition and binding . for dna binding , both the globular and n - terminal domains are important ( 15 ) . although the protein - only hypothesis is currently the most accepted explanation for the development of transmissible spongiform encephalopathies ( 1 ) , the evidence for an additional cofactor in the prp conversion is accumulating ( 710 , 17 , 42 ) . the physiological role of prion protein interaction with nucleic acids has attracted special attention in the last five years , with particular emphasis on the potential for new therapeutic strategies ( 27 , 43 ) . we have shown that recombinant murine prion protein binds to nucleic acid sequences and acquires higher -sheet content but remains soluble , assuming a scrapie - like form ( 8 , 44 ) . more recently , we have mapped the regions that are directly involved in dna binding by prp ( 15 ) , which revealed that both the globular and n - terminal domains participate in the binding event . this result clarified the importance of the prp globular domain for dna recognition , but it also showed for the first time that the n - terminal domain is involved in stabilization of the complex . since then , new information about the possible physiological relevance of prion - nucleic acid complexes in vivo has appeared ( 13 , 17 , 19 , 45 ) . the use of na aptamers for the diagnosis of prion diseases is another interesting approach that takes advantage of the selective prp - na interaction . it has been reported that some aptamers can interact with specific isoforms of the prion protein , recognizing either the cellular or the scrapie form of prp in vitro ( 20 , 23 , 46 ) . it remains a challenge to develop such an assay for prp identification in vivo . the following concentrations were used : rprp23231 at 0.115 mg / ml ( 5 m ) , n2arna at 0.057 mg / ml , rprp - n2arna ( 0.115 mg / ml:0.057 mg / ml ) complex , saf93 at 5 m , and rprp23231:saf93 ( 5 m:5 m ) complex . b , statistics of viability assay presented in a. for simplicity , the results are expressed as the percentages of living cells relative to the value for control cells ( cells in culture medium only ) . only rprp23231-n2arna complex is toxic to cultured neuroblastoma cells . the following concentrations were used : rprp23231 at 0.115 mg / ml ( 5 m ) , n2arna at 0.057 mg / ml , rprp - n2arna ( 0.115 mg / ml:0.057 mg / ml ) complex , saf93 at 5 m , and rprp23231:saf93 ( 5 m:5 m ) complex . b , statistics of viability assay presented in a. for simplicity , the results are expressed as the percentages of living cells relative to the value for control cells ( cells in culture medium only ) . here we show that rna sequences can also interact with the prion protein with high affinity but with different specificities and that they lead to different changes in the secondary and tertiary structure of recombinant prp compared with dna binding . the interaction of prion protein with ribonucleic acid has been investigated by other groups ; it has been shown that prp can acquire protease resistance upon rna binding ( 17 ) and that scrapie and cellular prp isoforms can bind with different affinities to some highly structured rna sequences ( 1921 ) . this property has allowed specific rna aptamers to be constructed to aid in the early diagnosis of prion diseases in humans and animals . although much effort has been devoted to identifying a key sequence or effect of rna interaction with the prion protein , this subject remains controversial . first , there is no single , specific sequence that binds to prp , nor is there a single nucleic acid - binding domain in this protein . moreover , the minimum number of bases required for efficient binding of prp by rna is unknown . therefore , the aim of the present work was to investigate in more detail what happens to the prion protein structure during interaction with rna and also to compare the observed changes with previous data on the prp - dna interaction ( 8 , 15 ) . we have seen that these interactions , although identical in some respects , display substantial differences . we have shown that prp interacts with rna with high affinity ( nanomolar range ) and that this interaction leads to an insoluble aggregated protein . it is noteworthy that prp is less prone to aggregation when it interacts with double - stranded dnas ( 8 , 15 ) . moreover , the prion protein ( n - terminal ) deletion mutants do not seem to interact with any of the rna sequences investigated . rna - induced aggregation of prion protein depends on the n - terminal region of this protein , indicating that at least part of the 5190 prp region is responsible for this process . it has been reported that truncation of the prp n - terminal region affects formation of protease - resistant prp ( prp - res ) in vitro ( 47 ) . reduced formation of prp - res with deletion of n - terminal residues relative to full - length hamster prp ( 47 ) may be related to rna binding ability in vivo because n - terminal deletion mutants do not bind rna and are also not toxic to cultured cells . this differs from what was observed with the prp - dna interaction ( 15 ) , suggesting that the role of these nucleic acids in prion biology must be different . in an early publication , gabus et al . ( 26 ) found that the human prion protein has dna strand transfer properties comparable with the retroviral nucleocapsid protein and that it forms large nucleoprotein complexes upon binding to dna . the same group found that the huprp(23144 ) fragment could bind dna and have strand transfer activity ( 26 ) . our previous nmr study demonstrated that most of the changes in chemical shift upon dna binding were in the n terminus domain and in helix 1 ( 15 ) . interestingly , the nmr hsqc measurements revealed that , although there is immediate aggregation of rprp23231 upon rna addition , a few days after incubation with rna , the rprp recovers most of its original fold ( fig . 4 ) . this result indicates that the changes induced by this synthetic rna are partially reversible , but most importantly , the appearance of additional cross - peaks in the hsqc spectrum indicates that part of the protein n - terminal domain is modified after interaction with rna ( fig . the nmr data lead us to conclude that , although the overall fold of rprp is recovered 3 days after rna addition , significant structural changes are now present and can reveal important features about the prp interaction with ribonucleic acids . we previously reported that -helical rprp is more hydrated and has a larger solvent - accessible surface area than aggregated -rprp as measured by pressure perturbation calorimetry ( 48 ) , and it may be that nucleic acid binding to cellular rprp leads to similar dehydration effects . these changes in structure of the prion protein when complexed to the nucleic acid would be typical of nucleic acid chaperones ( 10 , 41 , 42 , 49 ) . to investigate whether the aggregates formed upon rna binding are toxic to n2a cells in culture , we performed cell viability assays . interestingly , the aggregates formed upon prp incubation with total rna extract from n2a cells were significantly cytotoxic , leading to an approximately 60% loss in cell viability ( fig in contrast , we did not observe significant toxicity when recombinant prp aggregates were induced by small synthetic oligonucleotides ( oprna or saf ) . our findings obtained when prp protein binds either to dna or rna show that the heterogeneous mixture of rna extracted from neuroblastoma cells was the only one to trigger toxicity and massive aggregation . the small rnas were able to bind prp , also leading to protein aggregation ( fig . 5 , b and d ) , but the oligomeric species formed presented no toxicity ( figs . 6 and 7 ) . the component of the n2a rna extract that leads to aggregation and further toxicity remains to be identified , but we suppose that a specific rna sequence or structure present in this preparation may be responsible for this effect . the local secondary structures of the synthetic rnas , based on the prediction program genebee , were predicted to be hairpins . although the saf sequence has more guanine ( 39% versus 22% for oprna ) , they are otherwise similar in base content . the guanine base is expected to contribute to a greater number of hydrogen bonds with the binding protein ( 50 ) , but both rna sequences displayed a very similar pattern of interaction with the prion protein , and this base composition difference would also not explain the toxicity observed upon incubation with n2arna . moreover , because the n2arna pool is very heterogeneous , it is still not possible to infer whether there it contains a specific rna secondary structure responsible for rprp binding and conversion to a toxic species . altogether , our results reveal that there is selectivity in the species generated by interaction with different molecules of rna . because the catalytic effect of rna on the prp prp conversion may depend on the rna sequence , small rna molecules

conversion of the cellular prion protein ( prpc ) into its altered conformation , prpsc , is believed to be the major cause of prion diseases . although prp is the only identified agent for these diseases , there is increasing evidence that other molecules can modulate the conversion . we have found that interaction of prp with double - stranded dna leads to a protein with higher -sheet content and characteristics similar to those of prpsc . rna molecules can also interact with prp and potentially modulate prpc to prpsc conversion or even bind differentially to both prp isoforms . here , we investigated the interaction of recombinant murine prp with synthetic rna sequences and with total rna extracted from cultured neuroblastoma cells ( n2arna ) . we found that prp interacts with n2arna with nanomolar affinity , aggregates upon this interaction , and forms species partially resistant to proteolysis . rna does not bind to n - terminal deletion mutants of prp , indicating that the n - terminal region is important for this process . cell viability assays showed that only the n2arna extract induces prp - rna aggregates that can alter the homeostasis of cultured cells . small rnas bound to prp give rise to nontoxic small oligomers . nuclear magnetic resonance measurements of the prp - rna complex revealed structural changes in prp , but most of its native fold is maintained . these results indicate that there is selectivity in the species generated by interaction with different molecules of rna . the catalytic effect of rna on the prpcprpsc conversion depends on the rna sequence , and small rna molecules may exert a protective effect .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

interferon therapy which is used in hepatitis c therapy can activate one apha hydroxylase inside the macrophage and that can result in hypercalcemia . we think that internists should be aware of this rare but very serious complication of interferon therapy . a 48-year - old saudi male patient presented to our emergency room ( er ) with a one - week history of weakness , fatigue and vomiting . his medical history was significant for the diagnosis of hepatitis c. he denied any history of abdominal pain , hematemesis and melena or bowel problems . he gave no history of cough , expectoration , hemoptysis and dyspnea or weight loss . he was started on pegylated interferon alpha therapy 20 weeks prior to his presentation as well as ribavirin treatment for his hepatitis c. on physical examination , he was a well - developed gentleman with no cardiorespiratory distress , but looked confused . he had no lymphadenopathy and central nervous system examination was intact grossly with negative meningeal signs . his blood investigations revealed normal complete blood count , renal function tests and blood glucose . his calcium level was 15.2 mg / dl with albumin level of 3.5 gm / dl . he was managed by the emergency physician with aggressive normal saline hydration , intravenous pamidronate and hydrocortisone . laboratory investigation upon patient presentation in the emergency department endocrinology service was consulted and impression of non - parathyroid hormone related hypercalcemia was suggested . his work - up showed bilateral hilar opacity on chest x - ray , which was confirmed by a computerized scan of the chest showing bilateral hilar lymphadenopathy . his mediastinal lymph node biopsy showed non - caseating granuloma consistent with sarcoidosis . during his hospital stay , his mental status improved and his calcium level returned to normal level within 30 days . he was managed with 0.9% normal saline hydration , and furosemide with cessation of his hepatitis c therapy [ table 2 ] . the ct scan of his chest was repeated two months later and showed complete resolution of the hilar lymphadenopathy without the use of steroid , which confirmed this association . calcium level during admission this case describes symptomatic hypercalcemia as the initial presenting feature of interferon - induced sarcoidosis during hepatitis c therapy approximately 20 weeks after initiating therapy with pegylated interferon -2b and ribavirin . the association between interferon therapy for the treatment of chronic hepatitis c and the manifestation of sarcoidosis has been reported in the literature . multiple case reports have linked the development of sarcoidosis ( de novo reactivation ) to the use of interferon during or after the treatment of chronic hepatitis c. around 70 cases have been reported in the literature so far , the vast majority of which ( approximately 90% ) have been published since 2000 . a recent review of 50 cases was published by ramos -casals et al . in 2005 . the estimated prevalence of sarcoidosis in the general population ranges from 1 to 40 cases per 100,000 . thus , the frequency of sarcoidosis seems to be higher in hcv patients receiving antiviral therapy than in the general population . several case reports outside the setting of hepatitis infection have been reported in patients being treated with interferon for multiple sclerosis , leukemia , lymphoma , kaposi sarcoma and melanoma . abdi et al , in 1987 published the first case of pulmonary sarcoidosis following the use of interferon for renal cell carcinoma . patients may be asymptomatic or have vague non - specific symptoms i.e. , fatigue , weakness , malaise , fever and weight loss . the majority of hcv patients with interferon - induced sarcoidosis present with pulmonary , thoracic , mediastinal lymph node and/or cutaneous involvement . the mechanism is believed to be related to extra - renal production of calcitriol ( 1,25-dihydroxy - vitated d3 ) by activated macrophages.[1810 ] the resultant high serum calcitriol concentration induces increased intestinal calcium absorption.[1810 ] chronic hypercalcemia may cause a diverse array of signs and symptoms . the frequency of interferon - related sarcoidosis may be underestimated due to the lack of recognition . many symptoms of sarcoidosis are non - specific and overlap with the side - effect profile of interferon i.e. , generalized weakness , fatigue , fever , malaise , anorexia , weight loss and arthralgia . the data observed by ramos - casals et al , showed that nearly 85% of interferon - induced sarcoidosis triggered by an anti - hcv therapy improved or went into remission spontaneously upon cessation of therapy . severe sarcoidosis involvement ( for example , progressive pulmonary fibrosis , cardiac and neural involvement ) was observed in fewer than 5% . sarcoidosis was reported to be diagnosed during the first six months after the start of antiviral therapy in 31/47 ( 66% ) ; between 6 and 12 months in 9/47 ( 19% ) and after 12 months in the remaining 7/47 ( 15% ) . although less frequent , sarcoidosis may not become clinically apparent until after completion of antiviral therapy . reactivation of pre - existing sarcoidosis after initiation of antiviral therapy for hcv has also been reported in several instances ( 9/66 cases ) . ramos - casals et al . also reported a potentially higher prevalence of sarcoidosis in the treatment of nave patients with chronic hcv ( 18/68 ) . he did not find any significant difference when clinical manifestations were compared to the relationship with antiviral therapy . sarcoidosis is believed to be an immune - mediated process that is generally characterized by an exaggerated t - helper cell ( th-1 ) type 4 immune response with resultant non - caseating granuloma formation in susceptible individuals . it is believed that interferon provokes a similar exaggerated th-1 immune response both directly and perhaps indirectly by inhibiting th-2 activation . the th-1 immune response is characterized by the activation of cd4 t - lymphocytes and macrophages ( mononuclear phagocytes ) in affected organs . these t - lymphocytes also induce the release of th-1 cytokines ( interleukin 2 , interferon - gamma , interleukin-12 ) , a situation which has been repeatedly cited in the pathogenesis of sarcoidosis . the role of hcv itself as a possible co - factor in the development of the disease has also been postulated because of its own ability to induce a th-1 immune response . sarcoidosis has been observed in hcv patients who are not on therapy with increased frequency ( 18 cases reported ) suggesting a possible association between sarcoidosis and hepatitis c. this case reiterates previous recommendation that patients need to be carefully monitored for abnormal immunological reactions during ( as early as eight weeks ) and following interferon therapy within six months . the diagnosis of sarcoidosis may be masked by the insidious development of similar non - specific symptoms shared with the side - effect profile of interferon therapy . histopathologically , sarcoidosis is characterized by the accumulation of t - lymphocytes and mononuclear phagocytes that form widespread non - caseating epithelioid cell granulomas in the affected organs . trans - bronchial lung biopsy is recommended in most cases with a diagnostic yield of 4090% exclusion of alternate pathology cincture ( i.e. infectious , neoplastic cause ) is required . overall , the prognosis for ifn - induced sarcoidosis seems to be good when the medication is discontinued . decision to treat the data obtained in the ramos casals review show that nearly 85% ( 38/86 ) of sarcoidosis triggered by anti - hcv therapy improved or remitted spontaneously . improvement or remission was clear with the discontinuation of antiviral therapy with only 35% of patients requiring systemic corticosteroids . in most large series , 3050% of unselected patients with sarcoidosis were treated with corticosteroids although the symptoms requiring corticosteroid therapy remain controversial . antiviral therapy was discontinued in 14% ( 25/42 ) , while sarcoidosis appeared after completion of the therapy in the remaining 11 ( 26% ) cases . it has been suggested that continuation of interferon therapy or dose adjustment in the presence of a mild exacerbation of sarcoidosis may be safe in a minority of patients with non - critical organ involvement and close observation . although data is limited , similar viral response rates to anti - hcv therapy is observed in hcv patients with sarcoidosis compared with unselected hcv patients . this is the report of a rare case of severe hypercalcemic crisis with bilateral hilar lymphadenopathy in a man who was receiving interferon therapy for hepatitis c. gastroenterologists should be aware of this unusual but clinically important complication of interferon therapy .

interferon is being increasingly used in the treatment of chronic hepatitis c. several case reports have suggested an association between interferon therapy and sarcoidosis with hypercalcemia . we report a case of severe hypercalcemic crisis with bilateral hilar lymphadenopathy in a male patient who was receiving interferon therapy for hepatitis c. gastroenterologists should be aware of this unusual but clinically important complication of interferon therapy .

INTRODUCTION CASE REPORT DISCUSSION

the three main causes of anaemia are blood loss , lack of red blood cell production , and high rates of red blood cell destruction . blood loss is the most common cause of anaemia , especially iron - deficiency anaemia ( ida ) . between 2 and 5% of adult men and postmenopausal women in the developed world have ida [ 1 , 2 ] . in premenopausal women , menstrual blood loss is the most common cause for ida , but , in postmenopausal women and men , the commonest cause is blood loss from the gastrointestinal ( gi ) tract [ 36 ] . the most frequent causes of occult gi blood loss are nonsteroidal anti - inflammatory drugs , colonic cancer / polyp , gastric cancer , angiodysplasia , crohn 's disease , and ulcerative colitis . therefore , an upper and lower endoscopy is an important and necessary examination in the assessment of anaemic patients . apart from the more usual causes of ida , there are also less frequent and often overlooked causes of gi blood loss , such as paraoesophageal herniae ( ph ) . the prevalence of ida in patients with large ph is between 6 and 37% [ 811 ] , a clinical correlation first established by segal in 1931 . however , the exact cause of this association is not fully elucidated . in 1986 , cameron and higgins described the presence of linear gastric erosions at the waist of the hernia ( so called cameron lesions ) , which can cause chronic blood loss . although the association between cameron lesions and hiatal hernias is well described [ 14 , 15 ] , the pathophysiological mechanisms underlying these lesions remain unclear . however , these lesions do not fully explain ida , as only 1/3 or less of anaemic patients with a large ph might have cameron lesions at endoscopy [ 9 , 18 ] . although ida is a recognised complication of hiatus hernia , there are few published data regarding the incidence and outcome of severe , transfusion - dependent ida in patients with ph ( as opposed to mild anaemia not requiring treatment ) . the aim of this study was to specifically identify such patients and to assess resolution of the anaemia after laparoscopic repair of the hiatal hernia . between august 2006 and november 2010 , data were collected prospectively on all patients undergoing laparoscopic repair of ph . patients were followed up routinely with clinical assessment and blood tests to assess the resolution of anaemia and other hernia - related symptoms . patients with severe ida ( haemoglobin < 8 g / dl ) requiring red cell transfusion or iron infusion were retrospectively identified from the prospective database . all procedures were completed laparoscopically with the patient in the modified lithotomy position and the surgeon standing between the patient 's legs . the hernia sac was dissected away from its mediastinal attachments and the oesophagus dissected to ensure adequate intra - abdominal length . after the crural pillars were approximated with 0 ethibond sutures ( ethicon , johnson & johnson , cincinnati , ohio , usa ) , timesh titanized polypropylene mesh ( biomed biologics , nuremberg , germany ) was fixed to the crural muscle with laparoscopic helical screws ( protack 5 mm ; covidien , mansfield , ma , usa ) . seven patients ( 5.1% ; m : f 2 : 5 ; median age 62 years ( 5782 ) ) were identified as having a transfusion - dependent ( blood or iron ) anaemia at surgical presentation . amongst the group , the most common symptoms beside anaemia were chest pain ( n = 4 ) , shortness of breath ( n = 3 ) , regurgitation ( n = 3 ) , dysphagia ( n = 1 ) , and heartburn ( n = 1 ) . the median time duration between symptom onset and date of surgery was 12.0 months ( range 3120 months ) . appropriately , all patients had undergone preoperative diagnostic upper gi endoscopy , 6/7 had undergone diagnostic colonoscopy , and 3/7 had undergone capsule endoscopy . however , as ph was not considered as an explanatory cause for severe , transfusion - dependent ida or gastric ulceration , several patients waited significant lengths of time prior to being considered for surgery ( in one case , 10 years ) . during these delays in surgical treatment , 2/7 patients underwent unnecessary repeated endoscopic investigations after initial tests had yielded negative findings and a further patient underwent a second upper gi endoscopy to check for resolution of gastric ulceration ( cameron lesion ) before being referred for surgery . ( any follow - up endoscopy could have been performed at the time of surgery . ) in total , these 3 patients underwent the following : patient 1 : 10 upper gi endoscopies , 10 colonoscopies , and 2 capsule endoscopies , patient 2 : 3 upper gi endoscopies and 3 colonoscopies patient 3 : 2 upper gi endoscopies . patient 1 : 10 upper gi endoscopies , 10 colonoscopies , and 2 capsule endoscopies , patient 2 : 3 upper gi endoscopies and 3 colonoscopies patient 3 : 2 upper gi endoscopies . cameron lesions were historically identified in only 2 patients , but in 1 of these individuals the erosions had healed and disappeared prior to surgery ( patient 3 ) . apart from the ph identified endoscopically , no other mucosal abnormalities were identified within the gi tracts of these 7 patients to explain ida . haematological investigations also revealed no other potential causes of bleeding . during preoperative investigation and management , patients received either red cell transfusions ( n = 5 ) and/or iron infusions ( n = 2 ) . another one of the patients was due to have a red cell transfusion but this did not occur prior to the date of operation . in those patients receiving blood , the total number of units of packed red cells transfused ranged from 2 to 36 ( patient 1 received bimonthly blood transfusions for 6 years prior to surgery ) . all patients underwent laparoscopic mesh repair of ph with a fundoplication procedure ( 6 posterior 270 toupet , and 1 anterior 180 dor procedure due to an inability to mobilise the fundus sufficiently for a posterior wrap ) . the median postoperative stay was 3.5 days ( 25 days ) . during a follow - up period of 1 year paraoesophageal ( or rolling ) hiatal hernias may affect adults of any age and sex but are most common in elderly women . symptoms are due to the mechanical effects of the hernia and are therefore often resistant to pharmacological treatment . in contrast to the more common sliding hiatal hernia , a ph can lead to volvulus with pain and obstructive symptoms or , as our series demonstrates , they can be associated with ida , including severe , transfusion - dependent anaemia . in fact , severe anaemia appears to have a prevalence of 5% in these patients . such anaemia may occur even in the apparent absence of cameron lesions or obvious bleeding focal points at endoscopy . in our series , only 2 patients were ever noted to have gastric erosions . however , it is worth noting that , in 1 of these patients , the cameron lesions resolved , suggesting either that occult bleeding is an intermittent phenomenon that is not always present at the time of endoscopy or , less likely , that bleeding lesions are missed at endoscopy . nevertheless , other authors have found no correlation between cameron lesions and visible gi blood loss or haemoglobin levels . an alternative explanation for ida might be that large hiatus hernias interfere with the absorption and regulation of dietary iron . it is obviously vital that all patients with ida of unknown cause require upper gi endoscopy and colonoscopy . however , in our patient group , despite the severity of anaemia , patients experienced symptoms for a median duration of 1 year before being referred to surgical services for a hiatal repair . during this preoperative assessment stage , 3/7 patients underwent repeated endoscopic investigations and transfusions and were therefore unnecessarily exposed to the risk of procedure - related complications . repeated upper gi endoscopy is appropriate in those requiring endoscopic treatment for acute life - threatening haemorrhage from upper gi ulceration but not in those with chronic ida . it is therefore vitally important that physicians who assess patients with ida are cognisant of the correlation with hiatus hernia , particularly in the absence of erosive pathology at endoscopy . any failure to appreciate this association might represent a lack of awareness on the part of physicians and surgeons managing ida . for example , a review of 7 core european and australasian surgical textbooks reveals that only 3 textbooks make reference to hiatus hernias being a cause of bleeding or anaemia , and , in these books , bleeding is chiefly attributed to reflux oesophagitis [ 2026 ] . cameron lesions are described in only one of these textbooks , whereas ph as a cause of cryptogenic anaemia is not discussed in any of them . gastrooesophageal reflux disease ( gord ) and oesophagitis do frequently coexist with hiatal hernias , and therefore oesophagitis may well be the cause of anaemia in some patients . however , ph ( with or without cameron lesions ) should still be considered as an aetiological factor in severe ida . in our series , laparoscopic ph repair cured all patients of severe anaemia during a median follow - up period of 12 months . hayden and jamieson also included 3 patients with transfusion - dependent anaemia in their series of 11 patients with ph and ida . they also demonstrated resolution of patient anaemia during a median follow - up of 25 months . therefore , it would seem appropriate to offer surgical intervention to young , medically fit patients with ph and severe ida with no other cause found on endoscopic investigation . such an approach would also prevent the foregut and mechanical complications of ph , symptoms for which some authors would recommend routine surgical repair in any case . this recommendation contrasts with those of panzuto et al . , who demonstrated in a randomised control trial that proton pump inhibitor ( ppi ) monotherapy was as effective as surgery with ppi at resolving ida in association with large hiatal hernia . however , this study did not involve pure cohorts of patients with severe , transfusion dependent anaemia but also included individuals with mild anaemia . many patients with ph are elderly and frail and thus a nonoperative approach might be appropriate in this group . elderly patient age alone remains only a relative contraindication to surgery , however , as we have shown previously that laparoscopic repair is associated with acceptable rates of morbidity and a significant improvement in quality of life in such individuals . in summary , we have identified a small but significant number of patients with severe ida in association with ph , often in the absence of endoscopically identifiable cameron lesions . early recognition of the fact that ph may be an aetiological factor in severe ida will mitigate the likelihood of prolonged transfusion dependency and fruitless , repeated gi tract investigations as laparoscopic repair of ph resolves ida .

introduction . a paraoesophageal hernia ( ph ) may be one reason for iron - deficiency anaemia ( ida ) but is often overlooked as a cause . we aimed to assess the incidence and resolution of transfusion - dependent ida in patients presenting for hiatal hernia surgery . methods . we analysed a prospective database of patients undergoing laparoscopic hiatal repair in order to identify patients with severe ida requiring red cell / iron transfusion . results . of 138 patients with ph managed over a 4-year period , 7 patients ( 5.1% ; m : f 2 : 5 ; median age 62 yrs ( range 5782 ) ) with ida requiring red cell / iron transfusion were identified . preoperatively , 3/7 patients underwent repetitive and unnecessary diagnostic endoscopic investigations prior to surgery . only 2/7 ever demonstrated gastric mucosal erosions ( cameron ulcers ) . all patients were cured from anaemia postoperatively . discussion . ph is an important differential diagnosis in patients with ida , even those with marked anaemia and no endoscopically identifiable mucosal lesions . early recognition can avoid unnecessary additional diagnostic endoscopies . laparoscopic repair is associated with low morbidity and results in resolution of anaemia .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

the clinical presentation of these patients is varied , ranging from headache and focal neurological deficits to personality changes and/or mental deterioration , including memory loss , inattention , confusion , hallucination , and hyperphagia . as a result , these patients are often confused with encephalitis and its sequel , as happened in our patient . the role of surgery is limited , owing to the architectural complexity and depth of location of the lesion . an 8-year - old patient was referred to the pediatric emergency services with complaints of moderate- to high - grade fever for 10 days , 2 months back . on 5 day of fever , the patient developed choreoathetoid movements on right side of the body , which was more severe in the upper limb . few days later , he developed progressive loss of ambulation , cognition , and speech . on examination , he was in a state of altered consciousness ( e2v2m5 ) . however , his vitals were stable . the neurological assessment revealed , increased tone in all four limbs , with exaggerated deep tendon reflexes and bilateral extensor plantar reflex . fundoscopy revealed mild papilledema , pupils were of normal size and reacting to light . his complete hematological profile and serum chemistries were unremarkable . noncontrast computed tomography ( ct ) of the head revealed a bilateral thalamic iso- to hyperdense lesion with moderate hydrocephalus [ figure 1 ] . the mr examination of the brain showed significant swelling of thalamus with diffuse hypointense signal on t1-weighted ( t1w ) imaging and hyperintense signal on t2 and fluid - attenuated inversion recovery ( flair ) sequences [ figure 2 ] . hypointense signals on t1 and hyperintense signal changes on t2w and flair were also present in the basal ganglia , head of the caudate nuclei , and upper brainstem . findings were considered nonspecific and diagnosis of encephalitis was considered , however , bilateral thalamic glioma was the second possibility . noncontrast computed tomography of head reveals bilateral iso- to hyperdense thalamic masses with hydrocephalus and periventricular ooze t2-weighted magnetic resonance imaging reveals bilateral hyperintense thalamic masses with focal hypointensities t1-weighted axial post - contrast image reveals mild peripheral enhancement patient was managed on the lines of encephalitis and its sequelae . he was started on antiepileptic drugs and decongestants to decrease raised intracranial pressure and a low pressure ventricular shunt was inserted . histopathological examination of the biopsy specimen revealed a highly cellular tumor with foci of palisading necrosis [ figure 4a ] and microvascular proliferation [ figure 4b ] . the tumor cells showed marked nuclear pleomorphism and frequent mitotic figures [ figure 4b ] , including atypical forms . on immunohistochemistry , the tumor cells were immunopositive for p53 [ figure 4c ] and were negative for epidermal growth factor receptor ( egfr ) . mib - i labeling index was approximately 60% in the highest proliferating areas [ figure 4d ] . based on these features , a diagnosis of glioblastoma multiforme , world health organization ( who ) grade iv , was arrived at . photomicrographs showing ( a ) a highly cellular tumor with foci of palisading necrosis ( hematoxylin and eosin ( h and e ) , original magnification 400 ) and ( b ) microvascular proliferation ( h and e , original magnification 400 ) . ( b ) the tumor cells display marked nuclear pleomorphism and frequent mitotic figures ( h and e , original magnification 400 ) . ( c ) immunohistochemistry for p53 showed nuclear positivity ( immunohistochemistry ( ihc ) , original magnification 400 ) . ( d ) mib - i labeling index is 60% ( ihc , original magnification 400 ) tracheostomy was done in view of poor glasgow coma scale ( gcs ) considering the need of prolonged ventilatory support . although thalamic tumors constitute between 2 and 5% of all the pediatric intracranial tumors , it is only 1% in adult series . bilateral pediatric thalamic glioblastomas are extremely rare and only five cases are published in the english literature . the largest series on pure thalamic tumors to date is from the necker enfants malades hospital , paris , which reported 69 children treated over a 14-year period . lesions in the ventrolateral thalamus typically manifest with motor deficits , given their close proximity to the internal capsule . the visual signs and symptoms are present in as many as 50% of these patients and are due to involvement of the optic tracts and the lateral geniculate body . the oculomotor deficits are due to involvement of the retrolenticular segment of the internal capsule or the midbrain tegmentum . involvement of the mammillothalamic tracts or the fornices can lead to cognitive dysfunction and memory problems . bilateral thalamic lesions are associated with changes in personality , memory deficits , confusion , hallucinations , hyperphagia , and bradyphrenia . diffuse and bilateral involvement of thalamic nuclei by these tumors makes the surgical therapy very difficult . the role of surgery , hence , is limited and is performed for histological diagnosis as was done in our patient . the role of chemotherapy and radiotherapy is of questionable significance and are utilized as adjuvant therapies . bilateral thalamic tumors are typically treated with radiation therapy , regardless of whether they are histologically malignant . for bilateral thalamic tumors , the prognosis remains relatively poor regardless of tumor histology and treatment . in our patient , left thalamic tumor was biopsied under magnetic resonance imaging ( mri ) guidance . , documented p53 protein accumulation in 75% of high - grade childhood gliomas , and p53 mutation in 38% . pollack et al . , documented increased expression of p53 in about 35% of high - grade gliomas in children , which increased to 58% when only glioblastomas were considered . the authors determined that this feature was an adverse prognostic factor in a cohort of children who were treated uniformly with surgery , radiotherapy , and chemotherapy . on the other hand , egfr amplification is much less common in pediatric glioblastomas compared to adult primary glioblastomas and the reports on egfr protein expression have been quite varied . , who found only very small foci of tumor cells showing egfr immunopositivity in four of 13 glioblastomas . thalamic lesions continue to be a challenging entity to the neurosurgeons , particularly owing to their deep location and complex circuitry . unilateral tumors are increasingly being tackled by the neurosurgeons with an improved patient outcome . however , bilateral thalamic glioblastomas have a very poor prognosis , irrespective of the modality of treatment .

bilateral thalamic tumors are rare . pediatric bilateral thalamic glioblastomas are even rarer , only five cases reported in the english literature till date . the clinical presentation , natural history , and prognosis of pediatric thalamic tumors are still relatively obscure . in this article , we report an 8-year - old patient with large bilateral thalamic glioblastomas and briefly discuss its clinical presentation , possible modalities of management , and prognosis , in the light of available literature .

Introduction Case Report Discussion Conclusion

genetic studies of human diseases have been marked by an explosion in the number of susceptibility loci identified through genome wide association studies ( gwas ) in the past several years . similar to other complex disorders , neuropsychiatric diseases , too , had a share of their genetic risk loci discoveries , with for example28 published studies to date on alzheimer s disease ( ad ) , 18 on bipolar disorder and 22 on schizophrenia , according to the catalog of published gwas accessed on 21 aug 2012 . the translation of this success to viable therapies and biomarker discovery depends on the identification and characterization of the actual disease genes and functional variants at these susceptibility loci . furthermore , despite the large number of discovered loci , a substantial component of genetic susceptibility remains unexplained for complex diseases [ 2 ] . to overcome these major hurdles in the post - gwas era requires a multitude of alternative approaches including the use of endophenotypes , which are biologically - relevant , quantitative and heritable phenotypes [ 3 ] ( fig . the graded arrow represents the continuum of ad with darker colors symbolizing greater clinical expression of disease . the brackets and text above them depict the different uses of endophenotypes at various disease stages ( color figure online ) definition ( a ) and utility ( b ) of endophenotypes . the graded arrow represents the continuum of ad with darker colors symbolizing greater clinical expression of disease . the brackets and text above them depict the different uses of endophenotypes at various disease stages ( color figure online ) the endophenotype approach was initially advocated in psychiatric genetics [ 4 , 5 ] due to the need to have an objective and quantifiable outcome in genetic studies , given the relatively imprecise nature of the clinical diagnosis , which is thought to result in heterogeneity . the rationale for using endophenotypes , instead of or in addition to the binary disease phenotypes , stems from the following assumptions : ( 1 ) endophenotypes represent an intermediate outcome between genes and clinical diagnosis of a disease , and given their closer proximity to the genetic variation than the disease outcome , the genetic component influencing the endophenotype will be larger and therefore easier to detect [ 6 , 7 ] ; ( 2 ) endophenotypes are under the influence of a smaller number of genes than the more complex disease outcome ; ( 3 ) given its quantitative nature , using an endophenotype as an outcome variable will be statistically more powerful than the binary case / control approach in detecting genetic associations ; ( 4 ) since they are objectively quantifiable , endophenotypes constitute a more homogeneous and accurately measurable phenotype than disease outcome ; ( 5 ) the endophenotype approach can allow inclusion of individuals with and without a given diagnosis , which will increase power , particularly in family studies or for traits that are age dependent [ 7 , 8 ] ; ( 6 ) the endophenotype approach will provide information about the underlying mechanism of action for the gene and variant of interest and might therefore more readily enable downstream functional investigations , including the generation of animal models with quantifiable outcomes . while the accuracy of these assumptions needs to be established , the endophenotype approach has begun to generate hypotheses for novel genetic loci and pathways implicated in human disease and to enable the downstream characterization of disease variants and genes , as exemplified in this review ( fig . ad , the most common dementia in the elderly , is especially amenable to the endophenotype approach , for a number of reasons . first , ad has a distinct neuropathology characterized by accumulation of amyloid ( a ) in senile plaques and hyperphosphorylated tau in neurofibrillary tangles , both of which are quantifiable phenotypes , and the latter correlates with clinical disease severity . second , discoveries of mendelian mutations in the amyloid precursor protein ( app ) , presenilin 1 ( psen1 ) and psen2 genes in early - onset ad that lead to elevations in secreted a and their modeling in animals harboring these mutations ( reviewed ) bolstered the amyloid cascade hypothesis . the ability to measure a levels in the serum and cerebrospinal fluid ( csf ) of ad patients and their relatives , and the determination that a levels are heritable enabled the first studies utilizing a levels as endophenotypes in genetic studies that discovered genetic loci and variants influencing ad risk and a [ 8 , 1517 ] . this was followed by investigations of csf a and tau levels as endophenotypes in ad genetics studies . third , the availability of prospective , elderly cohorts with rich clinical , neurocognitive and neuroimaging measures [ 2022 ] , knowledge that many of these measures are heritable [ 6 , 7 , 23 ] , and detection of preclinical changes in these measures ( reviewed [ 24 ] ) advocate their use as endophenotypes in genetic studies of ad . fourth , the advent of technology that allows measurement of gene expression levels for all known transcripts ( transcriptome ) , development of methodologies that allow analysis of this data at the whole - genome level , significant heritability attributed to gene expression levels ( reviewed [ 3 ] ) and the availability of well - characterized brain tissue from neuropathologic ad and other patients in which transcriptome can be measured , empowered the use of gene expression levels from brain and other tissues as endophenotypes in ad . this review focuses on three types of endophenotypes in ad : gene expression levels , neuropathologic measures and cognitive measures . these diverse endophenotypes span the vast spectrum of biological insights that can be gained by this quantitative approach : genetic associations with transcript levels , the most proximal of these traits to the susceptibility allele , may uncover the initial mechanism for the functional consequences of the allele . neuropathologic phenotypes can relate a variant to the known neuropathology of the disease and might enable the dissection of pathophysiologic pathways influenced by the polymorphism of interest . finally , the use of cognitive endophenotypes can uncover genetic risk factors governing distinct aspects of human cognition and the clinical expression of the disease . we recognize that there are many other endophenotypes that are currently utilized or are excellent candidates for genetic studies of ad , including a and tau levels , neuroimaging measures such as hippocampal volume and magnetic resonance spectroscopy levels and methylation patterns . although a comprehensive assessment of all these endophenotypes is beyond the scope of this review , the generalizations that can be drawn from this synopsis could potentially be applicable to many other quantitative phenotypes in ad research . gene expression levels constitute a special group of endophenotypes for a number of reasons . first , because the tested phenotype is the level of the expressed gene transcript(s ) , genetic studies of gene expression endophenotypes ( also known as expression quantitative trait loci or eqtl studies ) directly identify the gene under the influence of genetic variants . this is in contrast to any other phenotype , where genetic studies merely implicate a locus of interest without definitive information about the affected gene . this first characteristic of the gene expression endophenotype can be utilized to uncover plausible disease genes via combined assessment of gene expression endophenotype and disease phenotype as discussed below . second , genetic factors identified via eqtl studies provide guidance about the underlying mechanism of action of the functional variants at the locus of interest . , for example by focusing on regulatory variants that influence whole transcript levels or splice isoforms . furthermore , such information can guide downstream in vitro studies that are more relevant to the underlying genetic variation . third , the ability to assess concerted expression level changes or eqtl associations at the transcriptome level via pathway analysis can lead to identification of novel biological networks that may underlie disease pathophysiology . below , we discuss the utility of gene expression endophenotypes in gene discovery and characterization in ad , highlighting examples that take advantage of these special characteristics of this approach . the utilization of gene expression endophenotypes in gene discovery in ad first began with transcriptome profiling ( or mrna profiling ) studies , which are recently comprehensively reviewed . the underlying premise of these studies is that mrna from patients with disease will have changes in comparison to controls ; and that these changes may underlie disease pathophysiology . the most important caveat in this assumption is that the detected gene expression changes may be a consequence of the disease and non - specific , rather than a causal event [ 3 ] . this pitfall is especially concerning if the transcriptome profiling is performed in tissue affected by the disease ( such as the temporal cortex in ad ) . indeed , in a detailed microarray - based transcriptome profiling study of 14 different cerebral cortex regions and the hippocampus , from 69 autopsied ad subjects of varying clinical and pathologic severity versus 18 controls ( maximal number of subjects utilized in the study ) , haroutunian et al . identified the greatest number of gene expression changes in regions from the temporal cortex across the disease stages , with increasing changes occurring in later disease stages and stronger correlations between gene expression and disease severity seen in more advanced disease . collectively , these results could imply that the progression of disease and cell loss may be driving these changes , rather than vice versa . these authors and others attempted to overcome this concern by analysis of autopsied ad subjects with mild neuropathology and concluded that gene expression changes that occur in regions prior to the development of neuropathology are unlikely to be a consequence of the disease process . analyzed 49 prefrontal cortex samples from subjects with alzheimers - type neuropathology to identify correlated changes in gene expression which varied with advancing braak stage . synaptic activity genes between braak stages ii and iii , which is prior to or just at the onset of ad - type neuropathology when the subjects were clinically non - demented . the authors also noted that levels of several genes correlated with increasing intracellular a levels during these braak stages , leading them to postulate that expression changes in genes of synaptic activity may be a coping mechanism against increased a that occurs prior to clinical and neuropathological ad . while it is not possible to draw definitive conclusions about the longitudinal cascade of events , including gene expression changes , based on cross - sectional assessment of brain tissue from small numbers of distinct subjects , these results nevertheless generate intriguing hypotheses about ad pathophysiology via correlative analysis of transcriptome and neuropathology data . another approach in utilizing gene expression endophenotypes in gene discovery is combined transcriptome profiling and ad risk association studies . in a small hippocampal mrna profiling study of six ad versus two control brains , li et al . detected lower gsto1 ( glutathione s - transferase omega-1 ) levels in the ad brains , followed by significant associations with age - at - onset of both ad and parkinson s disease with variants in both gsto1 and its nearby homologue gsto2 . this prompted follow - up genetic studies with disease risk and/or age - at - onset phenotypes with mixed results [ 2933 ] . in a gwas of brain gene expression ( brain egwas ) levels in ~800 tissue samples from ~400 brains [ 34 ] , we identified strong associations with variants at this locus and brain gsto2 but not gsto1 levels , consistent with results from another brain eqtl study . in our study , we determined that the same variant associated with both lower brain levels of gsto2 as well as increased ad risk in older subjects , which is biologically consistent with the antioxidant functions of this gene . furthermore , pathway analysis of the significant genes in our brain egwas showed significant enrichment for glutathione metabolism genes , suggesting there may be additional genes in this pathway with potential influence on ad and other neurodegenerative diseases . other genes which were detected by expression profiling studies of ad versus control tissue , followed by significant associations with ad risk , include pou2f1 and il-33 . these studies highlight the potential utility of the gene expression endophenotype in identifying gene(s ) and pathways that may harbor regulatory variants that influence disease risk . more recently , joint assessment of disease gwas with eqtl studies have been advocated to prioritize suggestive results from disease gwas and/or identify novel candidate disease genes , based on the premise that disease variants will be enriched for regulatory variants that influence gene expression and vice versa [ 39 ] . indeed , in a comparison of eqtl results from lymphoblastoid cell lines from hapmap samples with human disease / trait gwas summary data , nicolae et al . identified significant enrichment for snps that influence expression ( esnps ) amongst human disease / trait associating variants . combined assessment of brain expression endophenotype associations with disease gwas showed enrichment for eqtls amongst schizophrenia risk alleles . this approach , combined with pathway analysis led to nomination of novel genes for diabetes in another study . we have applied this approach for the first time to a large ad gwas , by combining with our brain egwas data and detected an enrichment for significant esnps amongst suggestive ad risk snps [ 34 ] . these results suggest that ad , like other complex human diseases , may at least in part be influenced by regulatory variants . the novel genes detected in the grey zone of disease gwas by this approach warrant further studies for identification of functional variants and to demonstrate their downstream regulatory effects . gene expression endophenotypes can also be used to characterize the effects of disease risk variants and their downstream consequences on the disease gene . a prime example of this is mapt , which has rare variants leading to frontotemporal dementia with parkinsonism linked to chromosome 17 , as well as common variants within a haplotype block that associate with multiple taupathies ( reviewed ) . these variants have been shown to influence either splicing or transcriptional activity of mapt [ 4548 ] . another example of gene expression endophenotype explorations for a known risk gene is apoe , which has common missense polymorphisms , leading to three isoforms apoe 2 , 3 or 4 , where apoe 4 has clearly been shown to influence ad risk , whereas apoe 2 might confer protection from ad [ 11 , 49 ] . apoe isoforms have dual types of functions in the brain with roles in both maintaining neural health and also in promoting ad pathophysiology ( reviewed ) . in addition to the most well - studied isoforms , a number of promoter region polymorphisms have been identified for apoe that impart risk for ad , at least partially independently of the apoe isoform ( reviewed ) . although consensus is still lacking , the most well studied promoter region polymorphism 491aa appears to confer ad risk independent of apoe 4 and increase apoe transcriptional activity [ 52 , 53 ] , suggesting that both apoe isoforms and levels may play a role in ad pathogenicity . despite absence of conclusive evidence for the role of the promoter region polymorphisms in ad risk , the transcriptional complexity of apoe and its dual role in the central nervous system ( cns ) , apoe - directed therapeutics aimed at modifying its levels a recent study in animal models of ad demonstrated clearing of a and reversal of behavioral and electrophysiologic deficits upon treatment with a transcriptional inducer of apoe . it is similarly critical to characterize the novel ad candidate variants and genes that are being identified in late - onset ad ( load ) gwas ( reviewed ) with respect to their influence on gene expression endophenotypes . this information will provide focus for the downstream functional variant discovery , in vitro and in vivo studies and ultimately set the stage for the search of therapeutics targeting the appropriate mechanisms and pathways . we have begun to characterize the novel load gwas variants for their influence on gene expression endophenotypes using our brain egwas [ 56 ] and eqtl analyses of data generated from peripheral immune cells . the latter study demonstrated that ad - associated variants , such as the one in the picalm locus , influence gene expression in non - resident cns cells and suggest that infiltrating immune cells may play a role in the onset of ad . on the other hand , the brain data identified association between the top ad risk variants at the clu and ms4a loci with brain levels of clu and ms4a4a genes , implicating regulatory genetic variation for these genes in ad risk . furthermore , we detected additional strong gene expression associations for both clu and abca7 , some of which also confer ad risk , independent of the top gwas variants , suggesting that new regulatory ad variants might exist at these loci , in addition to the top snps already identified by disease gwas . [ 58 ] who determined that the ad - protective clu variant is also associated with higher clu1 isoform levels in human brains . the direction of the gene expression endophenotype effect is identical in these two studies , and indicate that therapeutic approaches aimed at increasing levels of clu in the brain might confer protection from ad . interestingly , valproic acid ( vpa ) , a well - known anti - epileptic and anti - depressant with histone deacetylase ( hdac ) inhibiting properties , was shown to induce clu expression in astrocytes . vpa was also previously highlighted as a potentially promising drug for ad due to its pro - neurogenesis and neuroprotective properties . although clinical trials of vpa in ad patients have yet failed to demonstrate a beneficial outcome [ 61 , 62 ] , evidence warrants further investigations along the clu induction axis as a potential therapeutic avenue in ad . the utilization of gene expression endophenotypes in gene discovery in ad first began with transcriptome profiling ( or mrna profiling ) studies , which are recently comprehensively reviewed . the underlying premise of these studies is that mrna from patients with disease will have changes in comparison to controls ; and that these changes may underlie disease pathophysiology . the most important caveat in this assumption is that the detected gene expression changes may be a consequence of the disease and non - specific , rather than a causal event [ 3 ] . this pitfall is especially concerning if the transcriptome profiling is performed in tissue affected by the disease ( such as the temporal cortex in ad ) . indeed , in a detailed microarray - based transcriptome profiling study of 14 different cerebral cortex regions and the hippocampus , from 69 autopsied ad subjects of varying clinical and pathologic severity versus 18 controls ( maximal number of subjects utilized in the study ) , haroutunian et al . identified the greatest number of gene expression changes in regions from the temporal cortex across the disease stages , with increasing changes occurring in later disease stages and stronger correlations between gene expression and disease severity seen in more advanced disease . collectively , these results could imply that the progression of disease and cell loss may be driving these changes , rather than vice versa . these authors and others attempted to overcome this concern by analysis of autopsied ad subjects with mild neuropathology and concluded that gene expression changes that occur in regions prior to the development of neuropathology are unlikely to be a consequence of the disease process . analyzed 49 prefrontal cortex samples from subjects with alzheimers - type neuropathology to identify correlated changes in gene expression which varied with advancing braak stage . synaptic activity genes between braak stages ii and iii , which is prior to or just at the onset of ad - type neuropathology when the subjects were clinically non - demented . the authors also noted that levels of several genes correlated with increasing intracellular a levels during these braak stages , leading them to postulate that expression changes in genes of synaptic activity may be a coping mechanism against increased a that occurs prior to clinical and neuropathological ad . while it is not possible to draw definitive conclusions about the longitudinal cascade of events , including gene expression changes , based on cross - sectional assessment of brain tissue from small numbers of distinct subjects , these results nevertheless generate intriguing hypotheses about ad pathophysiology via correlative analysis of transcriptome and neuropathology data . another approach in utilizing gene expression endophenotypes in gene discovery is combined transcriptome profiling and ad risk association studies . in a small hippocampal mrna profiling study of six ad versus two control brains , li et al . detected lower gsto1 ( glutathione s - transferase omega-1 ) levels in the ad brains , followed by significant associations with age - at - onset of both ad and parkinson s disease with variants in both gsto1 and its nearby homologue gsto2 . this prompted follow - up genetic studies with disease risk and/or age - at - onset phenotypes with mixed results [ 2933 ] . in a gwas of brain gene expression ( brain egwas ) levels in ~800 tissue samples from ~400 brains [ 34 ] , we identified strong associations with variants at this locus and brain gsto2 but not gsto1 levels , consistent with results from another brain eqtl study . in our study , we determined that the same variant associated with both lower brain levels of gsto2 as well as increased ad risk in older subjects , which is biologically consistent with the antioxidant functions of this gene . furthermore , pathway analysis of the significant genes in our brain egwas showed significant enrichment for glutathione metabolism genes , suggesting there may be additional genes in this pathway with potential influence on ad and other neurodegenerative diseases . other genes which were detected by expression profiling studies of ad versus control tissue , followed by significant associations with ad risk , include pou2f1 and il-33 . these studies highlight the potential utility of the gene expression endophenotype in identifying gene(s ) and pathways that may harbor regulatory variants that influence disease risk . more recently , joint assessment of disease gwas with eqtl studies have been advocated to prioritize suggestive results from disease gwas and/or identify novel candidate disease genes , based on the premise that disease variants will be enriched for regulatory variants that influence gene expression and vice versa [ 39 ] . indeed , in a comparison of eqtl results from lymphoblastoid cell lines from hapmap samples with human disease / trait gwas summary data , nicolae et al . identified significant enrichment for snps that influence expression ( esnps ) amongst human disease / trait associating variants . combined assessment of brain expression endophenotype associations with disease gwas showed enrichment for eqtls amongst schizophrenia risk alleles . this approach , combined with pathway analysis led to nomination of novel genes for diabetes in another study . we have applied this approach for the first time to a large ad gwas , by combining with our brain egwas data and detected an enrichment for significant esnps amongst suggestive ad risk snps [ 34 ] . these results suggest that ad , like other complex human diseases , may at least in part be influenced by regulatory variants . the novel genes detected in the grey zone of disease gwas by this approach warrant further studies for identification of functional variants and to demonstrate their downstream regulatory effects . gene expression endophenotypes can also be used to characterize the effects of disease risk variants and their downstream consequences on the disease gene . a prime example of this is mapt , which has rare variants leading to frontotemporal dementia with parkinsonism linked to chromosome 17 , as well as common variants within a haplotype block that associate with multiple taupathies ( reviewed ) . these variants have been shown to influence either splicing or transcriptional activity of mapt [ 4548 ] . another example of gene expression endophenotype explorations for a known risk gene is apoe , which has common missense polymorphisms , leading to three isoforms apoe 2 , 3 or 4 , where apoe 4 has clearly been shown to influence ad risk , whereas apoe 2 might confer protection from ad [ 11 , 49 ] . apoe isoforms have dual types of functions in the brain with roles in both maintaining neural health and also in promoting ad pathophysiology ( reviewed ) . in addition to the most well - studied isoforms , a number of promoter region polymorphisms have been identified for apoe that impart risk for ad , at least partially independently of the apoe isoform ( reviewed ) . although consensus is still lacking , the most well studied promoter region polymorphism 491aa appears to confer ad risk independent of apoe 4 and increase apoe transcriptional activity [ 52 , 53 ] , suggesting that both apoe isoforms and levels may play a role in ad pathogenicity . despite absence of conclusive evidence for the role of the promoter region polymorphisms in ad risk , the transcriptional complexity of apoe and its dual role in the central nervous system ( cns ) , apoe - directed therapeutics aimed at modifying its levels a recent study in animal models of ad demonstrated clearing of a and reversal of behavioral and electrophysiologic deficits upon treatment with a transcriptional inducer of apoe . it is similarly critical to characterize the novel ad candidate variants and genes that are being identified in late - onset ad ( load ) gwas ( reviewed ) with respect to their influence on gene expression endophenotypes . this information will provide focus for the downstream functional variant discovery , in vitro and in vivo studies and ultimately set the stage for the search of therapeutics targeting the appropriate mechanisms and pathways . we have begun to characterize the novel load gwas variants for their influence on gene expression endophenotypes using our brain egwas [ 56 ] and eqtl analyses of data generated from peripheral immune cells . the latter study demonstrated that ad - associated variants , such as the one in the picalm locus , influence gene expression in non - resident cns cells and suggest that infiltrating immune cells may play a role in the onset of ad . on the other hand , the brain data identified association between the top ad risk variants at the clu and ms4a loci with brain levels of clu and ms4a4a genes , implicating regulatory genetic variation for these genes in ad risk . furthermore , we detected additional strong gene expression associations for both clu and abca7 , some of which also confer ad risk , independent of the top gwas variants , suggesting that new regulatory ad variants might exist at these loci , in addition to the top snps already identified by disease gwas . [ 58 ] who determined that the ad - protective clu variant is also associated with higher clu1 isoform levels in human brains . the direction of the gene expression endophenotype effect is identical in these two studies , and indicate that therapeutic approaches aimed at increasing levels of clu in the brain might confer protection from ad . interestingly , valproic acid ( vpa ) , a well - known anti - epileptic and anti - depressant with histone deacetylase ( hdac ) inhibiting properties , was shown to induce clu expression in astrocytes . vpa was also previously highlighted as a potentially promising drug for ad due to its pro - neurogenesis and neuroprotective properties . although clinical trials of vpa in ad patients have yet failed to demonstrate a beneficial outcome [ 61 , 62 ] , evidence warrants further investigations along the clu induction axis as a potential therapeutic avenue in ad . despite recent achievements by various gwas consortia [ 43 , 6366 ] , a large proportion of the genetic contribution to alzheimer disease still remains to be identified . the utility of the conventional approach that relies heavily on clinical diagnosis ( i.e. , a dichotomy of cases vs. controls ) is dampened by contamination of the control group with persons with pre- or sub - clinical disease . while the majority of persons clinically diagnosed with ad have ad pathology [ 6769 ] , ad pathology is also common among persons without dementia [ 7073 ] . neuropathologic abnormality have been reported in persons both with and without cognitive impairment [ 74 , 75 ] , suggestive of a disease process involving pathologic change of brain structure . moreover , the phenotypic heterogeneity of dementia reflects a broader spectrum of neurodegenerative conditions other than ad , including cerebrovascular infarctions , neocortical lewy bodies , and tar dna - binding protein 43 ( tdp-43 ) , just to name a few . each of these diseases independently contributes to the clinical dementia phenotype [ 76 , 77 ] . first , compared to the more distal clinical phenotypes , neuropathologic traits lie directly in the pathway connecting genetic actions to the clinical expression of ad dementia . in other words , genetic variants do not directly cause cognitive decline and ad , but rather contribute to a series of events associated with neuropathology ; these , in turn , result in cognitive decline and ad . thus , utilization of neuropathologic outcomes increases statistical power to discover genetic variants that influence ad - related processes . using the well - known apolipoprotein e genotype ( apoe ) , we demonstrated that among a group of only about 500 community based elderly with european ancestry , quantitative pathologic ad phenotypes provide considerably more power than phenotypes of clinical ad diagnosis or cognitive function [ 79 ] . in this analysis , the association of the protective apoe 2 allele with clinical ad and level of cognition were not significant . however , it has a strong association with a measure of overall burden of ad pathology ( p = 10 10 ) . we subsequently showed that measures of ad pathology mediated the association of allele status with cognitive decline illustrating that ad pathology is in the causal chain linking the genetic variant with cognitive decline . second , genetic associations with clinical outcomes are confounded by misclassification of pre- and sub - clinical subjects . these are people that are harboring genetic variants that link to ad pathophysiology , but these persons have not yet reached the threshold for a clinical ad diagnosis . as a result , the magnitude of the association can be diluted due to the discordance between ad neuropathology and diagnostic status . for example , in the study outlined above , we found that apoe 4 was associated with ad pathology among persons without dementia , i.e. , in analyses restricted to the control group of a case control study [ 79 ] . this issue is further complicated by individual differences in cognitive or neural reserve . both structural and neuropsychological components of reserve have been shown to influence the level of resilience in the face of accumulating disease pathology , such that a greater reserve capacity reduces the deleterious effect of ad pathology on clinical symptoms . without directly assessing genetic influences on disease pathology , such influences could be easily masked by the modifying effect of reserve . in a recent gwas of ad pathology , kramer et al . [ 85 ] discovered that polymorphisms in reln were associated with higher burden of neurofibrillary tangles ( nft ) among older persons without dementia , and they hypothesized a potential role of reelin in tau phosphorylation and that upregulation of reelin may be a compensatory remedy to tau - related stress . third , a compelling rationale for using an endophenotype is to refine and partition a generic phenotype into ones that are associated with very specialized pathways . the fine - tuning helps to address the complexity of the disease biology and can amplify the association of contributory loci along the targeted pathway . substantial evidence shows that ad tends to be co - existent with other brain lesions like cerebrovascular infarctions , lewy bodies , and tdp-43 , suggesting that not all ad - associated alleles will work through the pathologic accumulation of a and phosphorylation of tau , the pathologic hallmarks that characterize ad . the disease also involves many other biological processes such as oxidative stress , chronic inflammation , alteration in lipid metabolism and depletion of molecular chaperones . it is essential to disentangle distinct genetic risk factors for the different intermediate traits in order to understand the underlying biological mechanisms that contribute to the onset of ad . sleegers et al . presented a conceptual model for the implications of recently discovered loci on ad susceptibility highlighting the influence of these novel loci on different aspects of ad pathophysiology . clusterin ( clu ) was hypothesized to share many properties of apoe in regulating a formation and lipid transportation ; complement receptor 1 ( cr1 ) on the other hand likely contributed to chronic inflammation and c3b - mediated clearance ; and phosphatidylinositol binding clathrin assembly protein ( picalm ) was implicated in maintaining synaptic function and mediating endocytosis in app recycling . pathologic phenotypes offer the promise of assessing different mechanistic hypotheses of action for each risk allele . for example , we showed that cr1 , but not clu and picalm , was significantly associated with deposition of neuritic plaques , and this association further mediates , in part , the effect of the cr1 locus on cognitive decline [ 91 ] . however , the cr1 locus also affects the accumulation of cerebral amyloid angiopathy and may therefore also function through an effect on the cerebral vasculature . beyond demonstrating the utility of leveraging intermediate phenotypes to build a causal chain of events leading from a risk factor to a clinical syndrome , cr1 also illustrates the strategy of using an intermediate phenotype to perform fine mapping of a susceptibility locus , which can help to locate the causal variant and to find additional variants that have an effect on ad and its pathology . these types of studies are not unique to apoe and cr1 , as evidence supporting an association of the cetp ad susceptibility allele with ad pathology has recently been reported . further , an interesting study from brazil reported that an individual s proportion of african ancestry was associated with a lower burden of neuritic plaque pathology , although no specific variants were reported . in parallel to the neuropathologic phenotypes , cognitive endophenotypes ( i.e. , level of cognitive function and rate of decline in cognition ) serve as another promising alternative in gene discovery . these quantitative measures share the strengths of the neuropathologic phenotypes as presented above . in particular , they are independent of diagnostic status and can be assessed in persons with and without the clinical manifestation of the disease , which helps to overcome the obstacle of confounding due to pre- and sub - clinical contamination in the control group . we and others have shown that cognitive decline begins years prior to a clinical diagnosis of ad or mci . further , because of their quantitative nature , statistical power to capture heritable variation is improved . it is now clear that a better understanding of the earlier stage in the disease progression holds great promise for effective prevention and intervention strategies , and cognitive phenotypes can help to detect genetic risk factors attributable to the preclinical and subclinical change in cognition that are not likely to be captured in conventional case is that they can be measured within the individuals longitudinally throughout life . compared with cross - sectional data ad is the result of a sequence of pathophysiological events from a deposition to synaptic dysfunction , to tangle formation , to other structural changes [ 99 ] . trajectories of change in cognition characterized by repeated assessments of cognition provide objective evidence about how ad manifests over time . endophenotypes such as cognitive decline have been increasingly used [ 91 , 93 , 94 , 96 , 100 , 101107 ] to explore the genetic linkage to these trajectories in two important ways . first , the ad loci discovered so far in cross - sectional susceptibility studies are interestingly not in concordance with those that account for the disease progression . in a recent genome - wide scan , none of the known ad susceptibility variants , except apoe and cr1 , were found to be significantly associated with the rate of cognitive decline [ 100 ] . on the other hand , the study reported a highly suggestive association with a locus near the pde7a and mtfr1 genes which regulate inflammation and oxidative stress , respectively . similar non - findings were reported by a separate gwas effort , where minor allele homozygosity of multiple novel variants were found to be associated with a faster rate of disease progression in subjects with mild cognitive impairment , but none of these variants matched those identified in previous susceptibility studies . however , it should be noted that the sample sizes of these cognitive decline gwas are a fraction of the size of the case / control studies , so it is too early to make definitive statements about known and novel susceptibility loci . estimates suggest that > 5,000 subjects will be needed to begin to have reasonable power to identify a given variant [ 100 ] . second , ad develops slowly over decades and the cognitive trajectories cover a wide spectrum from preclinical phase of ad all the way through the terminal decline in the last few years of life . specific ad susceptibility loci could be associated with different aspects of this cascade ; therefore , it is plausible that they differentially affect various stages of the cognitive trajectory . intermediate phenotypes like cognitive decline provide additional utility in dissecting the functional pathway in gene action . apoe is again illustrative on this point : the ad susceptibility allele apoe 4 was discovered decades ago , and so far little is known regarding where the polymorphism exerts its effect over the course of the disease . in particular , it is not clear whether the effect of the apoe locus persists after the onset of dementia or whether it differs in its magnitude of influence along the progression . most literature consistently reports the effect of 4 on the risk of incident ad [ 110113 ] and decline in cognitive performance in persons free of dementia [ 102 , 114117 , 118 , 119 , 120 ] . controversy arises on whether there is an 4 effect on cognitive decline in the late stages of the disease . some studies suggest that 4 is not related to decline after a diagnosis of ad [ 121125 ] , which would support the theory that apoe works primarily as a triggering factor . on the other hand , other studies have found that the 4 allele remains as an important predictor of the progression to ad after subjects experience cognitive impairment [ 127 , 128 ] and is associated with cognitive decline in the early stages of ad . to unravel these controversies , more complex analyses such as nonlinear mixed models could be considered for studies that have a sufficient number of cognitive evaluations over a long enough time . recently , using random change point models , we have showed that , among participants who were dementia free at enrollment but later developed incident ad , 4 carriers had a more rapid cognitive decline both before and after the onset of ad dementia . the capacity to incorporate these types of analyses into high - throughput gene discovery programs as the meta - analyses of ad gwas come to a close , we will have a number of validated and suggested susceptibility loci whose functional consequences can begin to be elucidated by leveraging pertinent intermediate traits such as the ones that we have discussed : rna expression , neuropathologic measures and cognitive measures ( fig . 2 ) . as illustrated by the apoe and cr1 loci , such studies can be critical in tying risk factors to a particular aspect of ad - related pathophysiology and can lead to the elaboration of a causal chain of events linking risk factors to a clinical syndrome such as ad . furthermore , gene expression studies in combination with disease association can nominate transcriptional regulatory mechanisms as a testable culprit for novel ad gwas loci , such as clu , abca7 and ms4a4a , as discussed . however , beyond gene discovery , the ad gwas have had an added benefit in that the genotyping they have performed included many thousands of subjects with pertinent intermediate traits , and these genotype data can now be repurposed for discovery studies targeting the intermediate traits [ 34 , 56 , 100 ] . a major limitation of such efforts is the fact that , in many cases , the intermediate phenotypes were not collected systematically in the same manner or using the same strategy across different cohort studies , which complicates the merging of results across individual studies and reduces our statistical power . nonetheless , such meta - analyses for gene expression , neuropathologic , cognitive and other traits is clearly an important goal for the near future as large sample sizes will be needed for these studies , as has been well demonstrated by the case / control approaches . while repurposing existing data is a valuable activity that will yield insights , we ultimately need to gather as many intermediate traits as possible from the same subjects , as this allows us to fully explore the relationship of these traits and how the effect of a risk factor ( genetic or environmental ) propagates to influence ad susceptibility . the ad neuroimaging initiative and prospective cohort studies of aging , such as the mayo clinic study of aging , and religious order study and the memory and aging project , which also have brain donations , are excellent illustrations of the type of resource that are needed to powerfully investigate the pathophysiology of ad and other neuropsychiatric diseases . such studies , if they were tenfold larger , would provide ideal platforms for communities of investigators in this field to explore the chain of events linking risk factors to a clinical syndrome . this information will be critical in the successful translation of gene discoveries to viable therapeutic approaches.fig . 2simplified model for the pathophysiology of ad and its endophenotypes : the flow of major pathogenic mechanisms from top to bottom represent the proposed consequence of events . the dotted arrows and boxes symbolize the pathogenic events that presumably precede the endophenotypes , and the endophenotypes , respectively . the examples of genes that are associated with the endophenotypes are taken from the text . while there are clearly many other plausible endophenotypes in this cascade , only those that are the focus of this review are shown simplified model for the pathophysiology of ad and its endophenotypes : the flow of major pathogenic mechanisms from top to bottom represent the proposed consequence of events . the dotted arrows and boxes symbolize the pathogenic events that presumably precede the endophenotypes , and the endophenotypes , respectively . the examples of genes that are associated with the endophenotypes are taken from the text . while there are clearly many other plausible endophenotypes in this cascade , only those that are the focus of this review are shown

uncovering the genetic risk and protective factors for complex diseases is of fundamental importance for advancing therapeutic and biomarker discoveries . this endeavor is particularly challenging for neuropsychiatric diseases where diagnoses predominantly rely on the clinical presentation , which may be heterogeneous , possibly due to the heterogeneity of the underlying genetic susceptibility factors and environmental exposures . although genome - wide association studies of various neuropsychiatric diseases have recently identified susceptibility loci , there likely remain additional genetic risk factors that underlie the liability to these conditions . furthermore , identification and characterization of the causal risk variant(s ) in each of these novel susceptibility loci constitute a formidable task , particularly in the absence of any prior knowledge about their function or mechanism of action . biologically relevant , quantitative phenotypes , i.e. , endophenotypes , provide a powerful alternative to the more traditional , binary disease phenotypes in the discovery and characterization of susceptibility genes for neuropsychiatric conditions . in this review , we focus on alzheimer s disease ( ad ) as a model neuropsychiatric disease and provide a synopsis of the recent literature on the use of endophenotypes in ad genetics . we highlight gene expression , neuropathology and cognitive endophenotypes in ad , with examples demonstrating the utility of these alternative approaches in the discovery of novel susceptibility genes and pathways . in addition , we discuss how these avenues generate testable hypothesis about the pathophysiology of genetic factors that have far - reaching implications for therapies .

Introduction Gene Expression Endophenotype Utility in Gene Discovery Utility in Gene Characterization Neuropathology Endophenotype Cognition Endophenotype Future Directions

extracorporeal shock wave lithotripsy ( swl ) has been the most common treatment of choice , especially for small renal stones ( < 2 cm ) , since its introduction by chaussy et al . the effectiveness of swl on kidney stones varies from 69.5% up to 99% [ 2 - 4 ] . failure to fragment by swl could result in unnecessary exposure of the renal parenchyme to shock waves and the requirement for an alternative procedure , which increases medical cost . therefore , it is important to identify patients who would benefit most from swl before treatment . there have been many reports on the factors predicting stone disintegration by swl . in particular , radiographic findings have been studied , such as the skin - to - stone distance ( ssd ) and the hounsfield unit ( hu ) for measuring the density of the stone on noncontrast computed tomography ( ncct ) . patient characteristics such as body mass index ( bmi ) have also been reported as significant predictors of the results of swl . in this study , we evaluated possible predictive variables for the outcome of swl of renal stones to help to better define the indications for swl treatment and to determine the efficiency with which we can check hu and stone size . between march 2008 and march 2010 , 115 patients ( 71 men and 44 women ) with solitary renal stones were evaluated . patients were included if they had a stone of 0.5 - 2.0 cm in the longest dimension in a plain film . patients who had multiple stones on the same side , patients with a stone size > 20 mm in maximal diameter , patients with radiolucent stones , cases followed up elsewhere , and cases that required a stent or developed steinstrasse and active urosepsis during the therapy were excluded . ncct was performed for all patients with a multislice helical computed tomography ( ct ) scanner ( 64-channel , multi - detector computed tomography , aquilion , toshiba , tokyo , japan ) . the images were obtained by use of the high - quality mode at 300 ma , 120 kvp , and 5 mm collimation . the patients underwent swl with the electromagnetic lithotripter ( compact delta , dornier , wessling , germany ) under fluoroscopy . in each treatment , the maximum number of shock waves was limited to 3,000 ( mean shock waves number : 2,900 ) at a maximum energy of 3.0 kvs increasing gradually from 0.1 kv . the result of treatment was evaluated by kub at 1 month after the last lithotripsy . if there were residual fragments larger than 3 mm after three sessions per week , the case was considered as a failure . multiple variables including patient characteristics such as sex and age and calculus characteristics such as location , size , density , and ssd were collected . stone location was categorized as lower calyx and non lower calyx . for the measurement of stone density , three regions of interest with a diameter of 2 mm were drawn on the stone at an axial plane of the ncct where the stone length was the longest . the mean number of hu calculated from the 3 regions represented the density of the stone on ncct ( fig . the ssd was calculated by measuring three distances from the stone to the skin at 0 , 45 , and 90 by using radiographic calipers , and the average of these three values was calculated to represent the ssd for each stone as described in the literature ( fig . 2 ) . statistical analysis was performed by using student 's t - test for continuous data and fisher 's exact test or chi - square test for categorical data . the impacts of variables were assessed by logistic regression analysis and those variables with a significant association with swl outcome were further evaluated by multivariate ( logistic regression ) analysis . a 5% level of significance was used for all statistical testing , and all statistical tests were two - sided . receiver operator characteristic ( roc ) curves were generated to compare the predictive power of the variables . the mean size and density of the stones were 10.1 + 5.0 mm and 799.5321.5 hu , respectively . of the 115 patients studied , 79 ( 68.7% ) patients had successful outcomes , whereas 36 ( 31.3% ) patients had residual stones . between the two groups , the mean hu of the success and failure groups were 675.29254.34 hu and 1,075.00290.41 hu ( p<0.001 ) , and the mean sizes of the stone were 8.343.58 mm in the success group and 13.575.41 mm in the failure group ( p<0.001 ) , respectively . however , patient age , ssd , and location of lower calyceal stones did not differ significantly between the two groups ( table 1 ) . in the univariate analysis , stone density and stone size were significant predictors of the outcome of swl ( table 2 ) . there were no significant differences in the prediction of outcome of swl according to ssd or lower calyceal stone location . in the multivariable analysis taking all factors into account , stone density and size were strongly associated with the outcome of swl ( table 2 ) . no other factors were significant . when sex and lower calyceal stone location were eliminated in the reduced model , stone density and size were still significant predictors of the outcome of swl ( table 2 ) . the roc curves of all parameters were obtained for the prediction of an unsuccessful outcome of swl ( fig . stone density showed that 863 hu was the ideal cutoff value for the prediction of failure of swl with sensitivity of 86.1% and specificity of 79.2% ( 95% confidence interval ) ( table 3 ) . stone size showed that 8.5 mm is the ideal cutoff value for the prediction of failure of swl with sensitivity of 83.3% and specificity of 70.1% ( 95% confidence interval ) ( table 3 ) . in the comparison of roc curves to test the statistical significance of the difference among the areas under different roc curves , the area under the roc curve ( auc ) was significantly higher for stone density and size than for the other parameters , but the auc between stone density and size did not differ significantly ( stone density : 0.874 ; stone size : 0.827 , p=0.388 ) ( fig . although ncct has become the most sensitive and accurate imaging modality for the diagnosis of urinary calculi [ 8 - 12 ] , the intravenous pyelogram ( ivp ) is still widely used in korea . until 2 years ago , the korean health insurance review agency prohibited the use of computed tomography as the initial imaging modality in the diagnosis of stone disease . however , ivp has many limitations in detecting urinary calculi owing to the interference with bowel gas or bony structures , and it also exposes patients to a risk of renal insufficiency and allergy by administration of contrast material . also , ivp has a limitation in diagnosis of radiolucent and small stones . by contrast , ncct is noninvasive and it can detect not only radiolucent and small stones but also other diseases of the urinary tract and other organs ( e.g. , renal mass , duplicated ureter , bladder mass , gall bladder stone , etc . ) . ncct can precisely localize the site of the stone without the use of contrast . besides , recent studies have used high - resolution ct protocols to predict the outcome of swl . for example , gupta et al concluded that the worst outcome was in patients with a calculus density > 750 hu and a stone diameter of > 1.1 cm , because 77% of those patients needed more than three sessions of swl and the clearance rate was 60% . the factors associated with the outcome of swl have been discussed in many studies over the past decade [ 16 - 21 ] . stone characteristics , such as size and location , have been reported as significant predictors of the results of swl by other authors . however , lower calyceal stone location was not a significant predictive variable in this study . also , the definition of success of swl in our study was to fragment the stones into pieces of less than 3 mm and not to excrete them all . this may be another explanation for the difference in results . there have been reports that the composition of the stones is related to the fragmentation of the stone by swl . however , knowing the stone composition before treatment is difficult and may not be sufficient to allow for prediction of the response to swl . therefore , pre - swl radiographic examination should focus on those radiographic characteristics that can influence swl outcome rather than stone composition . we also found that the density of the stone was a significant predictor of swl outcome . to date , a few studies have reported that stone density is a significant predictive factor for swl outcome . for example , pareek et al found that the mean hu values of stones were significantly higher in patients with residual stones , and joseph et al found a positive correlation between the number of shock waves required to treat a stone and its hu value . wang et al suggested cutoff values ( the stone density > 900 hu and volume > 700 mm ) for predicting swl failure . in the present study , the cutoff value was > 863 hu , which is lower than the cutoffs reported in other studies . el - nahas et al suggested that the differences in the cutoff values that predicted extracorporeal swl failure may be due to different inclusion criteria , the use of different ct protocols , or the measurement of different endpoints ( e.g. , failure of disintegration , the need for multiple sessions , or rate of residual stones ) in these studies . abe et al reported that older patients are likely to have difficulty in successful swl . in the univariate and multivariable analysis in the present study , however , age was not a significant factor . abdel - khalek et al also showed that age is not a significant predictor of swl outcome . other studies have reported that higher bmi and ssd are significant predictors for swl failure . in the present study , our patients ' relatively lower ssd than those of western patients could be the reason for that . also , we could not get exact information on the patients ' bmi for many patients in real practice . however , because this study was performed retrospectively and the number of patients studied was not large enough , further studies with large numbers of patients and a standardized ct protocol are needed to clarify this important point . ncct is noninvasive and useful for obtaining a lot of information about the patient and urinary calculi . in this study , stone density and size were significant predictors of the outcome of swl for renal stones less than 2.0 cm in diameter . we should consider hu and stone size when making decisions on the treatment of renal stones .

purposethe aim of this study was to evaluate possible predictive variables for the outcome of shock wave lithotripsy ( swl ) of renal stones in a single center.materials and methodsbetween march 2008 and march 2010 , a retrospective review was performed of 115 patients who underwent swl for solitary renal stones . the patients ' characteristics and stone size , location , skin - to - stone distance ( ssd ) , and hounsfield units ( hu ) of stone were reviewed . the impact of the possible predictors on the disintegration of the stones was evaluated by logistic regression analysis . receiver operator characteristic ( roc ) curves were generated to compare the predictive powers of the variables.resultsseventy-nine patients ( 68.7% ) had successful outcomes , whereas 36 patients ( 31.3% ) had residual stones . significant differences were found in the mean size and mean hu of the stones ( size : 8.343.58 mm vs. 13.575.41 mm , p<0.001 ; hu : 675.29254.34 vs. 1,075.00290.41 , p<0.001 ) . in the unadjusted model , age , stone size , and stone density were significant predictors . in the reduced model , stone density and size were significant predictors for the outcome of swl . the area under the roc curve ( auc ) was significantly higher for stone density and size than for the other parameters , but the auc between stone density and size did not differ significantly ( stone density : 0.874 , stone size : 0.827 , p=0.388).conclusionsstone density and size were significant predictors of the outcome of swl for renal stones less than 2.0 cm in diameter . we should consider hu and stone size when making decisions on the treatment of renal stones .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

complexity of a human biological system typically allows its relations to be expressed only in a nonlinear way . because of this complexity diabetes mellitus is a metabolic disorder of endogenous insulin allowing excessive amount of glucose to stay in blood . in general , blood glucose is transformed into energy required by human activities , such as , walking , and this transformation requires insulin functionality . however , in diabetes mellitus , since a human body fully or partially lacks the insulin functionality , unchanged glucose remains in blood . a condition of high blood glucose profiles results in several complications , such as , eye , kidney , and nerve damage , called hyperglycemia . thus , in order to avoid the hyperglycemia , a continuous supply of exogenous insulin is required , and the insulin - dependent diabetic therapy usually does this . on the contrary , too much insulin supply may lead to a condition of low blood glucose profiles resulting in drowsiness , mental malfunctioning , irritability , and loss of consciousness . thus , the insulin - dependent diabetic therapy must concern both hyperglycemia and hypoglycemia by providing an appropriate amount of exogenous insulin timely . at the beginning of the insulin - dependent diabetic therapy , it is required to obtain an approximation of the insulin - glucose relation . two methods are taken in this process , namely , empirical and fundamental methods . arguably , this process is most time consuming . based on mathematical equations representing the insulin - glucose mechanism , broadly , controlling the blood glucose levels is achieved by means of three strategies , namely , open - loop , closed - loop , and partially closed - loop schemes . in general , the fully and partially closed - loop schemes involves several medical devices but the open - loop scheme does not . while in the closed - loop scheme , a system is aimed to completely encompass the diabetic , open- and partially closed - loop require the physician 's contribution to complete the loops . therefore , typically any decisions of the insulin injections are made by a physician in open- and partially closed - loop schemes . this paper is the second part of our survey of insulin - dependent diabetes . our first paper , the first part of the survey , mostly spent its pages on the background of insulin - dependent diabetes therapy , such as , description of type 1 and type 2 diabetes , the insulin functionality , and medical devices involved in the insulin - dependent therapy . in this paper , we survey blood glucose control schemes which lie on the basics of the insulin - dependent diabetes therapies and systems . the rest of the paper is organized as follows . in section 2 , we briefly summarize diabetes mellitus for the sake of induction to the topic . in section 3 , we explain empirical and fundamental schemes to derive mathematical models of the insulin - glucose dynamics . from sections 4 through 6 , we explore the control strategies , especially for the insulin - glucose dynamics . in these sections , we provide several applications based on the controls . the world health organization ( who ) reported that there were currently nearly 180 million patients suffering from diabetes allover the world , and the number of the diabetics would increase more than 350 million people by 2030 . from the same report , approximately 1.1 million people died from diabetes in 2005 and among this number , people under 70 years old account for a half . in the united states , currently it costs 136 billion dollars annually to take care of 12 million diabetes patients [ 4 , 5 ] . in general , diabetes is considered as a condition that disproportionately affects developed countries . diabetes first emerged around 2000 b.c . while insulin and its functionality were discovered in 1921 . since the discovery of insulin , insulin - dependent diabetes therapies mostly concern how to delay the emergence of the complications in use of insulin supplement [ 6 , 7 ] . in short , diabetes is characterized in a condition that blood keeps high glucose levels unchanged into energy resulting in several complications . although insulin is largely concerned with this reaction , diabetes fully or partially lacks this functionality . diabetes eventually causes cardiovascular disease , chronic renal failure , retinal damage , nerve damage , and microvascular damage . besides , according to characteristics of diabetes , it is typically classified into two types , namely , type 1 and type 2 diabetes . in short , in type 1 diabetes , from the malfunction of the pancreas resulting from the destruction of the cells of the islets of langerhans , a supply of endogenous insulin completely stops . otherwise , the diabetic eventually falls into a condition of hyperglycemia . on the other hand , in type 2 diabetes , the insulin functionality gradually weakens , but does not completely stops . since the diabetic more or less has the endogenous insulin supply , diabetes therapies mostly focus on exercises or regimens consuming or suppressing excessive glucose in blood . however , both type 1 diabetes and type 2 diabetes are considered chronic and currently incurable . as mentioned before this is caused by the malfunction of the pancreas destroying the cells which are responsible for the endogenous insulin supply . it is considered a reason why the destruction of the cells occurs is due to the immune system which should react an infection by viruses , such as , the coxsackie virus family or german measles but mistakenly destroys the cells . type 1 diabetes is sometimes called childhood , juvenile or insulin - dependent diabetes although it does not only emerge during a childhood . on the other hand , type 2 diabetes does not stop the endogenous insulin supply , but instead it is characterized in insulin resistance , insulin deficiency , and hyperglycemia . although in type 2 diabetes , endogenous insulin still can facilitate its functionality , it is largely degraded and can not sufficiently change blood glucose into energy . thus , the amount of unchanged blood glucose will get larger resulting in hyperglycemia , a condition of high blood glucose profiles , causing eye , kidney , and nerve damage . however , since in the early stage of type 2 diabetes , the symptoms are not serious or noticeable , it is likely to miss its emergence easily . type 2 diabetes is sometimes inherited genetically , but in most of the cases it caused from irregular life styles , such as , the lack of exercise , obesity or a sedentary lifestyle . type 2 diabetes is also called non - insulin - dependent , obesity - related , or adult - onset diabetes . currently , diabetes can be treated at home by a patient himself or herself under the supervision of a physician . during the earlier years of the diabetes treatment , logs and tables of insulin injections and regimens were kept , and according to these records next insulin injections and regimens as well as exercises are determined by a physician . now , microcontrollers and sensors enable autonomous insulin - dependent diabetes therapies systematically adjusting the insulin supply . more precisely , according to feedback from one or more blood glucose sensors , a rate of insulin supply of an insulin pump is determined , which works like an artificial pancreas . the advantages of an artificial pancreas are safe , automatic , and nonintrusive . several control schemes are developed in order to achieve the optimal exogenous insulin supply suppressing the blood glucose levels within a safe range of nominal . for more details about diabetes fundamentals , to procure the mathematical models of the human insulin - glucose system , several approaches are taken by researchers . in these approaches , these approaches aim to describe the insulin - glucose dynamics as a couple of mathematical equations that should be easy to manipulate for the insulin therapies and should fully describe the characteristics of the internal insulin - glucose metabolism . basically , the empirical method uses a model structure ( formula or equation ) which is determined theoretically with several parameters . the behavior of this model structure is determined by only the input - output data of the system from a number of experiments . in this method , capturing the system behavior or data is the most time - consuming process . in an example of the linear structure of the insulin - glucose system , to represent glucose effects , two parameters are used , and to represent insulin effects , other two parameters are also used in order to close the model to the actual system . in addition to the input - output data , semiempirical method utilizes other physiological factors , such as dynamic behavior and kinetics to create a closer model of diabetic patients . in the fundamental methods , a mathematical representation of the human internal system which this system behavior includes kinetics and material transport . according to investigating the internal system , a lot of data from the literature can be used to determine the system parameters . in particular , in constructing a fundamental diabetes model , the authors in applied the insulin - release data of the cells of the pancreas from a number of examinations to a mathematical representation . there are several classes of solutions to this problem , ranging in complexity , prerequisite knowledge , and feedback . the open - loop system for the insulin - dependent diabetes therapy does not employ any glucose sensors . however , occasionally calling the open - loop system is not appropriate and more precisely , the system should be called the programmed insulin infusion system because of its incomplete openness . that is , the control loop can be closed by the physician and the diabetic when interacting on the system . one example of the systems is one that was developed by case - western reserve university , and this system is considered to be one of the most intelligent programmed insulin infusion products that deal with the noninsulin - dependent diabetics . the idea is that from an analysis of the insulin curve in the nondiabetic , it was turned out that the curve approximately traces a combination of a double exponential curve and a basal insulin infusion . according to this mathematical model , an intravenous insulin delivery system the system utilized a portable cart containing the control system , the insulin - pump , power supplies , and insulin reservoir so that the patient could move around with the devices . the insulin pump delivers low - concentration insulin and updates the insulin delivery rate every 30 seconds . because of its simplicity this research revealed the diabetics had the blood glucose profile to be improved considerably from a two - week examination , and , moreover , the improved conditions remained even several days after the examination . on the other hand , the researches of this programmed system so far did not indicate any hypocglycemic condition yet . in addition to the achievement of case - western reserve university , siemens and the finsen institute also developed a programmed insulin infusion system that employed a moderately complex delivery algorithm from another approach . the system is capable of manually inserting small pin connectors into the control unit in order to control the insulin delivery rate . like a product of case - western reserve university , the infusion rate follows an exponential curve , and the insulin infusion rate is updated every 30 minutes [ 1518 ] . the system to deliver insulin mechanically in order to regulate the glycemic profile is called the closed - loop system . as shown in figure 1 , the closed - loop system completes its operating cycle within the system and no external interaction to diabetic patients is required [ 19 , 20 ] . in other words , the closed - loop control uses the feedback from the output . typically , the closed - loop system for type 1 diabetes therapy utilizes the glucose sensor and schematically consists of three phases : blood glucose measurements , insulin demand calculation , and insulin injection . so far , along with the glucose sensor , the closed - loop system also employs an insulin pump which continuously infuses insulin via a subcutaneous root . basically , insulin delivery is controlled by these implanted blood glucose sensors and an insulin pump attached to a patient 's body [ 19 , 2123 ] . in short , according to measurements of glucose level from an implanted blood glucose sensor , an insulin pump continuously infuses insulin into a patient 's body . however , although implanted blood glucose sensors benefit a lot for the diabetes therapy , establishing reliable measurements of blood glucose is so difficult that many researches in this field are still under way by many biomedical researchers . currently many forms of blood glucose sensors exist , such as fingerstick types , implantable types , or noninvasive types . for example , in applying a fingerstick - type blood glucose sensor , blood glucose levels are measured three to seven times a day and according to the measurements , the amount of insulin supply by an insulin pump is updated manually . however , since with the fingerstick - type sensors , measurements are carried out by patients themselves on regular basis , managing patient lifestyle by themselves is rather troublesome . meanwhile , when using an implantable blood glucose sensor , glucose levels in blood are automatically monitored in a certain amount of period . in calculating the insulin infusion , many control models are developed by researchers until now : pole - assignment strategy , self - tuning adaptive control , or nonlinear predictive control . pole assignment is a standard control systems technique for designing an infinite impulse response filter [ 19 , 24 ] . this consists of a set of filter coefficients and a feedback loop in order to maintain a stable blood glucose level . in general , the closed - loop schemes of the insulin - dependent diabetes therapy utilize an insulin pump that automatically supplies insulin into the human body subcutaneously . usually the glucose levels are monitored by a needle - type glucose sensor through the subcutaneous ( sc ) route , and the insulin infusion rate ( iir ) is determined by continuous measurements of the blood glucose level . for example , in pole - assignment strategy , the iir in relation to blood glucose level , the insulin - glucose system , is determined by the following computation : ( 1)iir(t)=kpg(t)+kddg(t)dt+kcwithkp = amnvp , kdkp=1l+1m+1n+ba , kc = d+cakp , where g is blood glucose level and d is the insulin infusion rate through the intravenous ( iv ) route . parameters a , b , and c can be calculated from the relationship between plasma insulin i and blood glucose levels in a normal person , which are written as(2)i(t)=ag(t)+bdg(t)dt+c . moreover , other parameters n and l are from next equations which are the pharmacokinetics of insulin infusion through the sc route:(3)dx(t)dt = iir(t)+lx(t),dy(t)dt = lx(t)(p+o)y(t),dz(t)dt = py(t)nz(t),i(t)=z(t)v , where x , y , and z represent the insulin level in the two subcutaneous compartments and in the plasma , respectively . figure 2 shows such an x / y / z 3-level model . at last , this is a simplified approach , forgoing adaptive control for ease of characterization and implementation . for most situations , it will perform as desired , but if it encounters a situation that it handles poorly , it will handle that situation poorly every time it occurs again in the future . experiments showed that the combination of the pole - assignment strategy and lispro insulin generated a similar trend to the use of the iv route with regular insulin . however , the combination of the pole - assignment strategy and regular insulin generated much worse result . a difficulty of the pole - assignment strategy is to repeatedly evaluate model parameters in each computation of the iir . to avoid re - evaluations of the model parameters , the self - tuning adaptive control closed - loop scheme employs a recursive assessment of the model parameters so that the glucose level of time period k , that is , gk , is evaluated from the glucose levels of time period k 1 through k h , that is , { gk1 , , gkh } , and the insulin doses of time period k 1 through k p , { idk1 , idkp } , as well as some unknown parameters , which can be written as [ 2528](4)gk = m(gk1, ,gkh , idk1,idkp, ) , where p and h are time delays . since this method evaluates the blood glucose level of time period k from the previous evaluations and insulin doses , it can efficiently eliminate unnecessary re - evaluations of the model parameters . besides , according to glucose level gk , the next insulin dose is calculated as(5)j=(gkgk1)2ridk , such that i dk can minimize value j , where r is a weighting factor designed to control the amount of insulin dose . implementation of self - tuning adaptive control , shown in figure 3 , is quite similar to pole - assignment control , as it uses the same system modeling equations in order to compute the insulin infusion rate . the primary difference between the two methods is that another controller is used to constantly evaluate the system model , and may tune or redesign the pd controller parameters as needed to obtain more accurate results based upon minimum variance . one advantage of this control scheme is that it is comparatively easy for a physician to estimate the future trend of blood glucose levels from a set of the past inputs , where the model can be used to predict hypoglycemic or hyperglycemic events before they occur . a model predictive control ( mpc ) , or nonlinear predictive control ( nlpc ) algorithm attempts to learn what nominal means in a system [ 19 , 20 ] , shown in figure 4 . in the case of blood glucose management , a nonlinear mpc algorithm uses sensor data to track glycoregulatory system parameters in order to predict the levels of required insulin infusions . it then uses models of the human glucose metabolism to estimate the effects of the insulin injection . an example of a model used is a nonlinear autoregressive ( narx ) model , where previous blood glucose ( bg ) levels and insulin dosage levels are run through a nonlinear function , often obtained through neural network learning . bayesian learning is applied using the model - predicted effect of the insulin , and its actual measured effect . the learning process adjusts system parameters in order to increase the accuracy of its predictions as more iterations are performed . using this method , the system will become increasingly accurate , and will begin to understand how the patient that it is calibrated to will react to insulin injections of varying compositions and strengths . apparently , the insulin - glucose system is complicated , and the system is considered to be nonlinear . in , in order to follow this nonlinearity , one method utilizes a collaboration of a neural network ( nn ) and nonlinear model predictive control ( npc ) technique , that is , neural predictive control . more precisely , nns and an npc are used to simulate the glucoregulatory system . a schematic diagram of the neural predictive control is shown in figure 5 . basically nns approach the problem of blood glucose management without attempting to explicitly describe the exact model of the blood glucose - insulin system [ 3133 ] . this is particularly useful in situations where patients have a disease that complicates normal model description , or an abnormality exists which makes prediction difficult using just measured parameters and sensor data . like other control strategies , the main goal of the neural predictive control is to achieve regulation of the glucose profiles for the type 1 diabetics predicting a future glucose curve from the nonlinear model with time delays , so it can follow a similar curve to the metabolism of normal people . a feed forward neural network employing backpropagation can be trained offline using accumulated patient data , including daily blood glucose readings as well as insulin dosages . a neural network will then be able to learn based upon experience , much as a human brain learns . this will help it to predict nonlinear behavior , even multiple orders removed , imperceptible to standard data interpretation methods . this capability to be intuitive helps to drive a system in which unknowns or immeasurable parameters are still accounted for , and abnormalities are detected and intelligently handled . in addition to the control variables , it also designs a cost function in relation to the insulin - glucose model [ 19 , 30 ] . in the mechanism of the scheme , the neural predictive control makes consecutive control actions toward the glucose metabolism altering the control variables , so that the actions consequently minimize a designed cost function at each sampling time . however , the alteration of the control variables also changes the optimization problem at each sampling time . on the other hand , the model and its parameters can be of no difference during a whole examination . fortunately , in order to regulate the glucose profile , the so - called monomeric insulin ( mi ) analogs are currently available . these mi analogs have advantages that they are able to be absorbed through the subcutaneous route three or four times faster than human insulin resulting in that the rise of the plasma insulin concentration grows faster . besides , the mi analogs are more predicable than human insulin due to its less variability . in the forming of the control strategy , first develops a mathematical model of the insulin - glucose dynamics of the type 1 diabetics , which is mainly broken down into three parts : the subcutaneous insulin absorption model , glucose regulation model , and subcutaneous glucose model , shown in figure 6 . from the model , the subcutaneous insulin absorption is calculated in two steps : the subcutaneous mi analogs infusion and utilization from the subcutaneous depot . with respect to the glucose regulation , in order to model the system mathematically , adopts a compartmental model in which there are single compartments for glucose and glucagon , and three compartments for insulin ( liver and portal insulin , plasma insulin , and insulin in the interstitial fluid ) . also in the model , net hepatic glucose balance , renal glucose excretion , and glucose utilization are simulated to generate numerical values . from the glucose regulation model , the subcutaneous glucose model is generated by investigating transfer rates between the plasma and subcutaneous compartments . consequently , the subcutaneous glucose model forms a linear , first - order system with the transfer function . in the second step , using numerical data from simulations of the mathematical patient model , the paper develops the nonlinear system by nns in order to make future blood glucose predictions . to do this , the paper utilizes a nonlinear autoregressive model ( narx ) because of its popularity and usefulness . ,idknu)+ek , where g is a sequence of subcutaneous glucose profiles , especially gk is a future glucose prediction , i d is a sequence of subcutaneous insulin infusion rates , ek is a noise , and ny and nu are both durations of glucose utilization and insulin activation , respectively . at this point , however , the approximation of the nonlinear function f is a hard task . a reason why to approximate the nonlinear function is difficult is that the function is required to be made up from finite data although there are usually infinite solutions for it . to resolve this difficulty , approximation based on regularization techniques is used because it was proved that regularization principles consequently can derive networks with one layer of hidden units , that is , regularization networks . thus , the paper uses a function of radial basis function ( rbf ) networks , which are a subclass of regularization networks , in order to represent the nonlinear function f : ( 7)f(x(t))=i=1nwih(xxi0),h(xxi0)=1((xxi0)2+)2 , where h is a continuous function of rr , || || , represents the euclidean norm , xi are some proper center values selected from the data points , wi are some weight constants , and is a parameter representing the dispersion [ 19 , 30 ] . also the regularized orthogonal least squares ( rolss ) algorithm is used to determine rbf weights and centers in order for f(x(t ) ) to fit the data under some constraints . the third step to model thus , eventually controlling the glucose profiles is turned into resolving the minimization problem where a sequence of subcutaneous insulin infusions must sufficiently minimize the problem below(8)argminid j=[j = n1np(ejtej)+j=0nc1idt(t+j)uidt(t+j ) ] , where i d = [ idk , idk1 , , idknc1 ] and nc , np , n1 , e , u are parameters of the controller for tuning . there are three steps for the process of a fuzzy logic algorithm : fuzzification , rules , and defuzzification.fuzzification : the input of a controller is an exact number , like the concentration of glucose is 100 mg / dl . what the fuzzification do is to fuzzy the concentration such as low concentration , high concentration , and proper concentration . every exact number has the weight of all these low concentration , high concentration , and proper concentration.rules : after defining the fuzzy concept of input , rules are made to decide what the output should be : more drug , a little drug , or no drug.defuzzification : after the rule , the output of fuzzy concept is obtained , for example , more of 0.8 and little of 0.2 . but the output which is the object model 's input must be an exact number , that needs to be defuzzification . by defuzzification , the output gets an exact number . fuzzification : the input of a controller is an exact number , like the concentration of glucose is 100 mg / dl . what the fuzzification do is to fuzzy the concentration such as low concentration , high concentration , and proper concentration . every exact number has the weight of all these low concentration , high concentration , and proper concentration . rules : after defining the fuzzy concept of input , rules are made to decide what the output should be : more drug , a little drug , or no drug . defuzzification : after the rule , the output of fuzzy concept is obtained , for example , more of 0.8 and little of 0.2 . but the output which is the object model 's input must be an exact number , that needs to be defuzzification . by defuzzification , the output gets an exact number . in the paper , it is assumed that there are two different inputs of the concentration of glucose and the change rate of concentration , and one output of the dose of drug . overlow , good , high , and overhigh are defined for the concentration . the dose of drug is defined as zero , little , ten rules are defined such that : if ( rate is overlow ) , then ( dosage is zero);if ( concentration is overhigh ) and ( rate is low ) , then ( dosage is little);if ( concentration is overhigh ) and ( rate is highest ) , then ( dosage is most);and so forth . if ( rate is overlow ) , then ( dosage is zero ) ; if ( concentration is overhigh ) and ( rate is low ) , then ( dosage is little ) ; if ( concentration is overhigh ) and ( rate is highest ) , then ( dosage is most ) ; in a partially closed - loop scheme of the insulin - dependent diabetes therapy , measurements are conducted three to seven times per day , and insulin injections are also performed three to four times under the supervision of a physician . these decisions , for example , the number and type of insulin injections , insulin dosage , are made according to model - based or algorithmic - based decision support systems , such as dias , aida , and t - iddm . insulin injections are usually performed by using the subcutaneous ( sc ) route due to its management and safety . also there is an alternative route for insulin delivery that is ideal for control , the intravenous ( iv ) route . figure 7 shows a control flow of a partial closed - loop scheme . while in the closed - loop systems , the blood glucose levels are automatically monitored by an implantable sensor and according to the measurements , insulin infusions are carried out in the use of an insulin pump or three or four times of the insulin injections , in the partial closed - loop , the metabolic controls partly rely on the physician 's evaluations of the measurements of the blood glucose levels , the amount of insulin injections and physical exercises as well as glycosuria and ketonuria . moreover , as other metrics of the evaluation , medium period indicators , such as glycated hemoglobin ( hba1c ) , are captured , where the blood glucose levels of the past 60 days can be seen . in other words , the partial closed - loop scheme is made out of a collaboration of the feedforward and feedback controls , and usually feedforward controls are made by clinicians who determine it from the patient 's lifestyles . however , these evaluations largely rely on doctor 's experiences . from the objective of the insulin therapy that aims to reconstruct the artificial insulin metabolisms in relation to the levels of blood glucose in the body , it is typically an optimization problem that is viewed from four dimensions : the number of injections , time of injection , insulin type , and insulin dosage . basically , this four - dimensional space optimization is intrinsic . besides , occasionally a clinician provides him / her with a feedforward strategy from the patient 's lifestyle . the insulin regiment prescribed by a doctor , to be administered manually , constitutes partially closed - loop control [ 19 , 24 ] . a physician will dictate an insulin administration routine to a patient , variant upon a patient 's lifestyle . patients under such a system monitor their blood glucose level several times a day , administering insulin based upon prescribed tables according to their schedule and bg level . that has traditionally been used by insulin - dependent diabetics , but it performs poorly compared to other methods . partially , closed - loop control is far from real time and only updates its control routine at scheduled physician visits . furthermore , lifestyle events such as eating , sleeping , or working out must be accounted for by the patient in their interpretation of insulin tables , introducing the very real danger of human error . the bergman model is one of the virtual diabetes patient models represented in the literature . in the bergman model , the certain dynamics of the diabetes patient system can be represented as mathematical equations by employing three - order model : a glucose compartment , g , a remote insulin compartment , x , and an insulin compartment , i [ 4 , 36 , 37 ] . basically , the bergman model has a very simple form and is represented as follows:(9)dg(t)dt=(p1+x(t))g(t)+p1gb , dx(t)dt=p2x(t)+p3(i(t)ib),di(t)dt=(g(t)h)tni(t ) , where g(t ) represents plasma glucose and i(t ) represents plasma insulin at certain time t , which are initialized at t = 0 . x(t ) represents the effect of insulin causing net glucose disappearance , for example , the remote insulin concentration . gb represents a base value of plasma glucose and likewise , ib represents a base value of plasma insulin . moreover , p1 is a parameter representing glucose effectiveness , p2 fractionally represents the insulin - dependent increase rates and p3 fractionally represents the net remote insulin disappearance rates . h is a threshold where the plasma glucose levels are expected not to exceed . when the plasma glucose levels exceed the threshold , the second - phase of insulin secretion will be performed with additional insulin secretion . furthermore , insulin is removed from the plasma insulin space at rate of n. the bergman model is used for efficiently predicting the certain diabetes patient system dynamics . the automated insulin dosage advisor ( aida ) is a virtual diabetes patient model that was originally designed for the educational purpose so as to help patients and clinicians learn effective glycemic control [ 4 , 38 ] . basically , aida is used for estimating effects of insulin injections and regimens in type 1 diabetic therapy . more precisely , aida is a simulation program that models the insulin - glucose dynamics based on the physiological rules around metabolism of a single glucose compartment [ 19 , 38 ] . to model the insulin - glucose dynamics , metabolism around the glucose compartment is carried out such that delivering glucose into the compartment is conducted both by being absorbed by the intestine and by being produced by the liver ( gluconeogenesis ) . on the other hand , removing glucose out of the compartment is carried out by insulin - independent and insulin - dependent glucose utilization . more precisely , by the insulin - independent glucose utilization , glucose is carried from the compartment into red blood cells , the central nervous system and viscera , whereas by the insulin - dependent glucose utilization , glucose is carried from the compartment into the liver and periphery . to hepatic and peripheral glucose utilization , aida is designed to have a capability of adding different insulin sensitivities by modeling them separately . besides , the renal threshold is defined in order to model renal glucose losses [ 38 , 39 ] . figure 9 shows a variation of the net hepatic glucose balance in terms of the changes of the amount of the active insulin and blood glucose ( bg ) levels in the form of nomograms . moreover , the aida model applies a process of insulin absorption derived by berger and rodbard to its pharmacokinetics of the model . in addition , insulin is separated into two compartments where one is for plasma insulin and the other is active insulin . on the basis of a physiological model , active insulin controls metabolism . on the other hand , hepatic degradation produces insulin from plasma insulin . in the use of aida , by comparing home - monitored blood glucose levels to a typical behavior of blood glucose of a patient , for example , the model day , metabolic problems are identified . according to particular metabolic problems , aida can generate several possible solutions , and among these possible solutions , one best solution will be selected according to a nonlinear dynamic model of the insulin - glucose system . at last , this nonlinear dynamic model of aida consists of four differentials , 11 algebraic equations and 17 parameters . from the data , the model will be constructed while simulations are performed for each therapeutic choice to optimize the following cost function:(10)j(g)=0t(g(t)g0)2dt , where g0 describes a set point . the diabetes advisory system ( dias ) is a nonlinear model of the blood glucose - insulin system based upon real - life parameters , versus simply bg measurements . it incorporates qualitative and quantitative input from the user , including bg levels , meals , and past insulin injections . the system uses a discrete - time finite - state model of the system based upon user input . the system uses what it understands about the system as a whole , including dormant compartmentalized insulin , predigested carbohydrates , and current bg levels to compute a bayesian estimate of future bg levels . it uses all known information in order to compute the value of the optimal dosage such that it minimizes an associated cost function ( i.e. , hypoglycemia is far more costly than hyperglycemia , due to its possibility of severe damage ) . over iterations , it will adjust its system model parameters to better account for patient specific reactions it detects . for example , in dias , the flow of the physiological calculation of carbohydrate is drawn by figure 10 . besides , dias has three different modes , for example , the learning mode , the prediction mode , and the advisory mode . in figure 10 , there are two associated state variables , for example , cho and bg , and both of them record how much carbohydrate content exists in the gut compartment and the blood compartment , respectively [ 42 , 43 ] . for example , in this model , carbohydrate content is , at first , taken in from meals and delivered to the gut and the blood compartment . during the delivery to the blood , some of carbohydrate content is absorbed by the gut and only the rest of it is delivered to the blood and transformed into the main energy . in addition , this flow of carbohydrate is redrawn more precisely by using difference equations , which are shown in figure 11 . in this redrawn model , the amount of carbohydrate content in both the gut ( cho ) and blood ( bg ) compartment is updated in every hour such that both increase and decrease of carbohydrate content are kept track of in each compartment . in figure 11 , the gut - abs process variable represents how much glucose is absorbed by the gut , and the rest of glucose remains in the gut and is recorded in the cho state variable . besides , the renal - cl process variable represents how much glucose is removed , the ins - indep - util process variable represents how much glucose is used independently of insulin , the ins - dep - util , on the other hand , represents the amount of glucose to be used for insulin - glucose dynamics , and the glu - prod process variable represents the amount of glucose produced by the liver [ 42 , 43 ] . in dias , there are also two input variables , for example , meal and ins - inj , where the meal input variable is carbohydrate intakes at given time from each meal , and the ins - inj input variable represents the amount of the external insulin injection [ 42 , 43 ] . furthermore , to adjust individual physiological differences , two other parameters are employed , for example , ins - sens and nph - max , where the ins - sens parameter stands for the insulin sensitivity that affects the active insulin ( act - ins ) variable , and the nph - max parameter represents the time when nph insulin achieves the maximum absorption or concentration , shown in figure 11 [ 42 , 43 ] . the + and symbols in figure 11 represent fluctuation of carbohydrate on the cho and bg state variables . for example , the cho state variable at hour 1 can be written as an equation such that cho ( hour 1 ) = cho ( hour 0 ) gut - abs ( hour 0 ) + meal ( hour 1 ) [ 42 , 43 ] . the transition among the states in a graph of difference equations in dias is defined by causal probabilities . more precisely , the graph representation of difference equations of dias is defined by a causal probabilistic network ( cpn ) or a bayesian network using the hugin approach [ 42 , 43 ] . for example , the transition probability of the amount of carbohydrate absorbed by the gut given the carbohydrate content in the gastrointestinal tract is p(gut - abs cho ) . as previously mentioned , there are three modes in dias to conduct a calculation of the optimal insulin dosage ( the decision support system ) : the learning mode , the prediction mode , and the advisory mode . at first , the learning mode is used to generate the two adjustable parameters , for example , the insulin sensitivity ( ins - sens ) and time - to - peak absorption of nph ( nph ) , from the collection of standard data , such as the amount of blood glucose , insulin injection , and carbohydrate content in the meals [ 42 , 43 ] . for instance , an example of a prediction of blood glucose transition from , from the measurements of blood glucose , the mixture of short - acting insulin and intermediate - acting insulin and the carbohydrate intake , the transition of blood glucose is predicted as a straight line . secondly , the objective of the prediction mode is to predict the resulting blood glucose concentration from given an intake of carbohydrate and insulin injection as well as two adjustable parameters estimated in the learning mode [ 42 , 43 ] . there is a risk of hypoglycemia around lunch time , the ratio of the insulin mixture is manually changed in the morning to handle the condition . as an example of the objective of the prediction mode of dias given from , there is a risk of hypoglycemia around lunch time , the ratio of the insulin mixture is manually changed in the morning to handle the condition . in dias , the advisory mode , which is considered as a special version of the prediction mode , is used to generate possible insulin therapies that avoid the overall risk of an excess or shortage of blood glucose by minimizing an utility measure . in the advisory mode , the manual changes of the insulin regimen conducted in the prediction mode , such as switching to a different mixture of insulin , are automatically replaced to an optimal way by the system . for example , a replaced version of an optimal insulin injection procedure is used by the advisory mode , where the mode recommends reducing the amount of nph insulin from 10 to 6 u before dinner resulting in avoiding hypoglycemia during bedtime [ 42 , 43 ] . as an example of a result calculated by the dias advisory mode , dias further generates an optimal solution of an insulin therapy automatically to avoid the overall risk of an excess or shortage of blood glucose by minimizing a utility measure . the eu developed telematic management of insulin dependent diabates mellitus ( t - iddm ) which was a telemedicine system that supported clinician 's decision - making for providing insulin for the insulin - dependent diabetics . basically the system consist of two modules , for example , a patient unit ( pu ) and a medical unit ( mu ) , and two decision support elements , for example , a rule - based reasoner ( rbr ) and a case - based retrieval system ( cbrs ) [ 19 , 44 , 45 ] . the system architecture is shown in figure 12 . with the system , the pu is basically responsible for monitoring changes of the blood glucose concentration in patients , and the physiological data are transferred to the hospital database via the internet . in addition to the hospital database , the pu also has a local database that enables patients to deal with their own diabetics cases autonomously . on the other hand , thus the mu can visualize incoming data from patients , analyze them , and generate optimal insulin treatments for particular patients . basically , the mu is a web - based application made up of collaborating five servers : a database server , temporal abstraction server , data analysis server , decision support system , and web server . data communication is made up between the two devices . from the perspective of the decision support for the therapy , the rbr and cbrs are designed to resolve the following premises . at first , the insulin - glucose dynamics is very complicated that only highly parameterized nonlinear models can be applied to it although it is usual to have no more than three or four blood glucose tests per day in the insulin - dependent diabetes therapy , which is not enough to model the dynamics . due to the limitation of the measurements , some parameters are required to be fixed in all cases whereas only a few parameters are free to be set up according to each patient case . however , this limitation of setting parameters must make the models and the quality of prediction inflexible and inaccurate . one goal of t - iddm is to refine the models to generate more precise prediction of the insulin - glucose models from the limited inputs . t - iddm provides the professionals with more systematical way to plan the therapy for particular insulin - dependent diabetics . at last , the same metabolic behavior occasionally generates different results . for example , either honeymoon effect or other troubles may cause the same number of hypoglycemia over a month . however , this type of wrong diagnosis may fall the diabetic into a critical situation . therefore , in independence of metabolic behaviors , t - iddm should employ context that can generate different results from the same metabolic behavior . also the introduction of context may reduce the search space from whole possible solutions . in the implementation of the rbr , to optimize the therapy , it runs four sequential tasks each of which is connected to a set of rules through a forward chaining mechanism . these four sequential tasks are the data analysis , problem identification , suggestion selection , and therapy revision . however , only the rbr is sometimes not enough to produce reliable suggestions of the therapy for poorly controlled patients . therefore , in consideration of that situation , in addition to the rbr , t - iddm employs the cbrs to improve the system to be more accurate . the objective of the cbrs is to search a pool of past cases for similar situations to the current condition and utilize them to help the user make an optimal decision for the current condition . a set of past cases is narrowed for searching according to very high - level view of the cases by a nave bayes strategy , and after that , in the proper retrieval step , cases having the closest to the current situation are effectively chosen and shown to the user . regarding the artificial insulin - glucose control system , designs a fuzzy logic reasoning system . there are mainly two modules ( e.g. , an analog signal conditioning board and a microcontroller board ) and other interface devices , such as a lcd display , a sixteen button keypad , and alarm system , along with the operating software to work the system . the software operates the system according to a fuzzy logic reasoning method . in the system , an analog signal conditioning board is responsible for generating electrical signals from vital parameters monitored by several biomedical sensors . these parameters include sweating , snoring , heart rate , and eeg . in the meantime the system works with batteries so that it can be portable . in the use of the system , several electrodes are placed on the particular segment of the human body capturing the four physiological parameters . basically , these electrodes are connected to an analog signal conditioning board that amplifies and filters the vital signs . since a 10 bit a / d converter interfaces a microcontroller board , the analog signals can be converted to the digital signals with the sampling rate of 100 samples / s . the microcontroller invokes the fuzzy logic reasoning algorithm that resides on the rom of the system to process the four parameters . this survey mainly focuses discussions on control methods for the insulin - dependent diabetes ( type 1 diabetes ) . three control methods are introduced in this paper , namely , open - loop , fully , and partially closed - loop control methods . in either of them , the objective of the control methods is to suppress the blood glucose profiles to avoid a condition of hyperglycemia . because diabetes is a metabolic disorder which is characterized as complete or partial lack of insulin functionality , therapies can be done by making up for the lack of insulin supply by exogenous insulin replacement . in the open - loop control method , the insulin replacement is programmed such that the amount of the insulin supply follows the non - diabetic insulin delivery . the open - loop control method usually does not count on utilization of blood glucose sensors , but instead , a transition of the insulin supply is captured by carefully examining the nondiabetic in advance . on the other hand , fully and partially closed - loop controls typically rely on feedbacks from the blood glucose sensor measurement . in the closed - loop control method , the system loop is fully closed so that it does not require any assessment by physicians . it is only based on feedbacks from one or more blood glucose sensors , and it usually requires continuous glucose measurements . thus from the measurements of blood glucose profiles , a rate of the insulin supply by an insulin pump is adjusted so that it can lead to neither conditions of hyperglycemia nor hypoglycemia . examples of the closed - loop control method are pole - assignment model , self - tuning adaptive control , model predictive control , and nonlinear predictive control . in addition to the blood glucose sensor feedbacks , partially closed - loop control also relies on feedforwards by physicians . in the partially closed - loop control method , the blood glucose measurements are conducted three to seven times per day and insulin injections are done three or four times per day . although both the blood glucose samples and insulin injections are discrete , these are compensated by physicians feedford assessment of insulin requirements . usually a calculation of the insulin supply utilizes a flow chart or table which describes complex relations between blood glucose reduction and insulin supply . examples of the partially closed - loop control method includes bergman model , automated insulin dosage advisor ( aida ) and diabetes advisory system ( dias ) . hence challenges of the blood glucose control methods are to delay the emergence of diabetic complications but not cure the patients from diabetes . in practice , three control methods introduced in this survey partially satisfy these requirements . however , our expectation is that the progress of the technology will enable the construction an artificial pancreas that follow the regular functionality of the actual pancreas , and this new technology must lie on the current control technologies .

we survey blood glucose control schemes for insulin - dependent diabetes therapies and systems . these schemes largely rely on mathematical models of the insulin - glucose relations , and these models are typically derived in an empirical or fundamental way . in an empirical way , the experimental insulin inputs and resulting blood - glucose outputs are used to generate a mathematical model , which includes a couple of equations approximating a very complex system . on the other hand , the insulin - glucose relation is also explained from the well - known facts of other biological mechanisms . since these mechanisms are more or less related with each other , a mathematical model of the insulin - glucose system can be derived from these surrounding relations . this kind of method of the mathematical model derivation is called a fundamental method . along with several mathematical models , researchers develop autonomous systems whether they involve medical devices or not to compensate metabolic disorders and these autonomous systems employ their own control methods . basically , in insulin - dependent diabetes therapies , control methods are classified into three categories : open - loop , closed - loop , and partially closed - loop controls . the main difference among these methods is how much the systems are open to the outside people .

1. INTRODUCTION 2. TYPE 1 AND TYPE 2 DIABETES 3. MODELING THE HUMAN INSULIN-GLUCOSE SYSTEM 4. OPEN-LOOP CONTROL MODELS 5. CLOSED-LOOP CONTROL MODELS 6. PARTIAL CLOSED-LOOP SCHEME 7. CONCLUSION

germ cell tumors often complicate uncorrected cryptorchidism with an incidence of 9.8% reported by martin . occurrence of pure yolk sac tumor however is uncommon in the undescended testis with only a few cases reported in literature . we present a case of yolk sac tumor in a 2-year - old boy in the undescended testis which was primarily diagnosed by fine needle aspiration cytology ( fnac ) . a 2-year - old boy presented to the pediatric surgical out patient department ( opd ) with 5 days history of a right sided abdominal mass . on examination the lump was 5 cm 5 cm in size , firm in consistency with restricted mobility . a tumor was noticed at the right renal hilum , causing ureteric narrowing with resultant hydronephrosis along with presence of multiple , large oval , heteroechoic lesions in the right iliac region and reteroperitoneum . possibility of lymphoma was suggested . due to the absent testis in the right scrotum , testicular neoplasm smears were highly cellular showing tumor cells in variably sized loose clusters with occasional cells forming glomeruloid structures [ figure 1 ] . the nuclei were round to oval with irregular nuclear membranes , coarsely clumped chromatin and 0 - 1 nucleoli . there was abundant mucoid material in the background with presence of intracytoplasmic as well as extracellular periodic acid - schiff ( pas ) positive hyaline globules . this was corraborated by the raised alpha feto - protein ( afp ) levels ( > 2000 beta human chorionic gonadotropin ( hcg ) levels were within normal limits ( 1.61 miu / ml ) . contrast - enhanced computed tomography ( cect ) of the chest and abdomen were performed and revealed an elongated nearly oval shaped heterogeneously enhancing lesion in the right lower abdomen . multiple heterogeneously enhancing retroperitoneal lymph nodes , right sided hydroureteronephrosis with left sided pleural deposits was also seen . in view of undescended testis , possibility of germ cell malignancy was suggested . it was gray to yellow in colour with some myxoid areas . also present in the specimen sections were taken extensively and showed features consistent with yolk sac tumor with cells arranged in microcystic - reticular , myxomatous and focal macrocystic , alveolar - glandular and papillary patterns [ figure 2 ] . the rete testis , epididymis and surgically resected end of the spermatic cord were free of tumor . intracytoplasmic and extracellular hyaline globules present in the tumor were pas positive and diastase resistant . cd30 , epithelial membrane antigen ( ema ) and carcinoembryonic antigen ( cea ) were negative ruling out embryonal carcinoma and choriocarcinoma . smear showing clusters of pleomorphic cells with abundant mucoid material in the background ( giemsa stain , 400 ) . inset shows pleomorphic cells with intracellular periodic acid - schiff positive hyaline globules section showing glandular , microcystic pattern and myxomatous stroma ( h and e , 100 ) . inset on the left shows glands and inset on the right shows papillae ( h and e , 400 ) fine needle aspiration cytology is a useful first line of investigation to diagnose testicular masses . it provides a triage of cases of testicular tumors into those who require surgery and those who do not . smears are cellular with presence of cohesive clusters of very immature cells having basophilic cytoplasm and a prominent nucleolus . presence of intra and extracellular pas positive hyaline globules and a myxoid background are characteristic features . reported yolk sac tumor of the undescended testis which initially presented with a mass in the left lobe of the liver . in the present case , fnac provided a precise diagnosis of yolk sac tumor of the undescended testis which was clinically suspected to be either a renal or lymph node mass . fnac in conjuction with raised afp levels is useful to diagnose yolk sac tumors and for planning further therapy . sinha et al . and cox et al . reported similar cases of yolk sac tumor in an undescended testis in small children . testicular malignancy should always be ruled out when a cyrptorchid child presents with an abdominal mass . mixed germ cell tumors occur frequently in the testis and only one component may be sampled on fnac . the type of components present in the mixed germ cell tumor has major implications on the prognosis of the patient . tumor markers like afp , hcg , lactate dehydrogenase and placental like alkaline phosphatase are useful in making the diagnosis . treatment and outcome of germ cell tumors in undescended testis is similar to those of germ cell tumors in general . reported that survival among patients with testicular malignancy in descended and undescended testis was similar . the present case highlights the use of fnac in diagnosing testicular masses , especially in our case where the lesion presented with multiple lymph node masses and renal hydronephrosis and was clinically and radiologically suspected to be a wilm 's tumor with metastasis or a lymphoma . fnac is a useful , rapid and inexpensive tool , making it a first line of investigation in many hospitals . however , mixed germ cell tumors may be missed on cytology and should be correlated with clinical and radiological findings and serum tumor markers prior to surgery .

yolk sac tumor is the most common germ cell tumor in infants and children . majority of them arise in the gonads . yolk sac tumor of undescended testis has been rarely diagnosed . we present a case of yolk sac tumor in right undescended testis in a 2-year - old child primarily diagnosed by fine needle aspiration cytology .

Introduction Case Report Discussion Conclusion

incomplete regression of the mammary ridges may result in aberrant breast tissue , which can be classified based on its components . among the various types of breast choristoma , ectopic breast tissue , comprised of only glandular tissue without a nipple or areola , although ectopic breast tissue does not have a secretory ductal system , it is histologically no different from anatomically normal breast tissue . ectopic breast tissue therefore shows physiologic and pathogenic processes similar to those of eutopic breast tissue . clustered microcalcifications on mammography are a relatively common feature of invasive carcinoma or ductal carcinoma in situ . however , the microcalcification of ectopic breast tissue is difficult to delineate on mammography , and is often not detected . we report herein a case of ductal carcinoma in situ originating from axillary ectopic breast tissue that had revealed clustered microcalcifications on screening mammography . a 52-year - old woman with bilateral accessory breast tissue attended a local breast clinic for annual mammography and ultrasonography screening , during which clustered microcalcifications were detected in ectopic breast tissue of the right axillary area . it was decided to maintain observation only with close surveillance , because the mammography findings were assessed as breast imaging reporting and data system category 3 ( figure 1a , 1b ) . after 3 months the patient was readmitted to the clinic with a palpable axillary mass . the mass was hard and fixed in the right side of axilla and an irregularly shaped axillary mass including microcalcifications , was identified on ultrasonography . a breast surgeon performed an excision biopsy of the axillary mass , and clustered microcalcifications were identified on specimen mammography ( figure 1c ) . histologic observations revealed that the mammary ducts , within the fibrofatty stroma , were filled with and distended by a solid epithelial proliferation . necrosis and multifocal microcalcification were noted ( figure 2a ) , and the epithelial cells showed a moderate to high degree of nuclear atypia ( figure 2b ) . the patient was diagnosed as having high - grade ductal carcinoma in situ and was referred to our department for additional procedures . preoperative evaluation via positron emission tomography and computed tomography did not reveal any metastatic lesions . the patient underwent a wide local excision of the axillary ectopic breast tissue with a safety margin around the cavity of 1 cm ; sampling of sentinel lymph nodes was also performed . for the evaluation of sentinel lymph nodes , 2 ml of a blue dye ( indigo carmine ) the weight of the excised ectopic breast tissue was 60 g ; the contained microcalcifications were identified on specimen mammography . in a section of the excised specimen , a cavitary lesion was noted along with focal fat necrosis due to the excision procedure . remnant tumor was not clearly detected on macroscopic examination , but on microscopic examination , remnant tumor measuring 0.5 cm in maximum diameter was observed around the cavity ; histologic findings were similar to those for the excision biopsy . and there was no indication of metastasis to the lymph nodes among seven sentinel lymph nodes biopsied . immunohistochemical assay showed positive and negative staining for the estrogen and progesterone receptors , respectively , and strong positive staining confirmed overexpression of the c - erbb-2 protein . the patient continues to undergo surveillance up to 3 months after surgery , with no specific problems . ectopic breast tissue , a choristoma of the breast , can occur anywhere along the mammary ridge , and almost every form of breast disease can occur in ectopic breast tissue . primary breast carcinoma arising from accessory breast tissue has been reported in 60% to 70% of all forms of ectopic breast tumor . and the invasive ductal carcinoma is the most frequent histologic variant of primary ectopic cancer . however , ectopic breast cancer is not easily detected because of the late expression of pathologic symptoms , and is commonly misdiagnosed as axillary lymph node disease , lipoma , or a sebaceous cyst . furthermore , we report a microcalcification in the axillary region , revealed on mammography ; such findings could lead to ectopic breast cancer being overlooked or misdiagnosed as a calcification of the axillary lymph nodes . as late detection of ectopic breast cancer may lead to a worse prognosis , a precancerous sign or symptom should not be ignored . we wish to report the present case because of its rarity , as well as to highlight the implications of microcalcifications being discovered during the regular surveillance of ectopic breast tissue when an abnormality is detected on imaging . when an abnormal image or symptom of ectopic breast tissue is noted , it should be evaluated in the same manner as for eutopic breast tissue . if a palpable mass is detected in ectopic breast tissue , a needle biopsy can be helpful for a differential diagnosis . if a malignant microcalcification is detected on mammographic imaging of aberrant breast tissue , it should be evaluated as to whether the calcification is located in a choristoma of the breast or a lymph node ; a biopsy should be performed for further evaluation . a malignant microcalcification on mammography strongly correlates with ductal carcinoma in situ or invasive cancer . when a malignant microcalcification is detected on mammography , a needle biopsy or excision biopsy this principle would also apply to ectopic breast tissue , because the components of such tissue are the same as those of an anatomically normal breast . when ectopic breast cancer has been diagnosed , it is classified according to the tnm staging system used for primary breast carcinoma . there is a clear distinction between ectopic breast cancer and occult breast cancer that presents in axillary lymph nodes ; this is the reason for a thorough examination to accurately establish the location of a microcalcification . ectopic breast cancer should be managed based on the treatment protocols for primary breast cancer , in contrast to the management of occult breast cancer . although francone et al . strongly recommended the preventive excision of ectopic mammary tissue , the incidence of ectopic breast cancer is only 0.2% to 0.6% of all breast cancers , whereas the incidence of postoperative complications ( e.g. , severe scarring , infection in the remaining sweat glands , pain , or discomfort ) is reported to be as high as 4% to 10% . however , preventive removal of ectopic breast tissue should be performed in cases that show abnormal signs or symptoms ; if it were not performed , routine screening would be sufficient . a wide and complete resection of ectopic breast tissue is indicated after ectopic breast cancer has been diagnosed , and an evaluation of axillary lymph nodes is certainly indicated if the ectopic breast cancer occurs in the axillary region . ectopic breast cancer can spread to the axillary lymph nodes more rapidly than a typical breast cancer . based on the tnm staging and characteristics of an ectopic breast cancer , adjuvant chemotherapy or radiotherapy can be administered , and hormonal therapy or targeted therapy can also be used , in accordance with the status of the relevant biological markers . although ectopic breast cancer is very rare , diagnostic modalities are usually the same as for typical breast cancer . however , it is not easily detected if a significant clinical symptom is not obvious , which may result in a delayed diagnosis and poor prognosis . a patient with aberrant breast tissue when an abnormal finding is detected , such as clustered microcalcifications on mammography or a clinically palpable mass in ectopic breast tissue , a thorough evaluation should be undertaken . despite its rarity , symptomatic ectopic breast cancer is relatively easy to diagnose because of its similarity to typical breast cancer . however , it is less easily delineated on diagnostic imaging , leading to a late diagnosis and poor prognosis . examination of accessory or ectopic breast tissue in affected patients should take place at the same time as the evaluation of eutopic breast tissue .

ectopic breast tissue can occur anywhere along the incompletely regressed mammary ridge . among the various types of breast choristoma , ectopic breast tissue , which has only glandular tissue without a nipple or areola , is most commonly detected in axillary areas . however , ectopic breast cancer is often not detected until significant clinical symptoms have been revealed , or diagnosis is delayed . furthermore , an examination of ectopic breast tissue tends to be omitted from a screening mammography . especially , the microcalcifications of ectopic breast tissue are difficult to delineate on mammography . herein , the authors report a case of ectopic breast carcinoma that showed clustered microcalcifications on screening mammography , and discuss the interpretation and implications of microcalcification in ectopic breast tissue .

INTRODUCTION CASE REPORT DISCUSSION

the broadband dielectric spectroscopy ( bds ) is considered as a promising method for monitoring biological properties of cells [ 14 ] . the bds monitoring can give the information on membrane capacitance , cytoplasmic conductivity and permittivity and the cell shape and structure . the effect of physical and chemical treatments on the cellular membrane can also be detected , including the degree of the disruption of cells walls [ 14 ] . regarding practical implementations of bds in the industry area , the possibility of monitoring of cell concentration in cultivation and fermentation is worth mentioning , along with the possibility of the use of these data for the estimation of the cell cycle . these applications are notably strengthened by unique features of the modern bds which can detect , in a single scanning process , up to 15 decades in frequency / time . moreover , modern bds spectrometers can operate with high resolution in a very broad range of electric capacitances and conductivities , which is particularly important for bio - samples . nowadays , the temperature and concentrational ( isothermal ) bds behavior of diluted and dense solutions of cells is relatively well evidenced experimentally , although models for the analysis of experimental data are still puzzling [ 14 ] . this can be associated with extraordinary complexity of living and active bio - colloids / bio - composites . one of notable gaps in experimental bds studies on bio - systems is the practical lack of results related to the impact of hydrostatic pressure . the latter influences the activation energy whereas the compression changes the density , the free volume and distances between species in a fluid system . on the practical side , significance of the pressure - related insight into properties of bio - systems indicates the boost of high pressure preservation ( hpp ) of foods technology , known also as the cold pasteurization , pascalization or bridgemanization [ 810 ] . for basic hpp industrial implementations the high pressure pulse ( 300600 mpa ) this can lead to the reduction of the count of microorganisms from 5 to 8 decades [ 811 ] . the high microbiological safeness of hpp - treated foods is inherently associated with a set of highly beneficial features [ 610 ] : ( i ) the extension of shelf - life from 1 to 3 to even 90 days , ( ii ) taste , flavor and texture of the fresh product , ( iii ) preservation of vitamins amounts , ( iv ) avoiding of chemical preservatives , ( v ) the method can be applied for already packed food , ( vi ) the application is not limited to fluids , ( vii ) hpp is an environment - friendly method , i.e. it needs much less energy than the thermal pasteurization and there is practically no waste . these unique advantages of the hpp technology coincides with expectations of the 21st century consumers with regard to high quality and healthy food products . nowadays , there are over 200 large industry - scale hpp processors with the internal volume of the pressure chamber from 100 to 300 l. their number increase by ca . notwithstanding , the fundamental biophysical base of hpp - related issues is still very limited . one of the most important cognitive gaps is related to studies in situ under high pressure . the comprehensive insight into properties of biosystems and foods in the pressure - temperature ( p - t ) plane can yield a base for a pressure - related modelling of novel foods . bds offers unique research possibilities for in situ tests under high pressure due to existing state - of - the - art experimental implementations developed during studies on soft matter systems [ 1217 ] . this report presents results of the first ever in situ high pressure bds investigations for saccharomyces cerevisiae ( baker s yeast ) , the unicellular eukaryotes which are widely used in biotechnological model processes [ 810 ] . twenty - four hour culture in ypg - broth ( 1 % yeast extract difco , 1 % peptone difco , 2 % glucose poch ) was centrifuged at 4 c for 10 min at 6000 g , and the sedimented cells were aseptically re - suspended into phosphate - buffered saline ( pbs , ph 7.2 ) and again centrifugated . the final suspensions of s. cerevisiae were prepared in pbs , reaching cell concentration of about 1010 cfu / ml ( colony forming units / ml , ie . living cells / ml).electron microscopy.cells after uhp treatment were centrifuged 8 min at 3500 g , washed carefully with distilled water and again centrifuged under the same conditions . the cells was suspended in 2 % solution of glutaraldehyde and stored over night at room temperature . the samples were centrifuged for 10 min at 3500 g and the cells were prepared for electron microscopy . ultrathin sections were observed under a transmission electron microscope jem 100c ( jeol , tokyo , japan ) . the cells treated at 300 and 400 mpa showed essential ultrastructural changes . about 80 % of cells population , showed cell wall disruption and electron - dense areas of amorphous material packed with crushed microsomes and ribosomes.uv analysis.for the spectroscopic studies , uv - visible 1800 shimadzu double beam spectrophotometer was used to record the spectra . the yeast samples after high pressure treatment were centrifuged at for 20 min at 6000 g , and the supernatants obtained were scanned in the 240280 nm uv region . the wavelength maximum was observed at 260 nm , which relates to the yeast cell nucleotides and aromatic amino - acids present in the supernatant as the result of the high pressure cell wall disruption .high pressure treatment.the precise scheme of the high pressure set - up is given ref . . it enabled changes of pressure up to p = 500 mpa , monitored via the tensometric pressure meter with precision + 0.1 mpa . the temperature of the pressure chamber was yielded via a large volume ( 25 liter ) thermostat with external circulation , enabling the temperature stabilization up to 0.02 k. temperature was measured via constantant - copper thermocouples inside the pressure chamber . pressure was measured with the precission 0.1 mpa using tensometric pressure meter nova swiss . the sensor was located near but outside the pressure chamber , to avoid any impact of temperature on measurements.the greatest challenge for any in situ measurements under high hydrostatic pressure is the total isolation of the tested sample from the pressurized medium , matched with forceless transmission of the pressure to the sample . moreover , multi - cycle increasing and decreasing of the applied pressure should be possible . the authors of the given paper developed the high pressure electric capacitor fulfilling all these conditions . the flat - parallel measurement capacitor applied in the given research had the gap d = 1 mm and diameter 2r = 16 mm ) . the pressure is transmitted from the pressurized liquid to the sample via the deformation of 50m stretched teflon film . unique construction of the capacitor is very solid and does not enable changes of any significant part during pressurization . it is notable that its body is made from invar , hence it is also not sensitive to temperature changes.broadband dielectric spectroscopy under pressure.for bds studies the alpha - a bds novocontrol ( montabaur , germany ) analyzer ( model year 2013 ) , yielding an insight for frequencies f = 0.001 hz to 10 mhz ( 9 decades ) with 56 digits resolution was used . generally , bds yields the complex dielectric permittivity ( ) = ' ( ) + i( ) , = 2f . the first component can be determined as ' ( f ) = c / c0 , where c0 and c are electric capacitances of the measurements capacitor and the capacitor with the tested dielectric sample . the second component is experimentally defined as ' ' ( ) = 1/rc0 = ' /rc , where r denotes the resistivity . for the so - called static domain , in liquids most often for 1khz < f < 1mhz , ' ( f ) is constant and referred to as dielectric constant ( ' ( f ) = ) . in this domain the temperature and pressure evolution of the dielectric constant allow to identify the dominating type of arrangement of permanent dipole moments [ 617 ] . twenty - four hour culture in ypg - broth ( 1 % yeast extract difco , 1 % peptone difco , 2 % glucose poch ) was centrifuged at 4 c for 10 min at 6000 g , and the sedimented cells were aseptically re - suspended into phosphate - buffered saline ( pbs , ph 7.2 ) and again centrifugated . the final suspensions of s. cerevisiae were prepared in pbs , reaching cell concentration of about 1010 cfu / ml ( colony forming units / ml , ie . living cells / ml ) . cells after uhp treatment were centrifuged 8 min at 3500 g , washed carefully with distilled water and again centrifuged under the same conditions . the cells was suspended in 2 % solution of glutaraldehyde and stored over night at room temperature . the samples were centrifuged for 10 min at 3500 g and the cells were prepared for electron microscopy . ultrathin sections were observed under a transmission electron microscope jem 100c ( jeol , tokyo , japan ) . about 80 % of cells population , showed cell wall disruption and electron - dense areas of amorphous material packed with crushed microsomes and ribosomes . for the spectroscopic studies , uv - visible 1800 shimadzu double beam spectrophotometer was used to record the spectra . the yeast samples after high pressure treatment were centrifuged at for 20 min at 6000 g , and the supernatants obtained were scanned in the 240280 nm uv region . the wavelength maximum was observed at 260 nm , which relates to the yeast cell nucleotides and aromatic amino - acids present in the supernatant as the result of the high pressure cell wall disruption . high pressure treatment . it enabled changes of pressure up to p = 500 mpa , monitored via the tensometric pressure meter with precision + 0.1 mpa . the temperature of the pressure chamber was yielded via a large volume ( 25 liter ) thermostat with external circulation , enabling the temperature stabilization up to 0.02 k. temperature was measured via constantant - copper thermocouples inside the pressure chamber . pressure was measured with the precission 0.1 mpa using tensometric pressure meter nova swiss . the sensor was located near but outside the pressure chamber , to avoid any impact of temperature on measurements . the greatest challenge for any in situ measurements under high hydrostatic pressure is the total isolation of the tested sample from the pressurized medium , matched with forceless transmission of the pressure to the sample . moreover , multi - cycle increasing and decreasing of the applied pressure should be possible . the authors of the given paper developed the high pressure electric capacitor fulfilling all these conditions . the flat - parallel measurement capacitor applied in the given research had the gap d = 1 mm and diameter 2r = 16 mm ) . the pressure is transmitted from the pressurized liquid to the sample via the deformation of 50m stretched teflon film . unique construction of the capacitor is very solid and does not enable changes of any significant part during pressurization . it is notable that its body is made from invar , hence it is also not sensitive to temperature changes . broadband dielectric spectroscopy under pressure . for bds studies the alpha - a bds novocontrol ( montabaur , germany ) analyzer ( model year 2013 ) , yielding an insight for frequencies f = 0.001 hz to 10 mhz ( 9 decades ) with 56 digits resolution was used . generally , bds yields the complex dielectric permittivity ( ) = ' ( ) + i( ) , = 2f . the first component can be determined as ' ( f ) = c / c0 , where c0 and c are electric capacitances of the measurements capacitor and the capacitor with the tested dielectric sample . the second component is experimentally defined as ' ' ( ) = 1/rc0 = ' /rc , where r denotes the resistivity . for the so - called static domain , in liquids most often for 1khz < f < 1mhz , ' ( f ) is constant and referred to as dielectric constant ( ' ( f ) = ) . in this domain the temperature and pressure evolution of the dielectric constant allow to identify the dominating type of arrangement of permanent dipole moments [ 617 ] . dipole couplings in fluid dielectrics pressure dependences of dielectric constant showing preferable type of dipole dipole couplings in fluid dielectrics when lowering the frequency values of ' ( f ) permanently increase , since the low frequency electric field can interact with large and inert ions , in dielectric and molecular physics often recalled as ionic impurities [ 6 , 17 ] . the translation of different types of ionic impurities present in a fluid dominates the bds output in this region . in the dominated region it is convenient to transform experimental data to the complex electric conductivity form ( ) = i0( ) = ' ( ) + i ' ' ( ) = 0 ' ' ( ) + 0( ' ( ) 1 ) , where the electric constant ( vacuum permittivity , permittivity of free space ) o 8.854pf / m the tradition and practical reasons caused that in dielectrics physics and in soft matter physics for ( ' , ' ' ) relative and dimensionless values are used . as visible from the above definition this is not the case of ( ' , ' ' ) . usually , in liquids and soft matter systems in the region dominated by electric charges motion ' ( ) is not affected but ' ( ) increases with decreasing frequency = 2f in such a way that the slope at the plot log10 ' ' ( ) vs. log10 is equal to 1 .. consequently , in this domain ' ( ) is independent from changes of the frequency and called dc conductivity ( dc ) and often simply electric conductivity . electric conductivity can be considered as the metric of translation of ionic dopants and the static dielectric permittivity = ' ( f ) const gives an insight into polarization and permanent dipoles arrangements . the imaginary part of dielectric permittivity ' ' ( f ) is associated with energy losses and offers and insight into relaxation processes , manifesting as peaks in ' ' ( f ) spectrum . in the low frequency and ion - dominated region one should expect its growing up described by ' ' ( f ) f with = 1 dependence , in agreement with the definition of dc electric conductivity above . in classical molecular liquids one should expect that the dc electric conductivity will decrease with increasing pressure . in the simplest case , for describing such process the barus equation , can be used : (p ) = 0p exp(vap ) , where va denotes the constant activation volume and 0p is the prefactor related the value of (p ) for p = 0 . it can be considered as the pressure counterpart of the simple arrhenius relation (t ) = 0 exp(ea / rt ) , where r denotes the gas constant and ea is the activation energy . in a limited range of pressures , the barus equation can be reduced to the linear dependence (p ) = 0 exp(vap ) 0(1 vap + ) = 0 ( 0va)p , due to the implementation of the taylor expansion . such approximation is valid only for relatively small values of the argument ( pressure p in this case ) , if for experimental data notable distortion from such simple relation appears one should use the functional , experimental form ( preferably ) . this report bases on in situ measurements of (f ) for selected pressures and the discussion of the pressure evolution of dielectric constant (p ) , dc conductivity (p ) and ' ' ( f , p ) spectra . bds studies has been carried out for high concentration of saccharomyces cerevisiae ( 1010 cells / ml ) to avoid a significant influence of the maxwell wegner ( mw ) effect which can lead to the parasitic polarization of the capacitor plates and which can notable bias output results . the key target of given studies was the preliminary test of the possibility of bds technique as the tool for monitoring the high pressure cold pasteurization - related processes in situ . second , on the higher level of complexity , one can expect the interruption of cell walls . the high pressure denaturation in the p - t plane is portrayed by an elliptic - like curve , crossing the p 0.1mpa ( atmospheric pressure ) line near t 80c , related to pasteurization [ 8 , 19 ] . the isothermal ( i.e. well below the thermal pasteurization limit ) compression , for 5c < t < 40c , leads to the denaturation at 400 < p < 600mpa . . 2the schematic presentation of proteins denaturation in the pressure temperature ( p - t ) plane [ 8 , 19 ] . the form of a protein prior and after the denaturation process is shown the schematic presentation of proteins denaturation in the pressure temperature ( p - t ) plane [ 8 , 19 ] . the form of a protein prior and after the denaturation process is shown it is notable that for such path of approaching the denaturation curve one can minimize the influence of the irreversible coagulation , always occurring for the classical , thermal pasteurization . it is notable that death curves of microorganisms portrayed in the p - t , strongly resemble plane by the curves in fig . 2 . despite this fact , as the key reason of the killing action of high pressures on microorganisms is recognized the action on more complex structure , mainly the cell wall . 3the electron microscope of yeast cell before ( ( a ) , p = 0.1 mpa ) and after ( ( b ) , p = 400 mpa ) pressurization the electron microscope of yeast cell before ( ( a ) , p = 0.1 mpa ) and after ( ( b ) , p = 400 mpa ) pressurization the question arises , how the pressure - induced destruction of cells manifests in the bds monitoring . the first one is the imaginary part of dielectric permittivity , shown in fig . this process is described by ' ' ( f ) f with the exponent = 1 , as shown in fig . 4 . for f < 10hz at p = 0.1mpa and f < 100hz at p = 0.1mpa , the impact of the ionic polarizability emerges . on further decreasing of the frequency the evolution of log10 ' ( f ) vs. log10f becomes progressively nonlinear , until reaching the domain described by the exponent s < 1 for lower frequencies . . these changes can be related to the so strong impact of permanent ionic impurities that under the external electric field from the bds spectrometer causes the parasitic polarization of plates of the capacitor . it is notable that this phenomenon appear for much higher frequencies for the pressurized yeast which indicates much higher content of ionic impurities , presumably associated with free ions of na , k , ca , mg , cl and molecular species related to organic acids .fig . 4the imaginary part of dielectric permittivity in native ( atmospheric pressure ) and pressurized yeast for t = 25 c isotherm . the key parts of plots follow the dependence ' ' ( f ) f , i.e. log10 ' ' ( f ) log10 f. the frequency f b denotes the crossover to the maxwell wegner effect - dominated region the imaginary part of dielectric permittivity in native ( atmospheric pressure ) and pressurized yeast for t = 25 c isotherm . the key parts of plots follow the dependence ' ' ( f ) f , i.e. log10 ' ' ( f ) log10 f. the frequency f b denotes the crossover to the maxwell wegner effect - dominated region it is also notable that for the pressurized yeast , a primary relaxation process with the peak at ca . 8 mhz emerges . figures 5 and 6 show the pressure dependence of the static dielectric permittivity and dc electric conductivity as the function of pressure at 5 and 25 c temperatures . threshold , near pt = 200 mpa for t = 5 c and pt = 300 mpa for t = 25 c . below pt the increase of pressure increases the electric conductivity and above pt further compression decreases the conductivity . for p liquids , where the pressurization decreases the electric conductivity , what is coupled with a notable increase of viscosity . the rise of conductivity (p ) on increasing pressure up to pt indicates the appearance of new free ions in the system , gradually released from a reservoir of free ions . this can be associated only with the break of cell walls and leakage of the intracellular fluid . above the threshold pressure , for p > pt , the evolution of electric conductivity is similar to the behavior observed in normal liquids where number of free ions is constant and does not change during the experiment [ 6 , 15 , 17 ] . this domain is described by the barus equation as discussed above and shown in fig . these threshold pressures are also clearly visible in behavior of the dielectric constant ' ( p ) = (p ) . up to p = pt the linear increase of ' ( p ) is very weak and in fact close to (p ) const , what suggests the virtual lack of the preferable arrangement of permanent dipole moment . for p > pt dielectric constant ' ( p ) strongly increases indicating the creation of a notable parallel arrangement of permanent dipole moments . it is worth stressing that threshold pressures values manifest clearly both for (p ) and (p ) evolutions.fig . 5the pressure evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t = 5 cfig . 6the pressure application time ( p = 400 mpa ) evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t = 25 c . the pressure evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t = 5 c the pressure application time ( p = 400 mpa ) evolution of the real part of dielectric permittivity and the dc conductivity in yeast at t = 25 c . figure 7 shows the impact of time on the pressurized yeast , monitored by the changes of (p ) and (p ) . after 1 hour of high pressure acting , the electric conductivity increases and the dielectric constant decreases by ca . these results indicate that despite different nature of processes monitored by dielectric constant and electric conductivity , they are strongly coupled.fig . 7the time evolution of the dc electric conductivity and the static dielectric permittivity in the pressurized yeast at t = 5 c the time evolution of the dc electric conductivity and the static dielectric permittivity in the pressurized yeast at t = 5 c the increase of the electric conductivity can be linked to the leakage of intracellular liquid , appearing due to the pressure - induced breaks of the cellular walls . one of methods to estimate the amount of the intracellular , naturally turbid , liquid , are spectrophotometric measurements of absorbance of clear supernatant at 260 nm after the removal of cells and cells debris by centrifugation . figure 9 shows the clearly correlation between the normalized absorbance and the dc conductivity on increasing pressure in tested samples.fig . 8the uv absorbance of supernatant leaking out from cells of s. cerevisiae after pressurizing from 0.1 to 500 mpafig . 9the correlation between absorbance at 260 nm of the supernatant after pressurizing of saccharomyces cerevisiae and electric dc conductivity the uv absorbance of supernatant leaking out from cells of s. cerevisiae after pressurizing from 0.1 to 500 mpa the correlation between absorbance at 260 nm of the supernatant after pressurizing of saccharomyces cerevisiae and electric dc conductivity results presented show that the evolution of the static dielectric permittivity and particularly the dc electric conductivity can be considered as notable and sensitive tools for testing characteristics of pressurized yeast cells . it is notable that bds experiments under pressure can yield high resolution results , hardly available by other physical method implemented for such challenging conditions . microbiological studies of pressurized solutions of yeast and other model cells indicated that conditions related to the pasteurization , i.e. the reduction of the number of microorganisms by at least five decades ( 5 log - units ) , needs at least 400 mpa for minimum 3 minutes [ 811 ] . moreover , deeper pasteurization takes place for pressurization under higher temperature . this paper lead to the conclusion that the break of cell walls may take place for lower pressure at lower temperatures ( 5 c ) , already at 200300 mpa . the effective process needs also more time than 35 minutes used in hpp technology . all these may suggest that the action of pressure leading to the cold pasteurization phenomenon for commercially most often used pressures p = 400600 mpa is associated not only with the break of cell walls but also the influence of intracellular structures and pressure - induced denaturation .

results of broadband dielectric spectroscopy ( bds ) in saccharomyces cerevisiae ( baker s yeast ) , in situ as the function of pressure are presented . they show a clear evidence of a threshold to the new pattern of the pressure evolution of the static dielectric permittivity and dc electric conductivity already for pt 200mpa at t = 5oc and pt 300mpa at t = 25oc . bds monitoring versus pressure tests up to p = 400mpa revealed particularly notable changes of properties after 30 minutes of compressing . finally , the correlation between the amount of the spectrophotometric maximum absorbance and the dc electric conductivity was found . all these indicate significance of bds as the tool for testing of pressure properties of cells assemblies , model foods etc . , in situ under high pressures .

Introduction Experimental Results and Discussion Conclusions

coronary artery disease ( cad ) is one of the most common causes of death and disability worldwide ( zaman et al . , 2000 ) . genetic predisposition and interaction with a host of environmental ( endogenous and exogenous ) factors can cause the disease . among the risk factors predisposing to cad are hypertension , obesity , hyperlipidemia , stress , smoking , age , gender and family history ( kasap et al . , 2007 ) . oxidative stress has also emerged as a major risk factor ( yung et al . , 2006 ) in cad arising through an imbalance between oxidants and antioxidants ( nojiri et al . , 2001 ) . the oxidative modification of low density lipoproteins ( ldl ) also plays a causative role in the pathogenesis of coronary artery disease ( chisolm and steinberg , 2000 ) . furthermore , consequences of oxidative stress can cause damage to genomic integrity as atherosclerotic plaques have been reported to exhibit extensive genetic damage ( martinet et al . , 2002 ) . in fact , reports have also documented the contribution of dna damage to the development and progression of coronary artery disease ( andreassi , 2003 ) . there has been an increase in heart disease in punjabi population which may be attributed to economic surplus and change in dietary and life - style patterns ( anonymous , 2013 ) . the changeover from extensive agrarian activities to a sedentary / near sedentary life - style and the adaptation of the western dietary habits across the lower , upper middle and upper socioeconomic status classes , have resulted in increased general and abdominal obesity ( who , 2003 ) . as no studies have come to attention in punjabi populations associating cad with genomic damage , in the present study , adults ( cad patients and healthy controls ) have been assessed for chromosomal and dna damage in their peripheral blood leukocytes ( pbl ) . lipid profile , hypertension , diabetes , obesity parameters ( body mass index , waist - hip ratio , waist circumference ) and oxidative stress ( lipid peroxidation , total oxidant status , total antioxidant capacity , oxidative stress index ) . for genetic damage assessment , chromosomal damage in peripheral blood lymphocytes using the cytokinesis - block micronucleus ( cbmn ) assay and dna damage in pbl using the single cell gel electrophoresis ( scge / comet ) assay have been carried out . the cbmn assay is a well known cytogenetic test - method to assess the genetic effects of spontaneously and mutagenically- induced dna damage ( fenech , 2007 ) and the comet assay is a sensitive biomarker of oxidative stress and dna damage ( balasubramanyam et al . , 2010 ) . the study proposal was approved by the institutional ethics committee and voluntary written informed consent was taken from all study participants ( n = 65 ) . patients with diagnosed cad were contacted from local hospitals through the cardiologists . exclusion criteria included cad patients with renal , liver or thyroid disorders , those with negative results on the treadmill test , and those not belonging to the punjabi population . the control group included healthy adults with no past or present history of heart or any disease and no exposure history , matched for gender , population group and socioeconomic status . the demographic and disease - related information for each patient was documented on a pre - designed questionnaire . anthropometric measurements ( height , weight , hip circumference , waist circumference ) were taken following standard methodology ( weiner and lourie , 1981 ) . derived obesity measures were body mass index ( bmi ) and waist hip ratio ( whr ) . using the criteria of who ( 2004 ) for bmi , the participants were classified as obese or non - obese regarding general obesity , and cut - offs as per snehalatha et al . ( 2003 ) for whr and waist circumference ( wc ) values for abdominal obesity . physiometric measurements ( using a mercury sphygmomanometer and stethoscope ) were taken after the subject had rested for ten minutes as per the recommendations of american heart association ( kirkendall et al . , 1981 ) . the average of three measurements were taken as the blood pressure values . the mean arterial blood pressure ( mbp ) was calculated from systolic and diastolic measurements ( prusse et al . , 1989 ) . venous blood ( 5 ml ) was then drawn from each participant and processed for the cbmn and scge assays . the cytokinesis - block micronucleus ( cbmn ) assay : - short term lymphocyte cultures were set up ( moorhead et al . , 1960 ) and the cbmn protocol of fenech ( 2000 ) was followed . briefly , the leukocyte - rich plasma ( 2 ml ) was added to 8 ml of rpmi 1640 medium ( himedia , india ) . the lymphocytes were stimulated to divide by addition of phytohaemagglutinin - m ( 0.5 g / ml pha - m , biological industries , israel ) . the cultures were incubated for 72 h at 37 c ; after 44 h cytokinesis was blocked by the addition of cytochalasin - b ( 6 g / ml , sigma ) and on completion of incubation of 72 h at 37 c , the cultures were harvested . following centrifugation ( 800 g , 10 min ) , the pellet was fixed in chilled fixative ( 3 methanol : 1 glacial acetic acid , qualigens , india and srl , india , respectively ) for 30 min . after centrifugation and two - three washings of the pellet , the cell suspension was used to prepare the slides . the air - dried slide preparations were stained with 5 % giemsa ( himedia , india ) for 10 - 20 minutes . for each sample , a total of 2000 binucleated cells ( 1000/slide ) were scored for the presence of micronuclei ( mn ) and micronucleated cells following the criteria of fenech ( 2007 ) . random observations by another scorer were done to verify the observations . the nuclear division index ( ndi ) , which is an index of cytostatic effects , was calculated ( eastmond and tucker , 1989 ) by using the formula : ndi = ( m1 + 2m2 + 3m3 + 4m4)/n where m1-m4 represent the number of cells with 1 - 4 nuclei and n is the total number of cells scored . the scge / comet assay : the trypan - blue dye exclusion test for cell viability was performed ( strober , 2001 ) and samples with > 95 % cell viability were processed for the comet assay . the leukocytic dna damage was ascertained using the alkaline comet assay ( singh et al . , 1998 ) . in brief , on pre - coated slides with 1 % normal melting point agarose ( nmpa ; srl , india ) , a second layer containing 30 l of blood sample mixed with 110 l of 5 % low melting point agarose ( lmpa , srl , india ) was poured followed by the sandwiching with a third layer of 120 l of 5 % lmpa after the second layer had set . cells were lysed in freshly prepared cold lysing solution ( 2.5 m nacl , 100 mm na2 edta , 10 mm tris hcl , ph 10.0 ; 1 % triton x-100 with 10 % dmso added just before use ) for at least one hour to allow dna unfolding . then the slides were placed in a horizontal electrophoresis chamber , covered with freshly prepared alkaline electrophoresis buffer ( 0.3 m naoh , 1 mm no2 edta , ph > 13 ) at 4 c for unwinding and expression of alkali - labile sites ( 30 min ) and subsequently electrophoresed ( 25 v , 300 ma , 0.7 - 1.0 -v / cm , 25 min ) . slide preparations were then washed with neutralizing buffer ( 0.4 m tris hcl ph 7.5 ) to remove alkali and detergents , and left for drying . the slides were silver stained ( nadin et al . , 2001 ) and coded . scoring was done using the freely available casp ( comet assay software programme , 3.1.2 version ) . for this , images were captured with an olympus camera ( e420 , tokyo , japan ) fitted on an olympus microscope ( cx41 , tokyo , japan ) . a total of 100 cells per sample were scored for per cent tail dna , tail moment and olive tail moment as indices of dna damage . statistical analysis was performed using the statistical package for the social sciences ( spss , version 16.0 for windows 7 ) . differences between controls and patients for genetic damage indices were analyzed by the student 's t - test . demographic variables of patients and control groups were analyzed using the chi - square test . the association between dna / chromosomal damage and other variables was assessed by the pearson 's correlation test . the study proposal was approved by the institutional ethics committee and voluntary written informed consent was taken from all study participants ( n = 65 ) . patients with diagnosed cad were contacted from local hospitals through the cardiologists . exclusion criteria included cad patients with renal , liver or thyroid disorders , those with negative results on the treadmill test , and those not belonging to the punjabi population . the control group included healthy adults with no past or present history of heart or any disease and no exposure history , matched for gender , population group and socioeconomic status . the demographic and disease - related information for each patient was documented on a pre - designed questionnaire . anthropometric measurements ( height , weight , hip circumference , waist circumference ) were taken following standard methodology ( weiner and lourie , 1981 ) . derived obesity measures were body mass index ( bmi ) and waist hip ratio ( whr ) . using the criteria of who ( 2004 ) for bmi , the participants were classified as obese or non - obese regarding general obesity , and cut - offs as per snehalatha et al . ( 2003 ) for whr and waist circumference ( wc ) values for abdominal obesity . physiometric measurements ( using a mercury sphygmomanometer and stethoscope ) were taken after the subject had rested for ten minutes as per the recommendations of american heart association ( kirkendall et al . , 1981 ) . the average of three measurements were taken as the blood pressure values . the mean arterial blood pressure ( mbp ) was calculated from systolic and diastolic measurements ( prusse et al . , 1989 ) . venous blood ( 5 ml ) was then drawn from each participant and processed for the cbmn and scge assays . the cytokinesis - block micronucleus ( cbmn ) assay : - short term lymphocyte cultures were set up ( moorhead et al . , 1960 ) and the cbmn protocol of fenech ( 2000 ) was followed . briefly , the leukocyte - rich plasma ( 2 ml ) was added to 8 ml of rpmi 1640 medium ( himedia , india ) . the lymphocytes were stimulated to divide by addition of phytohaemagglutinin - m ( 0.5 g / ml pha - m , biological industries , israel ) . the cultures were incubated for 72 h at 37 c ; after 44 h cytokinesis was blocked by the addition of cytochalasin - b ( 6 g / ml , sigma ) and on completion of incubation of 72 h at 37 c , the cultures were harvested . following centrifugation ( 800 g , 10 min ) , the pellet was fixed in chilled fixative ( 3 methanol : 1 glacial acetic acid , qualigens , india and srl , india , respectively ) for 30 min . after centrifugation and two - three washings of the pellet , the cell suspension was used to prepare the slides . the air - dried slide preparations were stained with 5 % giemsa ( himedia , india ) for 10 - 20 minutes . for each sample , a total of 2000 binucleated cells ( 1000/slide ) were scored for the presence of micronuclei ( mn ) and micronucleated cells following the criteria of fenech ( 2007 ) . random observations by another scorer were done to verify the observations . the nuclear division index ( ndi ) , which is an index of cytostatic effects , was calculated ( eastmond and tucker , 1989 ) by using the formula : ndi = ( m1 + 2m2 + 3m3 + 4m4)/n where m1-m4 represent the number of cells with 1 - 4 nuclei and n is the total number of cells scored . the scge / comet assay : the trypan - blue dye exclusion test for cell viability was performed ( strober , 2001 ) and samples with > 95 % cell viability were processed for the comet assay . the leukocytic dna damage was ascertained using the alkaline comet assay ( singh et al . , 1998 ) . in brief , on pre - coated slides with 1 % normal melting point agarose ( nmpa ; srl , india ) , a second layer containing 30 l of blood sample mixed with 110 l of 5 % low melting point agarose ( lmpa , srl , india ) was poured followed by the sandwiching with a third layer of 120 l of 5 % lmpa after the second layer had set . cells were lysed in freshly prepared cold lysing solution ( 2.5 m nacl , 100 mm na2 edta , 10 mm tris hcl , ph 10.0 ; 1 % triton x-100 with 10 % dmso added just before use ) for at least one hour to allow dna unfolding . then the slides were placed in a horizontal electrophoresis chamber , covered with freshly prepared alkaline electrophoresis buffer ( 0.3 m naoh , 1 mm no2 edta , ph > 13 ) at 4 c for unwinding and expression of alkali - labile sites ( 30 min ) and subsequently electrophoresed ( 25 v , 300 ma , 0.7 - 1.0 -v / cm , 25 min ) . slide preparations were then washed with neutralizing buffer ( 0.4 m tris hcl ph 7.5 ) to remove alkali and detergents , and left for drying . the slides were silver stained ( nadin et al . , 2001 ) and coded . scoring was done using the freely available casp ( comet assay software programme , 3.1.2 version ) . for this , images were captured with an olympus camera ( e420 , tokyo , japan ) fitted on an olympus microscope ( cx41 , tokyo , japan ) . a total of 100 cells per sample were scored for per cent tail dna , tail moment and olive tail moment as indices of dna damage . statistical analysis was performed using the statistical package for the social sciences ( spss , version 16.0 for windows 7 ) . differences between controls and patients for genetic damage indices were analyzed by the student 's t - test . demographic variables of patients and control groups were analyzed using the chi - square test . the association between dna / chromosomal damage and other variables was assessed by the pearson 's correlation test . the demographic and clinical characteristics of patients and controls are presented in table 1(tab . 1 ) . cad patients had been diagnosed by the cardiologists on the basis of clinical symptoms , ecg findings , treadmill test and echocardiography . there were 26 males ( mean age 49.62 1.73 y ) and 20 females ( mean age 57.30 1.31 y ) in the patient group . the control group included 13 males ( mean age 53.54 1.17 y ) and 6 females ( mean age 55.67 1.33 y ) . on the basis of bmi , there were 14 obese patients ( bmi 25.0 kg / m ) while the whr revealed that 26 male and 20 female patients were obese as per the cut - off values given by snehalatha et al . ( 2003 ) . on the basis of the waist circumference cut - offs ( snehalatha et al . , 2003 ) , 23 male ( > 85 cm ) and 19 female ( > 80 cm ) patients had abdominal obesity . alcohol drinking was observed in 52.20 % male patients and in 31.60 % male healthy controls . non - vegetarian diet was preferred by 58.70 % of the patients and 52.60 % of the controls and sedentary life style was observed by 71.70 % cad patients and only by 36.80 % of controls . regarding medication in the patients group , 67.40 % were taking aspirin ( anti - inflammatory ) , 58.70 % lasix ( diuretic ) , 52.20 % metoprolol ( beta - blocker ) and 47.80 % were taking ramipril ( ace - inhibitor ) . socioeconomic status ( ses ) there were almost equal percentage of patients ( 15.20 % ) and controls ( 15.80 % ) belonging to upper ses . similarly , 76.10 % patients and 13.70 % controls belonged to upper - middle ses , while 8.70 % among patients and 10.50 % among controls had lower - middle ses . information about the history of disease revealed that date - of - onset / diagnosis ranged from 35 - 72 y ( average 53.09 1.3 y ) and the patients were on medication from 1 - 15 y ( average 5.04 0.6 y ) . on chi - square analysis it was observed that the demographic variables of patients and controls matched for age , gender , alcohol drinking , diet preference , socioeconomic status , whr ( men ) and waist circumference ( women ) , while bmi , diabetes , dyslipidemia , physical activity , whr ( women ) , waist circumference ( men ) , blood pressure , mbp and medication showed differences being characteristic of patients . in table 2(tab . 2 ) , the information on genetic damage indices in patients and controls is presented . the trypan - blue dye exclusion test had revealed 95.60 - 99.80 % cell viability of all samples . the comet parameters used as metrics of dna damage were tail moment ( tm = tail dna% x tail length in arbitrary units , au ) , tail dna% ( td = amount of dna in comet tail in percentage ) and olive tail moment ( otm = tail mean - head mean x % dna in tail in au ) . a comparison between patients and controls for these dna damage indices revealed that the values were significantly ( p < 0.001 ) higher in patients compared to those in controls . however no gender differences for these values were observed among patients and hence the respective data were pooled for genders in both , the control and patient groups . separate analysis also showed highly significant differences ( p < 0.001 ) for both , male and female patients from their control counterparts for dna damage indices . for the assessment of chromosomal damage , the binucleated cells with micronuclei ( bnmn ) , number of micronuclei in binucleated cells ( mn ) and the nuclear division index ( ndi ) were studied . statistical analysis revealed that the bnmn and mn values were significantly ( p < 0.001 ) higher and ndi values lower in patients than in controls . as observed for dna damage , no gender differences for chromosomal damage were observed among patients and controls , though values in male and female patients for bnmn and mn were significantly higher ( p < 0.001 ) in patients while ndi values were significantly ( p < 0.001 ) higher in controls . on correlation and association analyses ( table 3(tab . 3 ) ) , significant positive correlation ( correlation coefficient = r ) was observed between bnmn and diabetes mellitus ( r = 0.297 , p = 0.045 ) and between ndi and mda levels ( r = 0.296 , p = 0.045 ) and with age ( r = 0.571 , p = 0.000 ) . positive correlation was observed between tail dna % and diabetes mellitus ( r = 0.442 , p = 0.004 ) and with whr ( r = 0.349 , p = 0.017 ) and between tail moment and age ( r = 0.312 , p = 0.035 ) . on multiple linear regression analysis ( table 4(tab . 4 ) ) , regression coefficient ( b ) values for age ( b = 0.076 , p = 0.035 ) , whr ( b = 25.859 , p = 0.018 ) and diabetes mellitus ( b = 2.251 , p = 0.004 ) were observed to be independent predictors for dna damage and ndi for chromosomal damage ( b = 0.007 , p = 0.001 ) . in order to discern if there was any association between dna damage and chromosomal damage parameters , correlation analysis was carried out but it revealed no significant associations ( table 5(tab . 5 ) ) . as oxidative stress and lipid profile biomarkers of this study group had earlier been reported to be significantly higher among the patients ( bhat et al . , 2012 ) , it was also thought appropriate to ascertain whether there was any correlation ( table 3(tab . 4 ) ) of these variables with genetic damage . correlation and regression analyses were hence performed for genetic damage ( dna / chromosomal damage ) with oxidative stress parameters of malondialdehyde ( mda ) , total oxidant status ( tos ) , total antioxidant status ( tas ) and oxidative stress index ( osi ) and also with lipid profile biomarkers ( total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , triglycerides , very low density lipoprotein ( vldl ) and low density lipoprotein ( ldl ) . these showed neither correlation nor association with genetic damage except hdl - c and mda levels which showed positive correlation ( r = 0.322 , p = 0.029 ; r = 0.296 , p = 0.046 respectively ) and regression ( b = 0.259 , p = 0.029 ; b = 0.013 , p = 0.046 respectively ) with per cent tail dna and ndi respectively . it has often been reported that genetic damage may be caused by both , extrinsic and intrinsic environments ( gray and bennett , 2011 ) . the dna damage can manifest as dna strand - breaks , alkali - labile sites , incomplete repair sites and cross - links while chromosomal damage appears as chromosomal aberrations ( gaps , breaks , translocations , rings , insertions ) as well as micronuclei ( mn ) . mn originate from chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division ( fenech , 2000 ) . in the present study , the scge assay was used to measure the level of dna damage ( unrepaired ) and the cbmn assay to measure the level of chromosomal damage ( repaired ) in patients and controls . the results from the comet assay demonstrated that patients diagnosed with cad had higher values of percentage of tail dna ( 2.1x ) , tail moment ( 3 x ) and olive tail moment ( 3 x ) , indicative of higher dna damage in peripheral blood leukocytes of cad patients than the normal controls . there were no gender differences for these indices on statistical analysis implying that in this study group , genetic damage levels ( both dna and chromosomal ) are similar in males and females among patients and controls . the results of the present study have shown significant increase in unrepaired and repairable genetic damage in peripheral blood leukocytes / lymphocytes of patients with coronary artery disease . reports in literature have implicated excessive oxidative stress and inadequate defenses in the pathogenesis of cardiovascular disease ( andreassi , 2003 ) besides genetic damage in atherosclerotic plaques ( martinet et al . , 2002 ) . as oxidative stress has been implicated as a major contributor in the progression of atherosclerosis ( norman et al . , 2001 ) , it could also be a causative factor for dna damage in coronary artery disease as dna damage occurs frequently in cells exposed to reactive oxygen species ( botto et al . , the cad patients of the present study also had significantly increased ( 3 folds ) oxidative stress as given in an earlier work ( bhat et al . , 2012 ) . this oxidative stress hence could be contributing to the observed dna and chromosomal damage in cad patients . as dyslipidemia is an important causative factor for coronary artery disease ( arca et al . , 2007 ) , the patients of the present study had earlier been assessed for their lipid profile and had higher levels of triglycerides and total cholesterol and low levels of high density lipoprotein - cholesterol despite treatment ( bhat et al . , 2012 ) . the hdl - c and mda levels showed positive correlation with genetic damage on correlation and regression analyses implying that dyslipidemia also induced oxidative stress ( higher mda levels ) and could also be contributing to the observed genetic damage in these patients . in literature , various studies have demonstrated increased genetic damage in patients with heart disease . increased production of oxidant free radicals was observed in patients with ischemic heart disease ( logacheva et al . , 2001 ) and with evidence of oxidative dna damage in cells within the atherosclerotic plaque ( mahmoudi et al . , 2006 ) implying that genomic damage may itself be inducing cad . in their study on indian cad patients , rajesh et al . ( 2011 ) reported increased dna damage ( per cent tail dna ) while botto et al . ( 2001 ) had also reported increased dna damage levels in patients with coronary artery disease . ( 2005 ) had also documented that dna strands breaks , oxidized pyrimidines and altered purines were significantly higher in leukocytes of cad patients . the key advantage of the mn assay lies in its ability to detect both , clastogenic and aneugenic events , and to identify cells which have divided once in culture ( fenech , 2002 ) . the results of the present study showed that bnmn ( 1.5 fold ) and mn ( 1.6 fold ) values were significant increased in patients while ndi ( a biomarker of cytotoxicity ) was significantly higher in controls ( p < 0.001 ) than in patients implying that the cell proliferation was reduced while genomic damage was elevated in patients . increase in bnmn among cad patients in an italian population had also been earlier reported ( andreassi et al . , 2005 ) . the results of the present study are also supported by those of botto et al . ( 2001 ) who had observed approximately 5.6 folds increase in mn levels in cad patients while andreassi et al . the observed dna damage in patients of the present study could also have been contributed by the action of the medications prescribed to these patients namely , asprin ( anti - inflammatory ) , lasix / furosemide ( diuretic ) , metoprolol ( beta - blocker ) and ramipril ( ace - inhibitors ) . studies on genotoxicity of asprin have shown positive effects in bacillus subtilis assay ( kawachi et al . , 1980 ) and invitro in the chinese hamster lung cells ( ishidate , 1983 ) though negative effects have also been reported in the ames test ( mccann et al . , 1975 ) and in the chinese hamster ovary cells ( stich et al . , 1981 ( 2012 ) has reported dna strand breaks in mice hepatocytes detected by comet assay . chromosomal aberrations in chinese hamster ovary and lung fibroblast cells and in mouse germ cells have also been reported ( jameela et al . , 1979 ; bucher , 1989 ) , yet the genotoxic effects of furosemide are not well characterized ( bucher , 1989 ; brambilla et al . , 2006 ) . recently , on performing mutagenecity testing ( ames test , comet assay ) the effects of degradation products of metoprolol revealed positive genotoxicity ( sojic et al . . therefore the genetic - damaging effects of these drugs can not be ruled out . furthermore , the carcinogenicity of cardiovascular drugs , including diuretics , beta blockers , calcium antagonistics and ace - inhibitors has also been rebated upon ( grossman et al . , 1999 ) and requires further research . the significantly increased dna and chromosomal damage observed in the pbl of cad patients of the present study is of concern as genetic damage is an early indicator of malignancy ( andreassi et al . , 2007 ; manikantan et al . , 2010 ) and can also promote ageing and neurodegenerative diseases ( farooqui and farooqui , 2009 ; muralidhar et al . , 2012 ) . hence there is a need for preventive management for these aspects in patients with coronary artery disease . michael fenech ( csiro human nutrition , adelaide , australia ) for providing cytochalasin - b . the present study was carried out with support out of the departmental ugc - sap grant .

dna and chromosomal damage in peripheral blood leukocytes of patients with coronary artery disease ( cad ) were investigated by using the single cell gel electrophoresis assay /comet and cytokinesis- block micronucleus ( cbmn ) assays , respectively . the case - control study comprised patients with cad ( n = 46 ; average age 53.0 1.27 y ) undergoing treatment at local hospitals , and healthy age - and sex - matched controls ( n = 19 ; average age 54.21 0.91 y ) from the general population . the results of the comet assay revealed that the mean values of dna damage were significantly ( p < 0.001 ) higher in cad patients than in controls ( tail dna% 11.55 0.38 vs. 5.31 0.44 ; tail moment 6.17 0.31 vs. 2.93 0.21 au ; olive tail moment 3.52 0.23 vs. 1.25 0.11 au ) . the mean values of chromosomal damage were also significantly higher ( p < 0.001 ) in cad patients than in controls ( binucleated cells with mn- 28.15 1.18 vs. 18.16 2.59 ; micronuclei 29.52 1.21 vs. 18.68 2.64 , respectively ) while nuclear division index ( 1.48 0.01 vs. 1.63 0.01 ) was significantly higher ( p < 0.001 ) in controls . the results of the present study indicate that coronary artery disease patients had increased levels of both , unrepaired ( dna ) and repaired ( chromosomal ) genetic damage which may be a pathological consequence of the disease and/or the drug - treatment . this accumulation of dna / chromosomal damage is of concern as it can lead to the development of cancer with increased chances of morbidity and mortality in the cad patients .

Introduction Materials and Methods Subjects Data and sample collection Statistical analysis Results Discussion Acknowledgement

in the complex mechanisms of the functional regulation of genome , the spatial distribution of dna loci plays a key role . actually , the genome nuclear architecture deeply affects genes transcription status and can determine the success or the failure of several cellular processes [ 18 ] . a vital , nontrivial genome arrangement is the result of a number of interactions between distant dna loci which can pair and/or associate with nuclear scaffolding elements ( e.g. , membrane , matrix , etc . ) . a wealth of examples can be cited : from the homologous chromosomes recognition and pairing , necessary , for example , during meiosis [ 911 ] , to the clustering of genes around specific nuclear structures , such as the longstanding unresolved issues are the mechanisms whereby distant dna loci are able to cross - talk in order to achieve the right space configuration , and how the cell is able to organize in space and time such events . here , we set in a statistical mechanics framework the most recent discoveries about the molecular bases of such dna cross - talk phenomena . as a result , we show how it is possible to build a general , thermodynamic - based model , which answers the above questions . we refer to a well - established biological picture , whereby a dna locus needs the presence of soluble binding molecules to target another dna segment . starting from these experimental evidences , we analyzed a physics model describing the interaction between a couple of polymers and a concentration c of binding molecules in a lattice . we showed that these two ingredients say , a set of soluble molecules able to bind a number of dna sites are sufficient to enable a dna cross - talk , which eventually results in the colocalization of the dna loci . by means of extensive monte carlo computer simulations we dissected in fine details how the dna cross - talk is influenced by changes of some parameters , such as the binding molecules concentration , the dna binding sites number , and their relative affinity . we observed a threshold effect : the dna loci colocalization is possible provided that the above mentioned parameters pass specific threshold values . only in this case the dna loci stop their brownian diffusion and , as a result of a thermodynamic phase transition , pair off . while this rationalizes the experimentally proved importance for dna cross - talks of the molecular factors concentration and of the length of the relative dna attaching regions ( see and below ) , it suggests some possible regulatory mechanisms by means of well - described cellular strategies , like the upregulation of a dna binding molecules or the modification of chromatin structure . moreover , our analysis of the dynamics of dna colocalization , which is currently under investigation in several wet experiments , provides some interesting , testable results . in the following , before introducing our model in more details , we briefly report on some of the many experiments , which point out the special role played by precise dna segments and specific dna binding molecules for the dna cross - talks . protein - mediated chromatin loopsa variety of examples are known of chromatin loops , with fundamental regulatory roles , which are the result of molecule - mediated interactions between different dna loci . a naive t cell can differentiate into a th1 or a th2 cell depending on which locus among the ifng on chromosome 10 or the th2 on chromosome 11 becomes activated [ 14 , 15 ] . the cell maturation is accompanied by a change in the chromatin architecture within the above mentioned loci . in particular , on th2 cell activation , the chromatin at th2 locus adopts a trascriptionally active structure consisting of a series of small loops . these loops are realized thanks to satb1 protein , which is rapidly expressed upon the cell maturation and binds 9 sites within th2 locus . another example is the occurrence of two chromatin loops at kit gene , which are achieved through the contacts between different dna loci mediated by gata proteins . the loop able to activate kit is produced by the binding of gata-2 protein to the loci - situated + 5 kb and 114 kb with respect to the kit promoter . instead , gata-1 intervenes to mediate the interaction between the loci + 58 kb and + 5 kb in order to form the loop which downregulates kit . so , the control of the active / downregulated status of kit gene , essential for erythropoiesis ( the production of red blood cells ) , is thought to be accomplished through the regulation of the relative expression level of gata-1 and gata-2 proteins . a variety of examples are known of chromatin loops , with fundamental regulatory roles , which are the result of molecule - mediated interactions between different dna loci . a naive t cell can differentiate into a th1 or a th2 cell depending on which locus among the ifng on chromosome 10 or the th2 on chromosome 11 becomes activated [ 14 , 15 ] . the cell maturation is accompanied by a change in the chromatin architecture within the above mentioned loci . in particular , on th2 cell activation , the chromatin at th2 locus adopts a trascriptionally active structure consisting of a series of small loops . these loops are realized thanks to satb1 protein , which is rapidly expressed upon the cell maturation and binds 9 sites within th2 locus . another example is the occurrence of two chromatin loops at kit gene , which are achieved through the contacts between different dna loci mediated by gata proteins . the loop able to activate kit is produced by the binding of gata-2 protein to the loci - situated + 5 kb and 114 kb with respect to the kit promoter . instead , gata-1 intervenes to mediate the interaction between the loci + 58 kb and + 5 kb in order to form the loop which downregulates kit . so , the control of the active / downregulated status of kit gene , essential for erythropoiesis ( the production of red blood cells ) , is thought to be accomplished through the regulation of the relative expression level of gata-1 and gata-2 proteins . x chromosomes dynamic arrangement during x - chromosomes inactivationan even more striking event , which entails the organization of two whole chromosomes , happens during x - chromosomes inactivation ( xci).xci is one of the most mysterious aspects in current mammalian x biology . it is the phenomenon occurring in the female mammalian cells , which leads to the silencing of one of its x chromosomes randomly chosen , to equalize the dosage of x products with respect to males , having only one x [ 1720 ] . the x chromosomes cross - talk and their spatial arrangement is fundamental for the success of xci.at the beginning of process , the xci regulatory sequences on the x 's couple , called xic ( x - inactivation center ) , have to be juxtaposed [ 21 , 22 ] . the interaction allowing this colocalization is mediated by a protein - rna bridge which includes ctcf protein , able to bind a cluster of about 50 sites in the tsix / xite sequence within xic . the random choice of the x to be inactivated follows and , then , while the active x moves to the nuclear membrane , the inactive x associates with the nucleolus , to maintain its silenced status . an even more striking event , which entails the organization of two whole chromosomes , happens during x - chromosomes inactivation ( xci).xci is one of the most mysterious aspects in current mammalian x biology . it is the phenomenon occurring in the female mammalian cells , which leads to the silencing of one of its x chromosomes randomly chosen , to equalize the dosage of x products with respect to males , having only one x [ 1720 ] . the x chromosomes cross - talk and their spatial arrangement is fundamental for the success of xci.at the beginning of process , the xci regulatory sequences on the x 's couple , called xic ( x - inactivation center ) , have to be juxtaposed [ 21 , 22 ] . the interaction allowing this colocalization is mediated by a protein - rna bridge which includes ctcf protein , able to bind a cluster of about 50 sites in the tsix / xite sequence within xic . the random choice of the x to be inactivated follows and , then , while the active x moves to the nuclear membrane , the inactive x associates with the nucleolus , to maintain its silenced status . on the basis of the experimental evidences , we built a schematic model for dna cross - talk , including a couple of dna segments endowed with a number of binding sites ( bss ) for a set of molecular factors ( mfs ) ( see figure 1 ) . for sake of simplicity , dna segments as well as mfs move in a lattice whose spacing is d0 , of the order of magnitude of the bs length ( see section 4 ) . the lattice dimensions are lx = 2l , ly = l and lz = l in units of d0 . they are formed by l nonoverlapping beads , each included in a single lattice site , which randomly diffuse under the constraint that two proximal beads must be on next or nearest next neighboring sites ( nonbreaking constraint ) . in each dna locus , a number n0 of beads are bss . mfs are represented by brownianly diffusing beads , and , to a first approximation , only a single mf at a time can be present in a lattice site . when an mf and a polymer bs are in neighboring sites , they interact via an effective energy e ; mfs can bind a couple of bss from both dna segments at the same time ( in resemblance of several known mediating proteins , e.g. , ctcf ; see figure 1 ) . the energy function of the system is the following : ( 1)=er,rnb(r)nm(r ) , where nb(r)=0,1 is the occupation variable of bss ; it is 1 if the lattice site at position r is occupied by a bs ; otherwise it is 0 ( a site can not be occupied by more than one kind of particle ) . the sum runs on all nearest neighboring lattice sites r and r. we studied equilibrium and dynamic features of this system by means of monte carlo ( mc ) computer simulations . our schematic model does not include all the complex dna regulatory phenomena taking place within the cell nucleus . however , the cross - talking mechanism we envisaged here is general , since it grounds on thermodynamic bases ( see next sections ) , and for this reason it is not affected by the complexity of the model . thus , while the approximations we use do not have any influence on cross - talking efficiency , they allow us to explore a wide range of parameters combinations , without the drawback of computational unfeasibility . as for the parameter values , we chose weak biochemical energy values for e ( ~0/5kt ) , typical for dna - protein interactions [ 2528 ] . the mf concentration , c , ( expressed as the ratio between the mfs number and the lattice sites number ) , spanned the range 0.01/1% , which corresponds to typical nuclear protein concentration ( see section 4 ) . the binding sites number n0 is 24 ( i.e. , of the order of magnitude of the known case of ctcf binding sites in tsix / xite ) ; however , we also varied it to verify the bss deletion effects ( see figure 4 ) . the lattice we considered has l = 32 and periodic boundary conditions ( see section 4 for further details ) . time is given in units of monte carlo lattice sweeps ; details about the conversion from mc time to real time are given in the section 4 . all the following results are averaged over up to 500 runs . when an mf concentration c is present within the lattice and the polymers have n0 bss each , an effective attraction is induced between the couple of polymers , whose intensity depends on the value of the mf - bs interaction energy e. this is shown by the normalized mean square distance between the polymers , defined as ( 2)d2(t)=1n0z=1n0r2(z , t)l2 , that is , the square distance between two beads at the same height z , r(z , t ) , averaged over the n0 bss and over different realizations ( as the symbols indicate ) . l is used for normalization . in figure 2 the plot of d(t ) with c = 0.2% and n0 = 24 for two values of the energy e is illustrated . at t = 0 the mfs were randomly placed within the lattice sites , and the polymers were in their straight vertical configuration , at a distance l from each other ( so d(0 ) = 100% ) . it is evident that the increase of e from 1.4 kt to 2.2 kt determines very different equilibrium states for the system : while at lower energy , d saturates at the value expected from a couple of independent random walk polymers ( d ~ 40% ) ; when e = 2.2 kt , d eventually goes down to ~0 , thus revealing that the effective attraction is now high enough to make dna loci pair off . d(t ) shows an initial linear behaviour , determined by the random diffusion of the polymers when they are far from each other , and a final exponential saturation to a plateau which is dependent , as we saw , on the values of e. a good fit function , which includes both the above mentioned time regimes , is the following ( superimposed fits in figure 2(a ) ) : ( 3)d2(t)=d2()+[d2(0)d2()+at1+bt]et/ , where d( ) is the final equilibrium value , while a , b , and are fit parameters which depend on e , c , and n0 . to get more insight into the dynamics of this cross - talk mechanism and into the key role of the molecular factors , we monitored the time behaviour of the average fraction f of the mfs attached to a single dna locus . the role of mfs as mediators of the dna loci interaction clearly emerges in figure 2(b ) , where the plot of f(t ) , for the same values of the parameters used above , is shown together with d(t ) fit functions . for both the energies e , f(t ) starts from the same value , due to the initial random distribution of the mfs in the lattice sites . when e = 1.4 kt , f(t ) simply saturates in a time scale which is about 4 orders of magnitude smaller than that of d(t ) ; actually , mfs have a size much smaller than polymers , so their dynamics is much faster . if the energy is increased to e = 2.2 kt and the dna loci pair off ( d saturates at ~0 ) , a more interesting time evolution is found . in fact , now , two time regimes are easily distinguished : during the first ~0.5 minute , f(t ) has a behaviour similar to that in the previous case , except for the higher plateau due to the higher mf - bs affinity . this first time regime corresponds to the initial mf binding to the dna loci which , still out of the action range of the mf - induced effective potential , independently diffuse . yet , just when d begins to exponentially fall down to ~0 , f(t ) rises to a second plateau : as soon as dna loci , during their random diffusion , get closer , some mfs start to stably bind both of them , and , as a result , f(t ) increases . this is evidenced in figure 2(c ) , showing that quite all the bound mfs actually form bridges between the dna loci after ~1 minute . keep together the two dna loci , so that pairing is finally produced ( see figure 1 ) . the f(t ) analysis , while allows an immmediate distinction between two dynamical regimes , makes the mf mediating role evident : we saw that colocalization takes place as a result of mf bridges realization , which , however , occurs at thermodynamic equilibrium only for certain values of the parameters . to better understand this issue , we now carry out an analytic , though approximate calculation for the pairing probability p. for sake of simplicity , we considered the two dna segments close to each other in their straight configuration . if a random distribution of a concentration c of mfs is presumed , a number cn0 of them will be present , on average , between the n0 bss of the dna loci . for each of these mfs two states the energy equal to 2e corresponds to the former case , while the energy is 0 in the latter . thus , according to the canonical distribution , p = exp(2e / kt)/(1 + exp(2e / kt ) ) and q = 1/(1 + exp(2e / kt ) ) are , respectively , the probability that a single mf bridge is formed or not . we can define the pairing probability p as the probability that at least one mf bridge is built between the dna loci . since cn0 is the total number of the possible mf bridges , the following formula holds : ( 4)p=1(q)cn0=11[1+exp ( 2e / kt)]cn0 according to which p increases with ( c , e , n0 ) . while this calculation , in its simplicity , helps understanding how the mfs presence induces an effective attraction at thermodynamic equilibrium which is stronger with higher ( c , e , n0 ) , in the following section we seek for the exact thermodynamics of the system by means of mc simulations . what emerged from mc simulations is that , even if the intensity of the effective potential always increases with ( c , e , n0 ) , the system reacts to a change in one of these parameters only if a threshold value is crossed : below this threshold , dna loci are independent , above they colocalize , as a result of a thermodynamic phase transition [ 13 , 31 ] . in fact , as we know from thermodynamics law , the equilibrium state of a system must correspond to a free energy minimum : so , the dna loci form a thermodynamic stable couple only when the energy gain coming from the mf bridges is able to compensate the entropy loss which results from colocalization . however , in our finite size system , a narrow intermediate regime , where dna loci couples are unstable , is found as well ; the existence of such a regime is indeed typical for phase transitions in finite system . figure 3 shows the thermodynamic equilibrium value of the mean square distance d figure 3(a ) and of the fraction of colocalized dna loci , p ( figure 3(b ) ) , as function of the interaction energy e ( here we fixed c = 0.2% and n0 = 24 , as before ) . while for e e * = 1.75 the random values for d and p were measured ( d ~ 40% and p ~ 0% , orange horizontal lines ) , as soon as e e * , d suddenly falls down to 0% , and , correspondingly , p increases to 100% ( blue horizontal lines ) . in the crossover region around e * ( defined by the criterion p(e * ) = 50% ) , intermediate values for p and d are found , since dna loci couples are continuously formed and disrupted . as we explain at the beginning of this section , in the thermodynamic limit ( i.e. , with infinitely large system ) , e * would mark the transition point between two phases ( a brownian phase below it , a colocalization phase above ) , and a power - law behaviour would be found around e * , for the order parameters d(e ) and p(e ) . we fitted the quantitative data from the simulations , and we observed that power - law functions are also compatible with p(e ) and d(e ) for e e * in our finite - sized system ( see dashed gray lines in figures 3(a ) and 3(b ) ) : ( 5)d2(e)=drand2(ee0ed2),p(e)=(ee0ep) , where drand 40% , e0 = 1.7 kt , ed = 1.31 kt , ep = 0.16 kt , and = 0.43 . since , in a cellular process , the time needed to accomplish each task is also a very important factor , we studied the behaviour of ( see formula ( 3 ) ) , which represents the characteristic time scale to reach the thermodynamic equilibrium ( and colocalization , above threshold values of ( c , e , n0 ) ) as function of e ( figure 3(c ) ) . an increasing behaviour is observed due to the slower dna loci diffusion when the mf - bs bonds get stronger . the jump at e * signals the phase transition occurrence . for fitting we used power - law functions ( superimposed gray lines ) , since this is the expected behaviour for the system relaxation time near a transition point , in the thermodynamic limit . however , in our finite - sized case , we found that exponential functions can fit the data as well . we also varied all the three parameters ( c , e , n0 ) , in order to find the 3d phase diagram which is plotted in figure 4 . the yellow circles mark the transition points between the brownian and the colocalization phase . it is shown that , as predicted by the approximate calculation of p ( see ( 4 ) ) , the intensity of the effective attraction depends as well on mf concentration c and bs number n0 ; hence , dna loci colocalization is also triggered when threshold values in c and n0 are passed . a power - law surface fits the data well ( see ): we found cen0 cost with = 4 and = 1.1 . we can conclude that colocalization is possible only if a triplet of parameters ( c , e , n0 ) which is above this surface is given . we presented a very general mechanism which can account for different phenomena involving spatial chromatin organization . the mechanism we envisaged can produce the association between two dna loci , without the intervention of any molecular motor able to actively move them ( e.g. , actin / myosin system ) , since the energy needed is provided by the surrounding thermal bath . the only required ingredients are those emerged in several experiments ( see section 1 ) : say , the presence of diffusive molecules ( molecular factors , mfs ) which are able to bind specific sites ( binding sites , bss ) within the dna loci ( figure 1 ) . we carried out a detailed dissection of a schematic but quantitative model , both in its thermodynamic and dynamical aspects , by means of extensive monte carlo computer simulations . we found that the dna loci cross - talk and the consequent colocalization are enabled only when the mf concentration c , the bs number n0 , and the mf - bs interaction energy e are above specific threshold values , satisfying the relation cen0 = cost ( = 4 and = 1.1 ) . one important consequence of this result is that a cell can trigger or release pairing , for example , simply by upregulating / downregulating the mf production ( i.e. , by tuning c ) or by means of chromatin modification ( i.e. , by changing e ) . moreover , while these threshold effects can explain the results of several experiments ( e.g. , the importance of mediator concentration , see section 1 and [ 13 , 31 ] ) , they are prone to be quantitatively tested , for example , by means of bs deletions . actually , for the first time , we introduced a model which describes such phenomena in a quantitative way : we are able to predict the likelihood of pairing and its probability distribution , once the controlling parameters ( c , e , n0 ) are given . understanding the pairing dynamics is interesting , especially because several experiments are currently investigating it . importantly , we measured the time scale needed to reach full colocalization , as function of the system parameters . we showed that (e ) is an increasing function ; analogously , it can be shown that always increases with c and n0 as well , for the same physical reasons ( see above and [ 13 , 33 ] ) . this results in a nontrivial , counterintuitive prediction : if one ( or more ) of the control parameters ( c , e , n0 ) is reduced ( e.g. , n0 could be reduced by dna deletions , e by chemical manipulations ) at a level still above the threshold for pairing , pairing not only would occur , but it would be speeded up . we think that the general mechanism we envisaged here , with its thermodynamic robust roots , can apply to lots of cellular processes involving dna loci spatial organization ( e.g. , meiosis , xci , chromosome territories , etc . ) and can mark very relevant progress in their comprehension . for mc computer simulations we used a lattice with l = 32 , and so with dimensions lx = 64 , ly = 32 , and lz = 32 , in units of d0 , the lattice spacing constant . in this lattice , we represented dna segments as directed polymers . these choices allowed faster simulations , though they do not affect the results we discussed : if such constraints are released , the free energy minimization , above specific thresholds in ( c , e , n0 ) , would determine the dna loci colocalization as well . by referring to the known case of ctcf binding in tsix / xite region on x chromosome [ 22 , 29 ] , we assume that the order of magnitude of d0 corresponds to the linear dimension of 20 bp ( the length of a ctcf binding site ) . however , we also checked that our results remain essentially unchanged in larger lattices ( l as large as 128 ) and with longer dna loci ( up to 128 beads ) . in the implementation of mc stochastic dynamics , we considered the probability for a particle to move to an empty next neighboring site , proportional to the arrhenius factor exp(/kt ) , being the energy change due to the move , k the boltzmann constant and t the room temperature [ 30 , 32 ] . to measure p , the fraction of colocalized dna loci colocalized two dna segments at a distance less than the 10% of the lattice linear size l. a rough calculation can be made to estimate the molecular concentration in real nuclei , corresponding to the mf volume concentrations c. since in our model the number of molecules per unit volume is c / d0 , the molar concentration is = c/(d0a ) , where a is the avogadro number . provided that d0 10 nm ( see above ) , we obtain that the typical nuclear protein concentration , ~ 1 mol / l , corresponds to c ~ 0.1% . recent experiments have proved that human dna loci , while at long times , show a constrained motion , brownianly diffuse at short times . the order of magnitude of their typical short time diffusion constant is d = 1 m / h . for sake of simplicity , we took the diffusion constant of a free polymer ( i.e. , with e = 0 ) in our lattice equal to d ; this gives the conversion factor from mc unit time to real time ( 1 monte carlo lattice sweep 30 microseconds ) .

the recognition and pairing of specific dna loci , though crucial for a plenty of important cellular processes , are produced by still mysterious physical mechanisms . we propose the first quantitative model from statistical mechanics , able to clarify the interaction allowing such dna cross - talk events . soluble molecules , which bind some dna recognition sequences , produce an effective attraction between distant dna loci ; if their affinity , their concentration , and the relative dna binding sites number exceed given thresholds , dna colocalization occurs as a result of a thermodynamic phase transition . in this paper , after a concise report on some of the most recent experimental results , we introduce our model and carry out a detailed in silico analysis of it , by means of monte carlo simulations . our studies , while rationalize several experimental observations , result in very interesting and testable predictions .

1. Introduction 2. Results and Discussion 3. Conclusions 4. Materials and Methods

the influenza pandemic of 19181920 was the most lethal natural event in recorded human history . within a few months , the influenza a / h1n1 pandemic spread worldwide and killed 4080 million people . , during the pandemic , influenzarelated mortality increased in all affected populations ; however , it markedly varied across locations and among demographic subgroups at specific locations . for example , during the pandemic , previously healthy young adults had relatively high mortality risk ; the unprecedented wshaped relationship between mortality and age remains unexplained . also , during the pandemic , influenzarelated mortality rates were extraordinarily high in some island and other isolated populations ( e.g. , arctic ) . this has led researchers to believe that mortality was because of a lack of crossprotective immunity provided by prior influenza virus strains . the analysis of isolated communities , and specifically demographic subgroups within those communities , can help to understand the outcome of infection . mortality differed by more than 10fold across apparently similar island populations and more than 30fold across demographic subgroups of specific island populations . the 1918 influenza a / h1n1 virus was efficiently transmitted but not inherently or inevitably lethal . most individuals who were infected with the pandemic strain had unremarkable clinical courses with complete recovery . nearly all pandemicrelated deaths were because of postinfluenza bacterial pneumonias rather than direct pathologic effects of the virus . in the absence of antivirals , antibiotics , mechanical ventilation , and intensive medical care , significant differences in mortality among various island populations were unlikely related to differences in medical care . also , because most epidemics on isolated islands were caused by point source introduction of the pandemic strain ( e.g. , via the arrival of a single ship ) , it is thought that nearly all humans were immunologically susceptible to infection with the pandemic strain in 1918 . the wide ranges of mortality across islands and across subgroups on the same islands suggest that the number and nature of prior respiratory infections ( with influenza viruses and other respiratory infectious agents ) may have significantly influenced clinical expressions and outcomes of infections with the pandemic strain . we hypothesize that exposures to few , and a limited diversity of , respiratory infectious agents prior to the 19181920 influenza pandemic enabled extremely high mortality in isolated communities , particularly islands , during the 19181920 influenza pandemic . for this report , we examined the influenza mortality records from several island communities , both civilian and military , to assess the relationships between prior influenza and other respiratory infectious disease experiences and mortality during pandemicrelated epidemics in 19181920 . data sources consisted of administrative , military , medical , and historical records , including official public health reports , mortality registries , commissioned reviews , and publications in medical journals during and following the 19181920 influenza pandemic period . some of the reports and original data were drawn from unpublished sources that are not archived in readily searchable systems such as pubmed ; thus , a systematic search strategy was not used to identify the useful and relevant data sources . lists of references and bibliographies were reviewed to identify the reviews , registries , and reports published in english or french that recorded prospectively collected data from island populations that documented pandemicrelated mortality and numbers and characteristics of island residents . on most islands , the numbers of island residents who died during influenza pandemicrelated epidemics were specifically reported ; it was not always possible , however , to determine the underlying causes of the deaths , for example , influenza versus all respiratory deaths ( including pneumonia and influenza ) . because of the massive social , military , and political disruptions during the first world war , the numbers of island residents during the pandemic period are necessarily estimates . to enable comparisons across islands and demographic subgroups , all mortality rates are presented as percentages . during pandemicrelated influenza epidemics from 1918 to 1920 , pneumonia / influenzarelated mortality rates on various islands were estimated as australia ( 04% ) , hawaii ( 04% ) , new zealand ( 07% ) , fiji ( 5% ) , mauritius ( 55% ) , tonga ( 64% ) , guam ( 66% ) , french polynesia ( 16% ) , nauru ( 18% ) , and samoa ( 22% ) . , , , , , , , , , thus during the period , respiratory illness related mortality across pacific islands varied by more than 50fold ( range , 0422% ) . on fiji mortality was 5% among residents overall , but varied in relation to raceethnicity from 14% among europeans to 57% among fijians ( table 1 ) . the colonial government of the time interpreted these figures through a racial lens seeing them as an indication of the relative fitness of different races in a social darwinian sense . it seems much more likely that epidemiological isolation was the differentiating factor between those with the highest mortality rates ( e.g. , fijians ) who had lived the greater part of their lives on small pacific islands and the lower mortality immigrants from both india and europe who had been exposed to a much larger number and wider range of respiratory pathogens , especially in childhood prior to immigration . variable civilian mortality in various island populations during the 19181920 influenza pandemic grouped by island and ethnicity using published and archival material , we were able to investigate the implication of isolation on responses to the 1918 influenza pandemic more closely . for example , although mortality was relatively low on hawaii compared with other pacific islands , it was higher among hawaiian ( 11% ) than caucasian and chinese ( 03% ) island residents ( table 1 ) . in 1918 , the crew / passengers of a us army transport ship were infected with influenza in the philippines and carried it to the isolated island of guam . during the ensuing epidemic , , in 1964 , another pandemicrelated epidemic affected the still very isolated western caroline islands . during the epidemic , there were many deaths , including among the adults , especially on the most isolated atolls ( e.g. , mortality % , by island : ulithi , 1% ; woleai , 3% ; ifaluk , 65% ) . during 1964 pandemicrelated epidemics on saipan and guam , there was no significant influenzarelated mortality . prior infection of the latter in 1918 and thereafter protected them against mortality from the 1964 pandemic strain . the benefit of exposure to diverse respiratory pathogens and/or environmental antigens is also observed when residents of isolated island communities were recruited into the us , french , and uk armed forces . , , the influenzarelated mortality experiences of islanders in military service are informative to the extent that they varied from the experiences of their counterparts who remained on their home islands . as expected , us soldiers serving in the philippines , hawaii , and puerto rico experienced lower influenzarelated mortality rates compared with their locally recruited counterparts . ( table 2 ) of interest though , mortality rates were generally lower among recruited philippino , hawaiian , and puerto rican soldiers than their civilian counterparts on the respective islands particularly if the soldiers were seasoned ( e.g. , had completed recruitment training and were not new in their current assignments ) . , the differences in the mortality risk from influenza among the individuals of the same ethnicity suggest that epidemiological factors in addition to host ( e.g. , genetic ) factors were significant determinants of risk of lethal outcomes after influenza illnesses . influenza mortality rates in us , french , and uk military units either operating in or coming from island areas during 19181919 pandemic this is supported by the data acquired for polynesian and melanesian soldiers recruited into the french army who experienced relatively low influenzarelated mortality while working in labor battalions in france ; there was a nearly 50fold difference in mortality between them and their civilian counterparts in french polynesia . ( 1 , 2 ) the distinguishing epidemiological factor in these examples is military service in europe which would have certainly exposed the soldiers to a high level of respiratory infections prior to 1918 . the experiences of new zealand and australia ( that were relatively isolated at this time ) differed in regard to the timing ( separated by 4 months ) and severity of their respective influenza epidemics . despite considerable historic and cultural similarities , influenzarelated mortality was twice as high among the residents of new zealand as australia , and on new zealand , mortality rates were much higher among the polynesian maori ( 42% ) than among the caucasian immigrants ( 06% ) . in the fall of 1918 , large numbers of soldiers from australia and new zealand were engaged in the first world war in europe . during the fall winter influenza epidemic period , caucasian soldiers from australia and new zealand had similar influenzarelated mortality rates ( approximately 07% ) ; however , mortality in the new zealand army s maori battalion was approximately four times higher nearly the same as among maori still in new zealand . , , ( table 2 ) the higher susceptibility of the maori population may reflect their relatively higher isolation prior to joining the military . the effects of isolation on responses to infection are not restricted to the 1918 pandemic influenza strain . in 1918 , the densely populated highlands of new guinea were unknown to the outside world . because of the rigid quarantine of australia , it is likely that the 1918 influenza pandemic did not affect new guinea . in 1964 , influenza outbreaks in isolated new guinea highland areas produced high mortality , particularly among the adult males . furthermore , through 1996 , isolated highland groups in the indonesia province of papua experienced high mortality during epidemics caused by influenza . during the 19181920 pandemic period , , , in the 19th century , the samoan islands were partitioned into western and american samoa ; the two geopolitical entities had ethnically identical populations . during the influenza pandemic period , more than onefifth of all residents of western samoa , but no residents of american samoa , died the survival of american samoans reflected the effectiveness of a maritime quarantine that was instituted by the military governor in pago pago ( table 1 ) . , quarantines of isolated islands can delay but not permanently prevent the introductions of pandemic influenza virus strains . in 1926 , a less virulent form of influenza arrived in american samoa ; during the epidemic , 25% of the residents became ill but < 01% died in sharp contrast to the experience on western samoa in 1918 . during the first world war , a new zealand battalion of mostly caucasian soldiers felt to be unfit for duty in europe occupied samoa . when influenza arrived in samoa , mortality was approximately 10 times higher among samoan residents than among the new zealand soldiers who were stationed there . many new zealand soldiers on samoa had close contact with the island residents , for example , during disaster relief and burial duties . of note , mortality rates were similar among the new zealand soldiers on samoa and those in recruit camps in new zealand ; however , mortality was much higher among the new zealand soldiers in recruit camps than among the civilians in communities adjacent to the camps . , , ( table 2 ) when a group of new zealand soldiers bound for europe encountered influenza at a west african port , 7% died on board the ship or shortly after landing in the uk . the experiences of new zealand soldiers overall suggest that their mortality risk during the 1918 pandemic period was not exclusively determined by their ethnic backgrounds but was related to prior respiratory infection experiences ( e.g. , isolation of their home communities ) as well as the epidemiological contexts in which their influenza infections occurred . during the pandemic period , there were large mortality differences in relation to the ethnicities of industrial workers on small pacific islands . for example , pacific islander ( from marshall and caroline islands ) and chinese laborers were employed on the phosphate island of nauru . when pandemic influenza arrived in 1920 , the mortality rate was very high among the pacific islanders , intermediate among the native nauruans , and very low among the chinese workers . ( table 1 ) the mortality experience on nauru in relation to ethnicity in 1920 was also reflected during a less lethal influenza epidemic in 1948 on ocean island , another phosphate island with contract mining workers where only the ellice islanders ( 17% ) and no chinese or europeans died from influenza . racial / ethnic group is thus very unlikely to be a key mortality determinant but probably is a surrogate for number and range of previous respiratory infections prior to arrival on nauru or ocean island . in some ways , the example of saipan can be seen as the exception that proves the rule . distinct ethnic groups on the same island had very different mortality rates possibly due to the differences in their isolation during the previous pandemic in the 1890s . in 1919 , a japanese ship introduced pandemic influenza to saipan in the northern mariana islands . the chamorro and caroline islander residents of the island lived in close proximity to each other in adjacent communities . during the pandemicrelated epidemic on the island , there was more than a 30fold difference in mortality among the chamorros and caroline islanders . ( table 1 ) saipan and guam are separated by 120 miles . in 1919 , chamorro people lived on both islands ( many had migrated to saipan from guam early in the 20th century ) . caroline islanders had migrated to saipan in the 19th century following their displacement from truk . , the influenza pandemic of 1890 likely spared the relatively isolated saipan which at the time had no regular maritime contact with guam . we were unable to find any record of influenzalike infection in this isolated population before 1919 , but surmise that the influenza epidemic of 1919 was likely the first exposure to influenza for most caroline islanders on saipan . in contrast , many chamorros on saipan had probably been exposed to influenza ( prior to 1900 ) while living on guam . during the 1919 epidemic on saipan , mortality was relatively low among the caroline islanders ( possibly during first lifetime infections with influenza virus ) but very high among the chamorros ( likely during second ever infections with influenza ) , suggesting that previous exposure to a particular respiratory pathogen is not always beneficial . pandemic influenza was introduced to french polynesia when a ship from california landed in tahiti . during the ensuing epidemic , > 16% of the total population died ; however , mortality varied across the islands from 119% on maketea to 178% on tahiti . new caledonia , another french colony in the pacific , escaped influenza entirely in 19181920 ; protection from the pandemic was attributed to the strict quarantine of ships leaving from australia . when influenza did arrive in new caledonia in july 1921 , a large proportion of the population was infected , but there were very few deaths as on american samoa in 1926 . in this review of experiences during the 19181920 pandemic period , influenza pneumoniarelated mortality varied by more than 50fold during pandemicrelated epidemics on various pacific islands ; mortality seemed correlated with the island s degree of contact with outside epidemiological sources as marked by the number of steamships arriving in the major island ports . in the early 20th century , islands were connected to the outside world by transoceanic steamships ( which travelled at < 14 knots ) ; obviously , the interconnectedness of island communities has enormously changed since then . the extreme globalization that occurred in the last century has enabled continuous circulation of antigenically diverse influenza viruses and other respiratory microbial agents ; hence , the epidemiological conditions that set the stage for the extraordinarily lethal 19181920 pandemic are unlikely to recur . other aspects such as socioeconomic status , along with living and environmental conditions , may have also played a major role in populationlevel mortality . in 1918 , the most isolated island populations may have had more virulent clinical expressions of influenza because their lung microenvironments were not well conditioned by prior inflammatory events . ( figure 1 ) epidemiological and animal model data demonstrate that infection with a respiratory pathogen alters subsequent immune responses to unrelated respiratory pathogens ; the effect is long lasting and does not depend on t and b lymphocytes . the alteration may arise from airway remodeling of damaged tissues , alteration or redistribution of lymphatics and vascular tissues , modification of epithelial cells , and/or resetting of the activity of innate immunity . islanders with a narrow range of exposures to respiratory infections prior to 1918 would have experienced the 1918 influenza virus with lungs qualitatively different from those who had had multiple respiratory infections prior to 1918 ( e.g. , military service veterans ) . the impact of infection exposure on the development of pathology during the 1918 influenza pandemic . if crossreactive t cells are present , they may be overrepresented ( a ) . in the absence of crossreactive antibody should specific t cells be present , they expand exponentially because other infections have not adjusted their memory clone size . nk cells are present , and airway macrophages are blunted in their responsiveness . during a subsequent influenza infection ( d ) , the whittled down crossreactive and bystander t cells enter the lung ( 2 ) but are exposed to a reduced inflammatory microenvironment because airway macrophages are less responsive ( 3 ) and memory nk cells have already reduced the viral load ( 4 ) . furthermore , lung remodeling ( 5 ) by prior infection has made the lung more resilient . repeated infections with antigenically diverse respiratory agents continuously alter and further partition the memory tcell pool ; as a result , no single specificity is overrepresented among the memory t cells . overrepresentation of a memory tcell clone may not benefit the host when reexposed to the cognate tcell epitope . for example , when cd8 + t cells lyse virally infected cells , they produce abundant quantities of proinflammatory cytokines with characteristic clinical manifestations , for example , cachexia , fever , vascular leakage , and respiratory failure . successive infections may alter the relative proportions of each memory tcell clone in an attrition process . there is limited space to accommodate memory t cells , and diverse respiratory infections progressively decrease preexisting tcell populations . a richly diverse tcell repertoire , as in persons with many previous respiratory infections , could lead to a smaller clonal burst size with less immunopathologic effects . the human immune system evolved when most people lived in small family / tribal groups in the context of social isolation . relatively recent social , political , economic , and cultural changes increased the mixing of human populations with different infectious disease experience on both the individual and population levels . thus , it is not surprising that there is a period of epidemiological adjustment once an isolated area becomes connected to new pools of infectious disease pathogens and human populations . for example , the linking of dense urban populations by trade routes enabled continuous chains of transmission and great epidemics of measles and smallpox . industrialization and warfare accelerated and enhanced the process . during the 2009 a / h1n1 pandemic , mortality was relatively high among the individuals who were pregnant , obese , or otherwise immunocompromised ( e.g. , chemotherapy ) ; however , the magnitudes , natures , and distributions of mortality during the 2009 and 1918 pandemics differed enormously even though both pandemics were caused by antigenically similar a / h1n1 influenza viruses . the influenza pandemic of 1918 remains a unique event with several unexplained clinical and epidemiologic characteristics ; the findings of this and other historical reviews suggest that the extreme mortality associated with the 1918 pandemic is unlikely to recur . more detailed and comprehensive understanding of the pathophysiologic mechanisms ( e.g. , immunopathogenicity , virusbacterial interactions ) that enabled the extreme mortality of the 1918 pandemic would inform preparations for and responses to future pandemics . the global emerging infectious disease and response system ( geis ) , which is part of the armed forces health surveillance center of the us department of defence , provided funding for this project . the opinions expressed are those of the authors and do not necessarily reflect those of the australian defence force or the us department of defense . gds conceived the epidemiological study , gathered the data , assisted in the analysis , and wrote the first draft of the manuscript . th and jb

please cite this paper as : shanks et al . ( 2012 ) epidemiological isolation causing variable mortality in island populations during the 19181920 influenza pandemic . influenza and other respiratory viruses 6(6 ) , 417423 . background during the 1918 pandemic period , influenzarelated mortality increased worldwide ; however , mortality rates varied widely across locations and demographic subgroups . islands are isolated epidemiological situations that may elucidate why influenza pandemic mortality rates were so variable in apparently similar populations . objectives our objectives were to determine and compare the patterns of pandemic influenza mortality on islands . methods we reviewed historical records of mortality associated with the 19181920 influenza pandemic in various military and civilian groups on islands . results and conclusions mortality differed more than 50fold during pandemicrelated epidemics on pacific islands [ range : 04% ( hawaii ) to 22% ( samoa ) ] , and on some islands , mortality sharply varied among demographic subgroups of island residents such as saipan : chamorros [ 12% ] and caroline islanders [ 04% ] . among soldiers from island populations who had completed initial military training , influenzarelated mortality rates were generally low , for example , puerto rico ( 07% ) and french polynesia ( 013% ) . the findings suggest that among island residents , those who had been exposed to multiple , antigenically diverse respiratory pathogens prior to infection with the 1918 pandemic strain ( e.g. , less isolated ) experienced lower mortality . the continuous circulation of antigenically diverse influenza viruses and other respiratory infectious agents makes widespread high mortality during future influenza pandemics unlikely .

Introduction Methods Results Discussion Conflicts of interest Funding Disclaimer Contributors

the term parkinson 's disease refers to a neurodegenerative disease that affects several regions of the brain , including the pigmented nuclei in midbrain and brainstem , the olfactory tubercle , the cerebral cortex , and elements of the peripheral nervous system . parkinson 's disease was first described in a medical context in 1817 by james parkinson , a general practitioner in london in his essay on the shaking palsy . even earlier many physicians have picked up some of the features of parkinson 's disease and described them in their writings , for example , franciscus de le be ( 16141672 ) who described tremors and franois boissier de sauvages de la croix ( 17061767 ) who described patients with running disturbances of the limbs . wilhelm von humboldt ( 17671835 ) , the celebrated academic reformer and writer who lived in the era of parkinson , has described his own neurological condition in a series of letters . reviewing the multiple clinical findings in pd will show us the need of more researches to reach a possible curative therapy . a group of neurobehavioral abnormalities can be found in pd such as apathy , fearfulness , anxiety , emotional liability , social withdrawal , increasing dependency , depression , dementia , bradyphrenia , a type of anomia termed the tip - of - the - tongue phenomenon , visual - spatial impairment , sleep disturbance , psychosis , and other psychiatric problems . the variable presentations of pd often cause diagnostic confusion and a delay in treatment . in the early stages , parkinsonian symptoms are often mistaken for simple arthritis or bursitis , depression , normal aging , alzheimer 's disease , or stroke . characterized a prodromal phase 46 years before the main manifestation in pd patients . during this period , pd patients , compared with normal controls , had a higher frequency of mood disorder , fibromyalgia , and shoulder pain . a sum of possible pd manifestations would be tremors at rest , rigidity , bradykinesia , and loss of postural reflexes which represents the cardinal manifestations besides possibility of the following . the basal ganglia include the neostriatum ( caudate nucleus and putamen ) the external and internal pallidal segments ( gpe , gpi ) , the subthalamic nucleus ( stn ) , and the substantia nigra with its pars reticulata ( snr ) and pars compacta ( snc ) . they participate in anatomically and functionally segregated loops that involve specific thalamic and cortical areas . striatum and stn receive glutamatergic afferents from specific areas of the cerebral cortex or thalamus , and transfer the information to the basal ganglia output nuclei , gpi and snr . the nigrostriatal projection terminates predominately at the necks of dendritic spines of the striatal medium spiny output neurons ( msns ) . this anatomic arrangement places the dopaminergic inputs in a position to regulate or gate the corticostriatal transmission . in parkinson 's disease , the degeneration of dopaminergic snc neurons and their projections to the striatum is a slowly evolving process that may take decades to develop . snc projections to the putamen degenerate earlier than projections to associative or limbic portions of the striatum . corresponding to this time course of degeneration , the motor symptoms and signs of parkinson 's disease develop before the nonmotor signs . recognizable motor or nonmotor signs appear only after substantial degeneration of the nigrostriatal neurons ( affecting at least 70% ) , this is due to the remarkable compensatory capacity within the dopaminergic system , or in the circuits it modulates . there may be mild frontal atrophy is some cases , but this is variable . histologically , there is neuronal loss in the substantia nigra pars compacta along with compensatory astrocytic and microglial proliferation . although biochemically there is loss of dopaminergic termini in the striatum , the striatum is histologically unremarkable . hyaline cytoplasmic inclusions or lewy bodies and less well - defined pale bodies are found in some of the residual neurons in the substantia nigra . lewy bodies are proteinaceous neuronal cytoplasmic inclusions . in some regions of the brain , such as the dorsal motor nucleus of the vagus , lewy bodies tend to form within neuronal processes and lewy bodies are accompanied by a variable number of abnormal neuritic profiles , referred to as lewy neurites . lewy neurites were first described in the hippocampus , but are also found in other regions of the brain , including the amygdala , cingulate gyrus , and temporal cortex . at the electron microscopic level , lewy bodies are composed of densely aggregated filaments and lewy neurites also are filamentous , but they are usually not as densely packed . some authors suggested that pre - parkinsonian manifestations would be nonmotor ( e.g. , autonomic dysfunction and anosmia ) and that the late stage would be associated with cortical lewy body dementia . synuclein has gain interest for its role in pd neurodegeneration since the demonstration that -synuclein is the major component of lewy bodies and lewy neuritis in idiopathic pd . the exact mechanisms of how conformational changes of -synuclein induce cell death are currently the subject of intense investigation , but are still poorly understood . it has been suggested that -synuclein oligomers might form pores on intracellular membranes such as the plasma membrane , and may increase cation permeability . -synuclein has been reported to negatively regulate the activity of tyrosine hydroxylase by maintaining the protein in the dephosphorylated inactive form , thus modulating dopamine biosynthesis in catecholaminergic neurons . in addition , -synuclein was proposed to negatively regulate the function of the dopamine transporter in dopaminergic nerve terminals by controlling the amount of the transporter at the plasma membrane . tau is a microtubule - binding protein that stabilizes microtubules and promotes their polymerization in neurons . while pd is the best known of the -synucleinopathies , tau pathology is also seen in many pd cases . staining of lewy bodies with multiple tau antibodies has been reported , suggesting cellular colocalization of these two pathologies . both of the proteins ( uchl1 and parkin ) are involved in the ubiquitin - proteasome pathway of abnormal protein degradation which is considered to be a cellular quality control . thus , it is possible that mutations in the parkin and uchl1 genes may lead to malfunction of the pathway and damaged proteins are not degraded . instead , they form aggregates that ultimately lead to cell degeneration with an unknown mechanism . the direct relation between mitochondrial dysfunction and pd came from the postmortem description of complex i deficiency in the substantia nigra of patients with pd . subsequently , the deficiency was also seen in the skeletal muscle and platelets and there was a decrease in complex i proteins in the substantia nigra of patients with pd . . da metabolism and auto - oxidation combined with increased iron , decreased total glutathione levels , and mitochondrial complex i inhibition can lead to cell death by exceeding the oxidative capacity of da - containing cells in the region . it has been an attractive hypothesis for decades that excitotoxicity and disturbance in calcium homeostasis are mechanisms leading to neurodegeneration ; disturbed calcium homeostasis is done through the activation of calpains , a family of cysteine proteases . these are elevated in the mesencephalon of patients with pd but not in other neurodegenerative disorders involving the mesencephalon . david park and colleagues have shown that calpains are activated and required for mptp - induced neuronal death in mice . in one of his dialogues , the greek philosopher plato speculated that our minds contain a block of wax , the size , hardness , and consistency of which varies from person to person . everything we think or perceive leaves an impression on the wax , and the quality and duration of this impression is the basis of our memory and knowledge . this metaphor was used by di monte , to describe the relation between genetics and environmental risk factors in developing pd . pd is considered a multifactorial disease resulting from the effect of environmental factors and genetic susceptibility . despite the mysterious cause of pd , the pathologic and neurochemical basis of parkinsonian signs and symptoms have been in part unraveled as discussed earlier . toxic insults could both modify the structure of -synuclein and interfere with the ubiquitin - proteasomal pathway , thus , promoting -synuclein aggregation and impairing the process of degradation of the abnormal protein . barlow et al . found that even in utero exposure with subsequent genetic damage can be a risk factor for neurodegenerative diseases , for example , alzheimer and pd . this can explain the finding of tsai et al . who described what he has called a young onset parkinsonian disease ( yopd ) which is a peculiar group of patients developing pd before 40 years on contrary to normal age group of more than 50 years . the reason for trying to identify toxic causes of pd ( besides future protection ) is the search for a perfect pd model . scientists are trying to unravel the exact cause of pd ; also we are trying to find out a perfect therapy and both the therapy and cause need a perfect model to act on . finding a toxin that can be a true cause of pd would solve the problem . since the discovery of mptp , parkinsonian inducing effects this arouse the possibility of other similar compounds relevant to mptp , for example , paraquat to induce pd . although pd existed long before the introduction of these pesticides , the thought is that pesticide exposure has contributed to the increased incidence of the disease . fumigantsthe fumigant class encompasses a variety of agents most commonly used to control insects or fungi in grains , soil , or other various consumables . fumigants , such as ethylene dibromide , are highly toxic to humans but most adverse actions are nonneuronal . although there is some evidence that carbon disulfide - based fumigants can induce parkinsonian - like neurotoxicity . this chemical class is not suitable for induction of pd in an animal model . the fumigant class encompasses a variety of agents most commonly used to control insects or fungi in grains , soil , or other various consumables . fumigants , such as ethylene dibromide , are highly toxic to humans but most adverse actions are nonneuronal . although there is some evidence that carbon disulfide - based fumigants can induce parkinsonian - like neurotoxicity . this chemical class is not suitable for induction of pd in an animal model . maneb , or manganese ethylene bis - dithiocarbamate , is one type with possible parkinsonian symptoms which may be secondary to exposure to the manganese metal core .in fact , maneb is one of the toxins used to induce pd in animal models but usually in combination with other agents specially paraquat . the maneb effect has been undoubtedly documented to increase the severity of pd models which make it one of the good candidates in pd research . maneb , or manganese ethylene bis - dithiocarbamate , is one type with possible parkinsonian symptoms which may be secondary to exposure to the manganese metal core . in fact , maneb is one of the toxins used to induce pd in animal models but usually in combination with other agents specially paraquat . the maneb effect has been undoubtedly documented to increase the severity of pd models which make it one of the good candidates in pd research . herbicides ( paraquat)the possibility that paraquat ( 1,1_-dimethyl-4,4_-bipyridinium ) may damage the nigrostriatal dopaminergic system and therefore contribute to the neuropathology of parkinson 's disease ( pd ) was first proposed in the mid 1980s following the observation that its chemical structure closely resembles that of mpp(1-methyl-4-phenylpyridinium ion ) .animal studies confirmed the ability of paraquat to induce selective dopaminergic nigrostriatal degeneration [ 3941].li et al . have developed subacute model for inducing pd in mice through using intraperitoneal injection of paraquat ( 10 mg / kg ) in old c57/bl mice . similar results were obtained 2 years later by kuter et al . using a similar approach.as described earlier , an improvement in modeling of paraquat induced pd was through adding maneb . a combination which improved the results . it also supported the multiple hit theory of pd .the presence of differences between paraquat and mptp has been questioned and the answer would come from a group of articles in toxicological sciences journal regarding mechanism of paraquat toxicity . richardson et al . proposed that paraquat would have different mechanisms for toxicity than mptp . later , ramachandiran et al . have shown that paraquat and mptp have divergent mechanisms of toxicity . another hot debate emerged on the same journal regarding the possibility of different mechanisms of neurotoxicity between paraquat and mptp [ 4548].despite the debate , there are certainly toxicokinetic and toxicodynamic differences which would give paraquat a unique pattern of neurotoxicity . despite the fact that paraquat models are still less validated than the previous well - established mptp models , combining maneb with paraquat would introduce a promising modeling technique that can be ( in our opinion ) a better model specially with the limitation in the other available toxic models . the possibility that paraquat ( 1,1_-dimethyl-4,4_-bipyridinium ) may damage the nigrostriatal dopaminergic system and therefore contribute to the neuropathology of parkinson 's disease ( pd ) was first proposed in the mid 1980s following the observation that its chemical structure closely resembles that of mpp(1-methyl-4-phenylpyridinium ion ) . animal studies confirmed the ability of paraquat to induce selective dopaminergic nigrostriatal degeneration [ 3941 ] . have developed subacute model for inducing pd in mice through using intraperitoneal injection of paraquat ( 10 mg / kg ) in old c57/bl mice . similar results were obtained 2 years later by kuter et al . using a similar approach . as described earlier , an improvement in modeling of paraquat induced pd was through adding maneb . a combination which improved the results . the presence of differences between paraquat and mptp has been questioned and the answer would come from a group of articles in toxicological sciences journal regarding mechanism of paraquat toxicity . richardson et al . proposed that paraquat would have different mechanisms for toxicity than mptp . later , ramachandiran et al . have shown that paraquat and mptp have divergent mechanisms of toxicity . another hot debate emerged on the same journal regarding the possibility of different mechanisms of neurotoxicity between paraquat and mptp [ 4548 ] . despite the debate , there are certainly toxicokinetic and toxicodynamic differences which would give paraquat a unique pattern of neurotoxicity . despite the fact that paraquat models are still less validated than the previous well - established mptp models , combining maneb with paraquat would introduce a promising modeling technique that can be ( in our opinion ) a better model specially with the limitation in the other available toxic models . many of the compounds in this class are , by design , neurotoxic . similarities between the insect and human nervous systems can lead to cross - toxicity of these compounds . organophosphatesalthough evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd .however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd rotenonedue to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53].chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs .rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . organochlorinesthe first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . pyrethroidsalthough pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system .pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine .although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism.the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . mptpthe potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) .mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin .one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . this recovery potential in young mice allows for modeling of recovery .various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues .in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.several mptp dosing regimens have been used . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks .the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death .one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) .strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl.an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . many of the compounds in this class are , by design , neurotoxic . similarities between the insect and human nervous systems can lead to cross - toxicity of these compounds . organophosphatesalthough evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd .however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd . rotenonedue to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53].chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs .rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . organochlorinesthe first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . pyrethroidsalthough pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system .pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine .although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism.the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . mptpthe potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) .mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin .one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . this recovery potential in young mice allows for modeling of recovery .various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues .in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent.several mptp dosing regimens have been used . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks .the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death .one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) .strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl.an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . although evidence supporting a role for ops in pd pathogenesis is scant , there are case reports of people with severe parkinsonism after op exposure ; however , the reversibility of the symptoms and lack of responsiveness to l - dopa are not consistent with pd . a recent family - based case - control study did implicate organophosphates and other insecticides in pd . however , it might be that the effects of op exposure probably involve disturbance of the balance between the dopamine and acetylcholine systems and not specific pathological changes in the dopamine system which makes them unsuitable in toxic modeling of pd . due to its ability of inhibiting mitochondrial complex i ( nadh dehydrogenase ) , rotenone has become one of the toxic models used to study pd in animals [ 33 , 53 ] . chronic systemic exposure to rotenone reproduced many features of pd , including nigrostriatal dopaminergic degeneration and the formation of cytoplasmic inclusions in these neurons . rats exposed to rotenone showed bradykinesia , postural instability , unsteady gait , and some evidence of tremors [ 54 , 55 ] . reported an improved rotenone model through giving 3 mg / kg daily with observation of behavioral changes including bradykinesia , postural instability / unsteady gait , and rigidity , which occurred maximally after 6 days of treatment where postmortem analysis ( immunohistochemistry of the th+ve neurons in substantia nigra ) confirmed the behavioral observations . others have shown the efficacy of rotenone as a model of pd in mice and suggested its use for assessing candidate anti - parkinson drugs . rotenone model can be used as an alternative to other classical pd models , for example , mptp and 6-ohda , especially when testing the neuroprotective effects of novel therapeutic modalities . a limiting factor of rotenone models would be the high mortality rate of the examined animals . another limitation would be the less - accurate results that can be obtained usually in cases of oral administered rotenone . the first clue of a relation between organochlorines and pd was the presence of them in postmortem specimens of pd patients . kanthasamy et al . have postulated possible mechanisms for the influence of organochlorines on pd patients . effects on dopamine and dopamine transportersthe assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum).sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin - induced oxidative stressdieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64].chun et al . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin and mitochondrial dysfunctiondieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line .neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons .pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669].dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . 2.4.1.4c.v . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation.these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . the assumption that organochlorines deplete brain dopamine was supported by the findings of miller et al . . they measured the expression and activity of the dopamine transporter ( dat ) and the vesicular monoamine transporter ( vmat2 ) in presynaptic terminals of dopaminergic neurons in the striatum . exposure of c57bl mice to an organochlorine pesticide ( increased the expression of both dat and vmat2 in the striatum ) . sanchez - ramos et al . showed that dieldrin is selectively toxic to dopaminergic neurons compared to striatal gabaergic neurons in primary mesencephalic cultures . dieldrin exposure was shown to induce ros production in a number of cell culture models including neuronal cell lines , mouse lung fibroblasts , and in mouse liver [ 63 , 64 ] . . showed that dieldrin exposure induces ros production in a mouse nigral dopaminergic cell line . dieldrin exposure has been shown to alter mitochondrial function and induce the release of cytochrome c in a human t cell leukemic cell line . neuronal death in pd seems to be mitochondrial dependent . through oxidative mechanisms the soluble pool of cytochrome c in the mitochondrial intermembrane space is increased . for the release of cytochrome c into the cytosol , an activation of bax to permeabilize the outer mitochondrial membrane appears to be necessary . recently caspase 3 has been identified as a critical factor in apoptotic cell death in dopaminergic neurons . pkcd is an important apoptotic substrate for caspase-3 , and subsequent studies have implicated caspase-3-mediated proteolytic activation of pkcd in apoptotic cell death [ 6669 ] . dieldrin treatment induced dose and time - dependent proteolytic cleavage of native pkcd ( 72 and 74 kda ) into two fragments , 38 kda regulatory and 41 kda catalytic subunits , in dopaminergic cells [ 70 , 71 ] . findings suggest that dieldrin can alter the function of the ubiquitin - proteasomal degradation machinery to promote protein aggregation . these encouraging data would increase the organochlorines chance of being a new toxic pd model , especially with their potential of accumulation and so chronic exposure which is one of the characteristics needed to reproduce the exact pathological findings in animal pd models . although pyrethroids are often considered environmentally labile , exposure of mice to the pyrethroid pesticides deltamethrin and permethrin has been demonstrated to increase dat - mediated dopamine uptake [ 7375 ] . acute toxicity of pyrethroids is primarily mediated through interaction with sodium channels , leading to prolonged depolarization and hyperexcitation of the nervous system . pyrethroids have also been shown to be potent releasers of neurotransmitters , including dopamine . although the mechanism by which pyrethroids are capable of increasing dat - mediated dopamine uptake is not clear , elwan et al . suggest that the effects of pyrethroids on dat are indirect and that longer - term exposures may be capable of damaging cells through an apoptotic mechanism . the use of pyrethroids in modeling of pd is still far from validation as frequent researches are needed to reach a final exposure protocol . the biometals , iron , copper , zinc , and manganese participate in many essential activities , and indeed deficiencies can be lethal . zinc and iron are increased and copper is decreased in the substantia nigra ( sn ) in parkinson 's disease ( pd ) . iron and pdof great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals .searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese : nontypical pd manifestationsmanganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . metals and -synucleinuversky et al . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation.the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . of great interest is the possible association between iron and nigral melanin as a source of free radicals . also , iron is postulated to interact with da or derived catecholic chrome derivatives discussed above , specifically by reacting with hydrogen peroxide that can be generated to produce short - lived hydroxyl radicals . searches for the mutation of genes associated with iron metabolism have revealed mutations in the ferritin - h , irp2 , and hfe gene in single pd patients . ceruloplasmin mutations causing protein instability and loss - of - function lead to extrapyramidal symptoms and parkinsonism , and are characterized by iron accumulation in the substantia nigra . manganese is an essential metal for life , yet chronic exposure to this metal can cause a neurodegenerative disease named manganism . despite similar clinical pictures , in parkinson 's disease the domaninergic neurons in the substancia nigra are damaged , while in manganism , mn accumulates in the globus pallidus and striatum , and it damages these two brain structures that control motor function . so despite having some similar manifestations , mn can not be ranked as one of the causes of pd . have shown that some metal ions can induce -synuclein fibrillation ( impaired metabolism of normal -synuclein , known to accumulate massively in lewy bodies ( lbs ) , is considered the primary cause of neurodegeneration in idiopathic pd ) . the most effective ions were al , fe , co , cd , mn , cu , co , in that order . moreover , they found that combination of metals and pesticides have synergistic effects on -synuclein fibrillation . the use of metals in toxic animal models however is difficult and did not gain any popularity due to difficulties in application and assessment . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice .however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . both ma and its derivative , 3,4-methylenedioxymethamphetamine ( mdma , ecstasy ) , are known to be toxic for dopamine nerve terminals , thus replicating striatal da loss occurring in parkinsonism . it has been shown that ma and mdma induce neuronal inclusions in the substantia nigra and corpus striatum of mice . however , in case of drug - induced pd , it is important to interpret neuronal damage using markers cautiously as there are usually compensatory changes in these enzymes which may not reflect neurodegeneration . the potent parkinsonian neurotoxin , 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) causes pd in animals and is one of the most accepted models . this may be due to its capacity to show some of pd pathological findings ( sn neuron loss and striatal da depletion similar to pd ) . mptp - treated animals ( including humans ) do not exhibit lewy bodies ( lb ) , a hallmark neuronal inclusion of pd , but old treated primates have inclusions that are somewhat similar . humans and many other animal species including nonhuman primates , guinea pigs , mice , and cats are susceptible to this neurotoxin . one interesting feature of the mptp mouse model of pd is the transient nature of the striatal damage in young mice . in contrast , administration of the drug to older mice would result in a permanent loss of nigrostriatal terminals , as well as cell bodies . various techniques can be used to modulate the action of mptp to induce the pathological findings needed to reach the desired pd model . one of these techniques is to give probenecid with mptp to prolong its persistence in the tissues . in sporadic pd , the generation of neural precursor cells in the subependymal zone is impaired by da depletion , which is the hallmark of pd . mptp - induced degeneration of dopaminergic snpc - svz fibres impairs nsc proliferation in primates and in mice in a similar fashion to pd . he et al . raised the possibility that migrating neuroblasts in the svz may be more vulnerable to mptp than nigrostriatal dopaminergic neurons in the sn . furthermore , they suggested that the death and subsequent loss of migrating neuroblasts in the acute or subacute model probably lead to a decreased potential for neurogenesis to some extent . the acute regimen consists of multiple systemic administration of mptp ( usually four doses at 2-h intervals per day ) . the subacute regimen consists of a single systemic administration per day for several consecutive days ( usually 5 days ) and the chronic regimen through several weeks . the comparison of these different models indicated clearly that different schedules of administration of mptp mimic distinct stages of the disease and might induce different mechanisms of neuronal death . one of the limitations of mptp models is their inability to induce behavioral deficits apparent on standard motor mouse tests . however , new techniques for behavioral monitoring have shown promising results , for example , grid test and its modification , the vertical grid test and modified horizontal grid test , swim test , or the automated behavioural apparatus , laborastm ( laboratory animal behaviour observation , registration and analysis system ) . strain differences regarding the response to mptp has been questioned where c57/bl mice show maximum response compared to balb / c mice [ 85 , 93 , 94 ] . , who found prominent changes in balb / c response to mptp as balb / c mice still show a possible alternative , though less effective to c57/bl . an important property of the mptp - lesioned mouse and nonhuman primate is the potential for intrinsic recovery . this capacity would be of great help in identifying new therapeutic targets for the treatment of pd . pd is a multifactorial disease , so despite role of environmental exposure , genetic predisposition in sporadic cases can be broadly grouped into four categories : genes involved in the metabolism of xenobiotics ( e.g. , cyp2d6 , nat2 , gsts ) , neurodegeneration ( e.g. , nos ) , the functioning of dopaminergic neurons ( e.g. , dopamine transporters and receptors ) , and linkage - derived genes ( e.g. , uchl1 , alpha - synuclein ) . there is increasing evidence that genes involved in inherited forms of the disease may act as predisposing factors in the sporadic forms of pd . so , new models of pd produced in vivo on transgenic animals and in vitro on transfected cell cultures are considered important because they may be helpful in discovering the pathophysiology of pd . meredith et al . have classified genetic models into three categories . first , mouse models based on the deletion of genes important for the development or maintenance of da neurons or their phenotypes . second , mouse or rat models based on expression or deletion of genes known to cause familial forms of pd . finally , a third class of genetic models is based on virally mediated expression of genes or mutations known to cause familial pd , usually in nigrostriatal da neurons . despite having some features of the human disease , genetic models do not provide an ideal choice for assessing regenerating capacity of new therapeutic approaches and so are of less use for pharmaceutical development . in our search for an ideal toxic model of pd , we could not find a perfect one . it seems that all toxins when used alone can precipitate only some characters of pd pathology but not the exact pd condition . these same findings may be the cause of the manning - bog and langston advice of using model fusion to overcome the single model limitation through combining genetic with toxic models . we would encourage the use of toxins combination in pd modeling as a new type of model fusion .

parkinson 's disease ( pd ) is one of the neurodegenerative diseases which we can by certainty identify its pathology , however , this confidence disappeares when talking about the cause . a long history of trials , suggestions , and theories tried linking pd to a specific causation . in this paper , a new suggestion is trying to find its way , could it be toxicology ? can we in the future look to pd as an occupational disease , in fact , many clues point to the possible toxic responsibility either total or partial in causing this disease . searching for possible toxic causes for pd would help in designing perfect toxic models in animals .

1. Introduction 2. Historical Background 3. Environment, Genetics, and PD 4. Conclusion

injuries in classical ballet are common and often challenging to treat for a number of reasons . many practitioners do not understand the physical demands of the sport or the terminology describing the common mechanisms of over use . classical ballet dancers subject themselves to repetitive loads that require progressive training over hundreds of hours both increasing the risk of overuse injury and complicating rehabilitation that involves any rest from dance . the ability to dance en pointe ( on the tips of the toes ) for instance , requires progressive development of the kinetic chain from the back to the toes , any disruption of which may result in overuse injury anywhere in the chain . foot and ankle injuries that are more common in classical ballet are both anterior and posterior ankle impingement , flexor hallucis longus tendonitis , and stress fractures at the base of the second metatarsal and fibula . this article will address the diagnosis and treatment of anterior ankle impingement including when performers should return to dance . successful treatment of injuries in classical ballet starts with an understanding of the basic positions and common movements which can lead to overuse injury . the five basic positions involve maximum turnout of the hips to achieve the foot position . inadequate hip turnout results in excessive knee or ankle external rotation and rolling in of the ankle to achieve the desired foot position ( fig . 1 ) . rolling in excessively stretches the medial ankle and compresses the anterior lateral ankle which can contribute to anterior impingement . pli ( fig . 2 ) is the common dance movement contributing to anterior impingement and the rolled in position of the foot exaggerates the lateral compressive forces worsening the problem . 2pli resulting in compressive force to anterior ankle right foot demonstrates excessive pronation of ankle in attempt to exaggerate turnout pli resulting in compressive force to anterior ankle anterior impingement in athletes is either secondary to hypertrophied soft tissue interposed in the anterior ankle joint or proliferation of osteophytes ( fig . 3 ) that limit the open space between the anterior lip of the tibia and the dorsal talar neck . different theories have been proposed to account for the pathologic changes . because impingement often occurs in athletes subject to forced plantar flexion ( soccer players kicking or dancers en pointe ( fig . 4 ) ) one proposed mechanism is that traction on the anterior capsule results in spurs that then impinge . tol et al . in a study of soccer players found that the spurs typically occur in the location of ball impact without excessive plantar flexion being achieved suggesting that the impact and not traction of the capsule results in the spurring . tol in a cadaveric study demonstrated that the location of the osteophytes does not correspond with the attachment points of the capsule . he also located hypertrophied soft tissue in the anterior joint overlying the capsule that was compressed between the tibia and the talus with ankle dorsiflexion . his conclusion was that traction could not be responsible for the spurs and that hypertrophied soft tissue in the anterior ankle was likely responsible for impingement symptoms . 3lateral ankle x - ray showing large talar osteophyte in patient with symptomatic anterior bony impingementfig . 4releve position lateral ankle x - ray showing large talar osteophyte in patient with symptomatic anterior bony impingement current thinking is therefore that osteophyte formation is secondary to repetitive ankle sprains or repetitive microtrauma as one would find with a ball impact or contact between the anterior tibia and talar neck with repetitive loaded dorsiflexion in ballet . once the osteophytes form , the space available for ankle dorsiflexion diminishes likely resulting in soft tissue impingement , inflammation , hypertrophy , and pain . the presence of osteophytes does not always correlate with impingement suggesting that soft tissue changes must also be necessary . this is supported by the finding that anterior osteophytes are very common in dancers occurring in 59% without symptoms of impingement . it is also well accepted that soft tissue impingement can occur without the presence of osteophytes . ankle arthroscopy has demonstrated anterior impingement from the anterior inferior tibiofibular ligament and from hypertrophied synovium in the absence of significant osteophytosis . athletes with anterior impingement often have a history of multiple ankle sprains and present complaining of pain and some swelling in the anterior ankle aggravated by loaded dorsiflexion of the ankle . experienced dancers may not have a history of ankle sprain but complain of increasing pain through training and performance especially with pli positions and landing from jumps . dancers are often very sensitive to body position and note a loss of dorsiflexion in the affected ankle usually noted as a loss of ability to pli on the affected side ( fig . 5 ) . osteophytes at the anterior lateral or anterior medial joint line can often be palpated with the foot in some plantar flexion . weightbearing full dorsiflexion of the ankle with the foot flat will often show some limitation on the affected side and reproduce symptoms . she is compensating by lifting heel nihall et al . in a review of 11 dancers with anterior ankle impingement treated arthroscopically provides a profile of the typical dancer presenting with impingement . the patients included 7 women and 4 men all professional or pre - professional dancers performing primarily ballet . the women had been dancing for a mean of 18 years and the men 10 years . describe a sign they found useful to diagnose synovial soft tissue impingement in the anterior lateral ankle in patients complaining of persistent pain following inversion injury . they repeat the maneuver ( for left ankle ) with right palm on calcaneus and thumb applying pressure to the lateral gutter . increased pain with this maneuver is theoretically secondary to the displacement of hypertrophic synovium in to the joint and denotes a positive test . forty - one had a positive test and 37 of those had synovial hypertrophy in the latter gutter ( sensitivity of 94.8% ) . of 32 patients with negative sign , 2 had synovial hypertrophy ( specificity of 88% ) . imaging of suspected anterior impingement starts with plain weightbearing radiographs of the ankle . as discussed above , the diagnosis is primarily clinical but imaging is necessary to rule out other causes of anterior ankle pain and confirm the presence and location of osteophytes that need to be removed to achieve the best surgical results . plain radiographs including ap and mortise views are less helpful for the diagnosis of impingement , but will demonstrate other causes of ankle pain including fracture , mortise disruption , ocd , and arthritis . the identification of arthritis is important to plan treatment because the surgical results with arthroscopy are significantly worse if arthritis is present . a lateral view will often demonstrate the tibial and talar osteophytes associated with anterior impingement . adding an oblique view with the beam 45 craniocaudal and the foot plantar flexed with the leg 30 externally rotated will increase the detection of medially located tibial and talar osteophytes [ 10[k1 ] ] . ct scanning can also be used to identify osteophytes contributing to impingement and in areas with easy access to ct , this is often the preferred method of identifying bony changes in the ankle , especially if plain films are non - diagnostic in the setting of clinical impingement . it is well accepted that anterior impingement can occur without the formation of osteophytes secondary to entrapment of hypertrophied synovium or torn anterior lateral ligaments [ 11[k2 ] ] . t1 and proton density axial views demonstrating thickened synovium in the anterior lateral joint space or hypertrophy of the anterior talo - fibular ligament are the findings most consistently associated with impingement at arthroscopy [ 12[k3 ] ] . the identification of chondral injury to the anterior aspect of the joint is also helpful as this finding decreases the likelihood that arthroscopic debridement of anterior osteophytes and hypertrophied tissue will result in significant improvement . relative activity limitation such as avoiding jumps , avoiding forcing of demi - pli , combined with attention to correct technique to reduce pronation may improve the symptoms of anterior impingement in dancers . physical therapy with focus on the entire kinetic chain , joint mobilization , and taping may resolve symptoms in some dancers . use of a night splint or removable walking cast brace can be tried briefly , and a single intra - articular corticosteroid injection may be used in select cases when other conservative measures prove ineffective . ankle arthroscopy is a useful tool both for diagnosis and treatment of persistent ankle pain following injury and it may be the only way to definitely diagnose and treat soft tissue impingement from an abnormal fibrous band [ 13[k4 ] ] . arthroscopy also provides highly effective treatment of bony and anterior soft tissue ankle impingement provided there is no significant associated chondral lesion in the anterior ankle . if initial radiographic studies fail to show osseous impingement , then an ankle arthroscopy is performed . thickened synovial tissue , and or the thickened distal fascicle of the anteroinferior tibiofibular ligament is debrided with a shaver . any articular cartilage damage is addressed with debridement , drilling , and/or microfracture . for those dancers with osseous impingement , open treatment is not recommended when an osteochondral defect is present , but for talar neck or distal tibial osteophytes without other intra - articular pathology , a small anteromedial arthrotomy can be used effectively . post - operative care includes use of an ankle cryo - cuff to control edema and pain , alternating with a removable boot for ambulation . patients are allowed to bear weight as pain allows unless an osteochondral defect has been drilled . in that case , patients are maintained strictly non - weightbearing for 6 weeks . after 1014 days ( for those with simple debridement or ostectomy ) , the patient is allowed to wean out of the boot . physical therapy is begun to gradually increase range of motion and strength , with attention to proprioception exercises . dancers may start pilates , floor barre , and return to class gradually as comfort allows . dancers are counseled that return to full performance may be limited for up to 6 months . relative activity limitation such as avoiding jumps , avoiding forcing of demi - pli , combined with attention to correct technique to reduce pronation may improve the symptoms of anterior impingement in dancers . physical therapy with focus on the entire kinetic chain , joint mobilization , and taping may resolve symptoms in some dancers . use of a night splint or removable walking cast brace can be tried briefly , and a single intra - articular corticosteroid injection may be used in select cases when other conservative measures prove ineffective . ankle arthroscopy is a useful tool both for diagnosis and treatment of persistent ankle pain following injury and it may be the only way to definitely diagnose and treat soft tissue impingement from an abnormal fibrous band [ 13[k4 ] ] . arthroscopy also provides highly effective treatment of bony and anterior soft tissue ankle impingement provided there is no significant associated chondral lesion in the anterior ankle . if initial radiographic studies fail to show osseous impingement , then an ankle arthroscopy is performed . thickened synovial tissue , and or the thickened distal fascicle of the anteroinferior tibiofibular ligament is debrided with a shaver . any articular cartilage damage is addressed with debridement , drilling , and/or microfracture . for those dancers with osseous impingement open treatment is not recommended when an osteochondral defect is present , but for talar neck or distal tibial osteophytes without other intra - articular pathology , a small anteromedial arthrotomy can be used effectively . post - operative care includes use of an ankle cryo - cuff to control edema and pain , alternating with a removable boot for ambulation . patients are allowed to bear weight as pain allows unless an osteochondral defect has been drilled . in that case , patients are maintained strictly non - weightbearing for 6 weeks . after 1014 days ( for those with simple debridement or ostectomy ) , the patient is allowed to wean out of the boot . physical therapy is begun to gradually increase range of motion and strength , with attention to proprioception exercises . dancers may start pilates , floor barre , and return to class gradually as comfort allows . dancers are counseled that return to full performance may be limited for up to 6 months .

anterior impingement is a common problem in dancers occurring primarily secondary to the repetitive forced ankle dorsiflexion inherent in ballet . symptoms generally occur progressively and may respond to conservative treatment including addressing biomechanical faults that contribute to the problem . as impingement progresses , movements essential to ballet may become impossible and arthroscopic ankle surgery is often effective for both diagnosis and treatment , allowing athletes to return to dance .

Introduction Terminology Pathophysiology Diagnosis Treatment Conservative Operative

a 57-year - old hiv - positive man was admitted to general internal medicine after presenting with acute - onset retrosternal chest pain accompanied by a 24 h history of fever . apart from longstanding hiv infection , his medical history was significant for charcot - marie - tooth disease with a chronic ulcer on his right first toe , osteoporosis , kyphoscoliosis and depression . two weeks before presentation , his cd4 count was 233 cells / mm with a viral load of < 40 copies / ml . the patient did not use intravenous ( iv ) drugs and was compliant with all of his medications including antiretroviral therapy ( darunavir , ritonavir , maraviroc and raltegravir ) , alendronate and buproprion . on examination , he was febrile ( 39.4c ) , with a blood pressure of 122/72 mmhg , heart rate of 100 beats / min , respiratory rate of 24 breaths / min and oxygen saturation of 100% on room air . his neurological examination was normal and there were no rashes , active joints or stigmata of endocarditis . the patient s chest pain was retrosternal , pleuritic , localized to the manubriosternal joint ( msj ) with some radiation to the scapula and was reproducible by palpation . his foot ulcer showed no obvious signs of infection and there was no history of recent cellulitis . complete blood count , electrolyte , creatinine and cardiac enzyme levels and a urinalysis were all normal . his chest x - ray was normal and computed tomography ( ct ) ruled out pneumonia , fractures , pulmonary embolism and aortic dissection . twelve hours after admission , two of two peripheral blood cultures were positive for gram - positive cocci in clusters . the patient was started empirically on iv vancomycin , which was changed to cefazolin when the antibiotic sensitivities of the s aureus isolate became available 60 h after admission . despite resolution of the patient s fever within 24 h of the initiation of antibiotics , he continued to experience severe central chest pain . by 48 h after admission , a new , tender mass was noted to have formed over the patient s msj . inflammatory markers analyzed on the third day of admission were significantly elevated ( c - reactive protein 48 mg / l and erythrocyte sedimentation rate 120 mm / h ) . although no obvious pathology of the msj was noted on the ct scan of the thorax , further imaging of this area was prompted by the patient s unresolving symptoms . thoracic magnetic resonance imaging ( mri ) six days after admission revealed phlegmonous regions anterior and posterior to the msj , with lobulated enhancements that were not initially appreciated on ct ( figure 1 ) . these findings were believed to be most consistent with septic arthritis of the msj . as a result he was discharged home after eight days with a peripherally inserted central catheter . on discharge , the mass at the msj could not be palpated two weeks postdischarge and he remained well four months later . acute chest pain is one of the most common reasons for presentation to the emergency department and , in the united states alone , accounts for approximately eight million visits per year ( 1 ) . determining the etiology of a patient s chest pain necessitates a broad differential diagnosis because more than one - half of these presentations are due to noncardiac causes ( 1 ) . to our knowledge , a total of 11 cases of msj septic arthritis have been reported previously ; the present case is the first to be documented in an hiv - positive patient . it is a secondary cartilaginous or symphysis joint , which has a lower risk of infection relative to synovial joints ( 2 ) . owing to the rarity of this condition , the epidemiology and risk factors for septic arthritis of the msj are based on case series , summarized previously in a report by peng et al ( 2 ) , with two additional cases ( including the present case ) having been reported since ( 3 ) . to date , the majority of patients with msj septic arthritis have been male ( 75% ) , with a mean age of presentation of 49 years ( median 46 years ; range 20 to 85 years ) ( 2,3 ) . a primary source of bacteremia outside of the msj ( eg , idiopathic bacteremia or cellulitis ) was the most common risk factor and s aureus was isolated in 67% ( eight of 12 ) of cases ( 2,3 ) . other reported risk factors include corticosteroid - induced immunosuppression , inflammatory joint disease and iv drug use ( 2 ) . a more recent report argued that derangements in the bony architecture of the thorax , such as in patients with advanced scoliosis , may transmit to the msj and result in joint subluxation and subsequent susceptibility to septic arthritis ( 3 ) . interestingly , our patient presented with significant kyphoscoliosis ; however , he had no previous history of subluxation or pain at the msj and imaging failed to demonstrate signs of anatomical abnormalities at the msj . it is unclear to what extent the patient s hiv status contributed to his clinical presentation , although hiv infection is a known risk factor for septic arthritis in general ( 4 ) . while there are no known pathognomonic findings for msj septic arthritis , almost universally , central chest pain is accompanied by a tender and/or palpable mass at the msj ( 2,3 ) . blood or synovial cultures were positive in 83% ( 10 of 12 ) of reported cases and provided sensitivities to guide antibiotic therapy ( 2,3 ) . of those reported , 62% ( five of eight ) of blood cultures were positive , compared with 90% ( nine of 10 ) of msj cultures . both blood and msj cultures were positive in 67% ( four of six ) of cases , when performed ( 2,3 ) . imaging aided in the diagnosis of several cases , with mri suggested as the optimal modality owing to superior visualization of tissue and bone planes at the msj ( 5 ) . our patient presented with all of the above signs / symptoms , with the exception of leukocytosis . diagnostic confirmation was obtained based on positive blood cultures and the mri findings after a ct scan failed to detect any abnormalities . medical therapy with iv antibiotics tailored to culture and sensitivity results ( either blood or msj aspirate ) occurred in almost all cases ( 2,3 ) . the duration of therapy was typically four to six weeks , consistent with the therapy for other similar forms of septic arthritis ( 2,3 ) . in all previous cases successfully treated medically ( 33% ) , prompt diagnosis led to early initiation of antibiotics . in the two culture - negative cases , both patients received surgical debridement in combination with broad - spectrum iv antibiotics ( 2,5 ) . this highlights the importance of blood or synovial cultures in tailoring the antibiotic regimen given the prolonged duration of therapy . the majority of reported cases ( 67% ) required surgical management in addition to antibiotics . of these cases , three followed a delay in diagnosis while one occurred following failure of antibiotics ( 2,3 ) . several surgical cases demonstrated evidence of msj destruction , highlighting the urgent need for appropriate treatment to prevent irreparable damage . similar to the medically treated patients , all surgical cases received a six - week course of iv antibiotics tailored to culture sensitivity results , with good clinical outcomes ( 2,3 ) . in previous cases of septic arthritis , serial imaging this may only be necessary if there is concern for failure of antibiotic therapy ( ongoing fevers , symptoms , worsening joint disruption ) . our patient was managed with six weeks of iv cefazolin based on the sensitivities of the blood culture s aureus isolate . given his rapid clinical improvement with antibiotics , surgical debridement was not required . we present a case of msj septic arthritis and the first in an hiv - positive patient . diagnosis was made based on the patient s fever , chest pain , positive blood cultures and mri findings . the patient was treated with six weeks of iv antibiotics with a good clinical outcome .

the authors of this article report a case involving a 57-year - old man with longstanding hiv infection who presented with fever and acute chest pain , which was determined to be caused by an infection of the manubriosternal joint . the course of treatment is described and a literature review summarizing the published cases of this rare infection is presented .

CASE PRESENTATION DISCUSSION CONCLUSION

post - herpetic neuralgia is a serious complication of herpes zoster that causes critical pain to patients even after skin lesions have healed . epidural blocks are achieved either by single injection of local anesthetic through the epidural needle or as continuous block by infusion pump through an epidural catheter . the epidural catheter can be inserted not only in thoracic and lumbar vertebra , but also in cervical vertebra . knotting of epidural catheters is very rare , but knotting in lumbar epidural catheters has been reported in a number of studies , and most of these cases involved removal difficulty . we report a case in which we inserted a cervical epidural catheter in a patient who was experiencing severe post - herpetic neuralgia and then removed the knotted catheter without complications . a female patient , 73 years old , was diagnosed with herpes zoster and received medical treatment at another medical clinic . however , when her serious pain did not subside after treatment , she visited our pain clinic . her pain was severe , 10/10 on the numeric rating scale ( nrs ) at the first visit . she appeared to have herpes zoster involving the fifth cervical spinal nerve root on the right and was diagnosed with post - herpetic neuralgia . we explained our plan to perform a cervical epidural block along with medical treatment and received informed consent . the patient was instructed to flex her neck so that her interlaminar space was easily distinguished . skin over the c6 - 7 interlaminar space was sterilized with betadine solution and topically anesthetized with 2 ml of 2% lidocaine . using the midline approach , we checked the epidural space through a 20-gauge tuohy needle using the loss of resistance method ; after assuring that there was no cerebrospinal fluid or blood aspirated , 5 ml of 1% mepivacaine and 2 mg of dexamethasone were injected . immediately after the injection , the patient reported that her pain temporarily decreased ; but after about 40 minutes , she said the pain was again as severe as it used to be . we explained the use of an epidural block with epidural catheter insertion and received agreement from the patient to proceed . the epidural space was checked using an 18-gauge tuohy needle at c6 - 7 using the same technique as for the cervical epidural block . the depth of the epidural space from the skin was 5.2 cm . a 20-gauge epidural catheter ( smiths medical asd , inc . keene , usa ) was advanced by 7 cm cephalad through the epidural needle ; after the needle was removed , the catheter was pulled out by 2 cm and inserted 5 cm from the epidural space . after insertion of the epidural catheter and confirming that no blood or cerebrospinal fluid had been pulled in , 3 ml of 1% mepivacaine was injected . the patient reported that her pain was subsiding , so she was given 2 ml of 0.18% ropivacaine per hour through an infusion pump . we prescribed pregabalin 225 mg , tramadol 150 mg , and amitriptyline 10 mg per day , and the patient was discharged . she visited our clinic every day for the next four days and received additional injections of 5 ml of 1% mepivacaine through the epidural catheter , maintained by infusion pump . after this , she reported that the pain was decreased to 56 on nrs , and that she could finally sleep at night . on the fifth day , she reported her pain to be 34/10 on nrs , and that she wanted to remove the epidural catheter , while remaining on her prescribed medication . we attempted to remove the catheter from the patient in a seated posture ; however , after it was pulled out about 3 cm , there was resistance , and it stopped moving . when asked if there was any pain or discomfort of the neck , the patient answered that there was not . after asking the patient to repeat bending and then straightening her neck a few times and pulling the catheter out with slightly stronger force , the catheter suddenly snapped out . the patient reported no pain in the neck or arm , so our medical team sterilized the area where the catheter had been installed and applied a dressing . when the removed catheter was thoroughly examined , a knot was discovered 1.5 cm from the tip ( fig . side effects or complications of epidural catheter placement include unsuccessful catheter placement , intrathecal or intravascular placement , catheter damage , knotting , and difficult removal . knotting is a very rare complication in epidural catheters , and the actual incidence is difficult to determine because most of the documentation is provided in the form of case reports . therefore , cases of knotting have been reported primarily in lumbar epidural catheters , with only one case involving a thoracic epidural catheter . in 12 cases , the knots arose less than 3 cm from the catheter tip , near the distal end . in one case , a double knot was found 1.5 cm to 3 cm from the tip of the catheter , while another case involved a butterfly - like knot 5 cm below the catheter tip . in our case , the knot was located 1.5 cm below from the catheter tip after cervical epidural catheter placement , a condition that has never been reported previously . the insertion of epidural catheters is performed through tuohy needles , with the clinician threading the epidural catheter into the epidural space and subsequently withdrawing the catheter through the desired level in the epidural space . the longer is the distance the catheter is inserted from the epidural space , the greater is the chance of knotting . therefore , several authors have recommended that the length of the catheter passed beyond the tip of the needle should be limited to reduce this risk . it has been recommended that the length of catheter insertion not exceed 5 cm , and less than 34 cm is considered optimal to prevent knotting . however , knots in epidural catheters have been reported even when no more than 3 cm had passed in the epidural space . in our case , when removing an epidural catheter , steady and gentle firm traction must be applied in the same direction as when the catheter was inserted . if the catheter begins to stretch too much or the patient complains of pain or paresthesia during withdrawal , the traction should be stopped at once . in most cases , difficult removal of epidural catheters occasionally occurs , and several maneuvers have been recommended in response : ( 1 ) slow , continuous , and steady pressure on the catheter to avoid catheter breakage ; ( 2 ) reapplication of force and traction several hours later ; ( 3 ) having the patient adopt the same position as when the catheter was inserted ; ( 4 ) having the patient adopt the lateral decubitus position ( as greater force is required to remove epidural catheters in the flexed sitting position than in the lateral position ) ; and ( 5 ) attempting removal after injection of normal saline through the catheter , which may also assist in determining whether the catheter is knotted . if necessary , radiological images can be checked while injecting radiopaque dye through the catheter , and computer tomography can also help to clarify the image . gadalla reported that catheters were more successfully removed with patients under general anesthesia with muscle relaxation . surgical removal can be also applied when a patient shows neurologic signs during traction or pieces of broken catheter are detected . after removing an epidural catheter , if a catheter fragment is retained in the body , it remains debatable whether the fragment should be removed surgically . some experts recommend that , as catheters are sterile and unlikely to cause neurologic damage , fragments should be left in situ , especially if no neurologic sequelae are noted . surgical removal may increase the risk of neurologic damage due to infection , fibrosis , or mechanical neural irritation . however , some reports have demonstrated adverse effects after leaving the sheared catheter in situ such as radiculopathy , weakness , granuloma formation , and fibrosis as a result of long - term retention of epidural catheters . in the present report our patient did not complain of discomfort during removal of the catheter , but mild resistance was experienced when our medical team attempted to pull out the catheter . the patient was placed into the same seated posture as she had assumed when the catheter was inserted and bent her neck down and up a few times , and the catheter was then successfully removed without any complications . a knot was found 1.5 cm from the tip of the catheter , which itself was 5 cm long . it is normally recommended that the length of epidural catheters be less than 5 cm , while catheters less than 34 cm in length are considered ideal to prevent possible knotting .

epidural block is achieved either by single injection of local anesthetic through an epidural needle or as a continuous block by infusion pump through an epidural catheter . complications associated with epidural catheters include breakage , entrapment , and knotting . knotting of epidural catheters is very rare , but knotting in lumbar epidural catheters has been reported in a number of studies , and most of these cases involved removal difficulty . we report a case in which we inserted a cervical epidural catheter in a patient who was experiencing severe post - herpetic neuralgia and then removed the knotted catheter without complications .

INTRODUCTION CASE REPORT DISCUSSION

approximately 3.7%7.5% of the total number of breast cancer patients diagnosed each year in the us [ 1 , 2 ] and western europe [ 35 ] are younger than 40 years . in saudi arabia , the proportion of breast cancer patients 40 years at diagnosis is dramatically larger with 25.1% . multiple retrospective series and subset analyses of larger randomized trials have shown that young patients with breast cancer have a poorer prognosis [ 716 ] compared to older age at diagnosis . breast cancer patients 40 years tend to have more triple - negative and fewer luminal a and b breast cancers [ 1719 ] , tumors of higher grade , more extensive intraductal component , more lymphovascular invasion , more likely estrogen receptor- ( er- ) negative tumors [ 2023 ] , and more often brca-1 or -2 germline mutations [ 13 , 2427 ] . although young women do appear to have tumors with more aggressive biological characteristics , younger age has been shown in several studies to be an independent predictor of adverse outcome [ 18 , 20 , 22 , 2831 ] . several current consensus guidelines have included age 35 years as an absolute indication for adjuvant systemic chemotherapy irrespective of other tumor characteristics [ 3235 ] . more research is needed to optimize the treatment for this patient group [ 14 , 36 , 37 ] . detailed data about prognostic factors and treatment outcome in breast cancer are scarce in saudi arabia and the middle east . the purpose of this study was to characterize the breast cancer patients treated with curative intend of this tertiary care hospital in saudi arabia 40 years of age at diagnosis compared to > 40 years , and to assess their prognosis . medical records were retrospectively reviewed of female breast cancer patients who consulted saad specialist hospital between 2004 and 2011 . eligibility criteria for the analysis were histologically confirmed diagnosis of invasive breast cancer or cancer in situ , surgical treatment with breast conserving surgery or mastectomy with curative intent . patients with distant metastases , synchronous , or metachronous cancer at diagnosis were excluded from the analysis . staging procedures included complete history and physical examination , laboratory assessments , and diagnostic bilateral mammogram . where indicated , ultrasonography of the breast and abdomen , chest radiograph , and radionuclide bone scan were performed . selected patients received magnetic resonance imaging ( mri ) of the breast , computerized tomography ( ct ) , or positron emission tomography - computed tomography ( pet - ct ) . patients were presented and discussed in an interdisciplinary tumor board meeting , and a treatment recommendation was generated usually based on the guidelines of the national comprehensive cancer network ( nccn ) . breast conserving surgery ( bcs ) consisted of wide local excision or lumpectomy and axillary dissection or sentinel lymph node biopsy in selected patients . after modified radical mastectomy , in selected patients breast reconstruction with tram - flap or latissimus dorsi - flap was performed . dependent on the t status , n status , hormone receptor status , age ( 35 years versus > 35 years ) , and menopausal status , four cycles of adriamycin / cyclophosphamide ( ac ) or six cycles of cyclophosphamide / methotrexate/5-fu ( cmf ) were prescribed for node - negative patients , and four cycles of ac followed by four cycles of paclitaxel or , alternatively , three cycles of 5-fu / epirubicin / cyclophosphamide ( fec ) followed by three cycles of docetaxel for node positive patients . herceptin was added according to the her2neu status and prescribed for at least one year . triple negative patients were usually treated with four cycles of ac followed by four cycles of paclitaxel . in selected patients a total dose of 50.4 gy in 28 fractions was prescribed , followed by a boost of 10 gy in 5 fractions in all patients younger than 50 years . postmastectomy radiotherapy of the chest wall was given in patients with at least one positive locoregional lymph node . usually three - dimensional conformal radiotherapy ( 3d - crt ) using opposed tangential beam was applied for the treatment of the whole breast or the chest wall . in selected patients , intensity modulated radiotherapy ( imrt ) was used to reduce the dose volume of the heart and lung . follow - up examinations were scheduled every three months in the first year , then every six months for 4 years . breast cancer was classified according to the international union against cancer ( uicc ) , with group clinical and pathological staging according to the american joint committee on cancer ( ajcc , 6th edition ) . data were entered into a computerized database ( ms access 2010 ) and analysed using a statistical software package ( spss 19 ) . sections with a thickness of four m were cut from paraffin blocks and used for immunohistochemical staining using the iview dab detection kit on benchmark autostainer ( ventana , tucson , az , usa ) . the clones of antibodies sp1 , 1e2 , and 4b5 were used to evaluate the er - a , pr , and her2neu status . the allred scoring system was used to assess the er and pr status . in summary , a total allred score was obtained by the summation of proportion score ( ps ) and intensity score ( is ) . ps is assigned depending on the proportion of positive cells ( 0 = none ; 1 < 1% ; 2 = 1% -<1/10 ; 3 = 1/10 -<1/3 ; 4 = 1/3 -<2/3 ; 5 2/3 ) , is ( 0 = none ; 1 = weak ; 2 = intermediate ; 3 = strong ) . a total score of 2 or more was considered as positive ; scores 0 and 1 were considered negative . the american society of clinical oncology / college of american pathologists ( asco / cap ) guideline recommendations were used to evaluate the her2neu status . briefly , score 0 indicates no staining in invasive tumor cells . score + 1 indicates weak incomplete membrane staining in any proportion of invasive tumor cells or weak complete membrane staining in < 10% of cells . score + 2 indicates complete membrane staining in nonuniform or weak but with obvious circumferential distribution in 10% of cells , or intense complete membrane staining in 30% of tumor cells . score + 3 indicates uniform intense membrane staining of > 30% of invasive tumor cells . scores 0 and + 1 were considered negative ; + 2 equivocal ; and + 3 positive . gene expression profiling studies have shown that immunohistochemistry of paraffin sections is a reliable surrogate for molecular classification of invasive breast cancers [ 4146 ] . based on this finding , patients of this study were categorized as follows : luminal a ( er+ , pr+ , her2neu ) , luminal b ( er+ and/or pr+ , her2neu+ ) , her2neu overexpressing ( er , pr , her2neu+ ) , and triple negative ( er , pr , her2neu ) . the z - test was used to test for statistically significant different proportions concerning disease- and treatment - related factors of patients 40 years versus > 40 years . the locoregional failure - free survival was estimated using the kaplan - meier method , and patient groups were compared using the log rank test . the locoregional failure - free survival was defined as the time between diagnosis and locoregional failure or death of any cause . patients who have not experienced a locoregional failure were censored at the time of their last follow - up . the simultaneous relationship of multiple prognostic factors to locoregional failure was assessed using cox 's proportional hazard regression analysis . the regression coefficients were estimated by the maximum likelihood method , and model selection was performed by a stepwise strategy using the likelihood ratio test . a 5% significance level was used and all tests are two - sided . no correction for multiple testing was used . the proportion of patients 40 years of all breast cancer patients who consulted saad specialist hospital was 22.6% , which is very much in accordance to the proportion of breast cancer patients 40 years published by the cancer incidence and survival report saudi arabia 2007 of 25.1% . two hundred and thirteen of all breast cancer patients met the eligibility criteria of this study and were analysed . of those , 158 patients were > 40 years of age at diagnosis and 55 patients 40 years . the patient and treatment characteristics are demonstrated in table 1 . the median follow - up time of the censored patients was 22 months ( mean 28 months , range 392 months ) . patients of 40 years at diagnosis exhibited statistically significantly less frequently the tumor type luminal a , and statistically significantly more frequently the tumor type triple negative compared to patients > 40 years ( table 1 ) . in addition , the body mass index ( bmi ) and the menopausal status were significantly different in the two age groups ( table 1 ) . the mean bmi ( standard deviation ) of all patients was 31.1 ( 6.0 ) , of patients 40 years 28.3 ( 5.2 ) , and for patients > 40 years 32.1 ( 6.0 ) . on univariate analysis , the age ( 40 versus > 40 years ) , t stage ( tis , t02 versus t3 - 4 ) , molecular tumor type ( luminal a versus luminal b , her2neu overexpression , triple negative ) , and lymphovascular status ( lv0 versus lv1 ) had a significant impact on the locoregional failure - free survival ( table 2 , figures 1 and 2 ) . the n status ( n0 versus n+ ) showed a statistical trend of an association with the locoregional failure - free survival ( p = 0.13 ) . on multivariate analysis , the age and t stage remained statistically significant ( table 3 ) . in saudi arabia , the proportion of breast cancer patients 40 years of age at diagnosis is about three times larger than generally reported in the west ( 25.1% versus 3.7%7.5% ) . according to the us census bureau , the proportion of females 40 years of the saudi arabian population is only 1.5 times larger than that of the us population ( 80.4% versus 52.2% ; year 2010 ) , suggesting that the larger proportion of breast cancer patients 40 years in saudi arabia may not be fully explained by the different age structures of the two populations . this observation and also the fact that some studies performed in asia and africa did not find a different prognosis of younger breast cancer patients compared to the older counterparts [ 48 , 49 ] suggest that regional differences may exist concerning the biology and prognosis of young breast cancer patients . detailed clinicopathological and prognostic data are scarce in saudi arabia and the middle east . our data show that young age is an independent negative prognostic factor for the locoregional control of breast cancer patients in saudi arabia . the same finding has been reported by another retrospective single - institutional study in saudi arabia . our findings are compatible with the notion that breast cancer arising in a younger host is a unique entity characterized not only by adverse prognostic features , but also by a diverse underlying biology against which novel therapeutics should be targeted [ 1 , 31 ] . several current consensus guidelines have included age 35 years as an absolute indication for adjuvant systemic chemotherapy irrespective of other tumor characteristics [ 3235 ] . the observation of a higher locoregional recurrence rate after bcs compared to mastectomy in patients 35 years of age in some studies [ 20 , 21 , 28 , 5156 ] raised the question about the optimal surgical approach of this patient group . however , a recently published large population - based analysis consisting of 1,453 early breast cancer patients 40 years showed that the 10-year overall survival was not impaired after bcs compared to mastectomy . in line with this observation , no difference of the two - year locoregional failure - free survival was detected in our study after bcs or mastectomy ( p = 0.59 ) . a pooled analysis of four eortc randomized controlled trials revealed that tumor size , nodal status , and molecular tumor subtype were independent prognostic factors for overall survival of the subgroup of young breast cancer patients . the authors concluded that future treatment guidelines concerning young breast cancer patients should be refined based upon tumor characteristics , probably derived from microarray driven translational research projects , and not based upon age alone [ 5961 ] . in our study , the molecular tumor subtype had a prognostic relevance for all patients on univariate analysis , but lost its significance on multivariate analysis . breast cancer in young women is probably the result of a complex interaction between genetic , environmental , and nongenetic patient related factors [ 24 , 25 , 27 , 31 , 6268 ] . however , no significant difference of the family history was found between breast cancer patients 40 years and > 40 years in our study . a striking difference to representative data from north america or europe was the significantly higher bmi of our study patients . the mean bmi ( standard deviation ) of our study population was 31.1 ( 6.0 ) compared to 24.8 ( 4.4 ) in north america ) and 25.5 ( 4.5 ) in europe . a closer look revealed that the main difference was confined to the subgroup of young patients . in our study , the proportion of patients 40 years with a bmi of 25 , 2530 , and > 30 was about 33% for each category , whereas in the western studies the corresponding proportions ranged from 57.0%73.2% ; 19.4%27.5% , and 12.1%17.1% , respectively [ 7175 ] . the differences of the bmi of older patients compared to the west were much smaller ( our study : 58.5% , 30.5% , and 11.0% ; western studies : 41.6%52.9% , 28.737.5% , and 11.5%20.9% [ 74 , 75 ] ) . studies have shown that the bmi is associated with poor prognosis in both premenopausal [ 7274 , 76 , 77 ] and postmenopausal patients [ 7480 ] . in addition , the bmi has been shown to be associated with an increased breast cancer risk in postmenopausal women [ 69 , 70 , 81 ] . in premenopausal women , a protective or no effect of the bmi on the breast cancer risk in contrast , several studies considering multiple surrogate markers for obesity have also reported an association of obesity with an increased risk to develop breast cancer in premenopausal women [ 69 , 87 , 88 ] . although the association between body weight and breast cancer appears to be complex , it represents an interesting factor to be evaluated in future studies concerning breast cancer in saudi arabia . patients 40 years exhibited more often triple negative and less frequently luminal a tumors compared to patients > 40 years . however , multivariate analysis revealed age 40 years as an independent adverse prognostic factor for the locoregional failure - free survival of breast cancer patients in saudi arabia .

background . this study was undertaken to evaluate the impact of prognostic factors on the locoregional failure - free survival of early breast cancer patients . methods . in this single - institutional study , 213 breast cancer patients were retrospectively analysed . fifty - five of 213 patients were 40 years of age at diagnosis . the impact of patient- or treatment - related factors on the locoregional failure - free survival was assessed using the kaplan - meier method . the simultaneous impact of factors on the locoregional failure - free survival was assessed using the cox proportional hazards regression analysis . results . the median follow - up time of the censored patients was 22 months ( mean 28 months , range 392 months ) . on univariate analysis , statistically significant factors for the locoregional failure - free survival were the age ( 40 versus > 40 years ) , t stage ( tis , t02 versus t3 - 4 ) , molecular tumor type ( luminal a versus luminal b , her2neu overexpression , or triple negative ) , and lymphovascular status ( lv0 versus lv1 ) . on multivariate analysis , age and t stage remained statistically significant . conclusions . being 40 years or younger has a statistically significant independent adverse impact on the locoregional failure - free survival of patients with early breast cancer .

1. Background 2. Methods 3. Results 4. Discussion 5. Conclusions

however , insufficient body water due to continuous dehydration caused by prolonged exercise leads to a decrease in plasma volume and cardiac output and an increase in plasma osmolarity and core temperature1 , 2 . furthermore , inefficient heat dispersion caused by a decreased rate of blood flow to the skin can accelerate fatigue and deteriorate exercise performance by retaining body heat3 , 4 . therefore , to improve exercise performance in athletes and to enhance the effects of regular exercise for health maintenance and promotion , it is important to develop a method that can alleviate exercise - induced rise in body temperature . far infrared electromagnetic wavelengths of 5.61,000 m can activate cells and promote metabolism when absorbed by the human body . previous studies verified the efficacy of ge - coated clothing5 , 6 , and observed that ge - containing fibers emitted far infrared rays and anions and had an antibacterial effect . these results suggested that ge - containing fibers could promote blood circulation and had the potential to suppress fatigue and relieve thermal stress by wearing ge - containing and/or ge - coated clothing during exercise . however , a scientific approach to proving such an effect has been very limited . therefore , the present study aimed to investigate the effects of wearing ge - coated functional clothing during exercise on tympanic temperature ; thermal sensation ; heat shock protein 70 ( hsp70 ) , which is involved in homeostatic regulation in exercise - induced thermal stress ; and lactate , an indicator of fatigue . nine healthy and untrained male subjects ( age : 20.2 0.4 years ; height : 175.7 2.8 cm ; weight : 73.1 10.1 kg ; body mass index : 23.7 3.0 kg / m ; muscle mass : 34.6 2.9 kg ; fat mass : 12.4 6.2 kg ; percentage body fat : 16.3 6.3% ; resting heart rate : 70.9 3.6 beats / min ; systolic blood pressure : 118.6 7.3 mmhg ; diastolic blood pressure : 72.6 8.4 mmhg ) volunteered as subjects for the present study . the study was conducted in accordance with the standards set by the latest revision of the declaration of helsinki , and all the subjects provided written informed consent in accordance with the department of sport for all studies at soongsil university guidelines . anthropometric measurements included height , body composition , resting heart rate , and blood pressure . height and body composition were measured using a stadiometer ( seca213 ; seca , germany ) and a bioimpedance analysis device ( inbody720 ; biospace , korea ) , respectively . resting heart rate and blood pressure were measured in a seated position , using a heart rate monitor ( polar a5 ; polar , finland ) and a mercury sphygmomanometer ( trimline ; pymah , usa ) , respectively . the 9 subjects participated in control and experimental tests , both involving 60 min of running on a treadmill at an exercise intensity of 75% heart rate reserve in a laboratory with temperature and humidity of 18.5 0.5 c and 36.0 1.0% , respectively . the clothing worn by each subject consisted of a half - sleeve t - shirt and short pants . during the control test , the participants wore conventional clothing ( which had not received functionality treatment ) ; during the experimental test , they wore ge - coated functional clothing ( which had been prepared by coating the same clothing used for the control test with ge ) . according to the result of a comparison test using fourier transform infrared spectroscopy ( ft - ir ) and a black body at 37 c , which is almost the same as body temperature , the far infrared rays emitted by the ge - coated functional clothing characteristically showed an emission rate of 0.893 m and a wavelength of 3.44 10 w / mm . tympanic temperature was measured using infrared ear thermometry ( thermoscan irt-4520 ; braun , germany ) as a surrogate for core temperature . thermal sensation was measured using a disc score7 . using a 22-gauge needle , a serum separator tube ( becton dickinson , usa ) , and an ethylenediaminetetraacetic acid - coated tube ( becton dickinson , usa ) , 8 ml of blood was collected from the antecubital vein of each subject immediately before exercise ( ibe ) and immediately after exercise ( iae ) . the collected blood samples were centrifuged for 15 min at 3,000 rpm , and then stored at 80 c until analysis . the serum hsp70 levels were determined using a high - sensitivity , commercially available enzyme - linked immunosorbent assay ( elisa ) kit ( assay designs , usa ) . the absorbance was measured at 450 nm with a microplate reader ( emax ; molecular devices , usa ) . the plasma lactate levels were determined using a clinical chemistry analyzer ( ektachem dt 60 ; eastman kodak , usa ) . statistical analyses were performed using spss software , version 21.0 for windows ( spss inc . data are presented as mean standard deviation ( sd ) . for identifying differences in normally distributed results , a two - way repeated analysis of variance ( anova ) when significant time by trial interactions occurred , simple main effects were assessed using the independent and paired t - tests . the tympanic temperature , disc score , hsp70 , and lactate levels are presented in table 1table 1.germanium-coated functional clothing effects on tympanic temperature , disc score , hsp70 , and lactate levelsvariablecontrol ( n=9)experimental ( n=9)ibeiaeibeiaetym t 36.73 0.3037.16 0.66disc score1.33 0.872.44 1.13*1.22 0.971.11 1.17hsp70 ( ng / ml)0.184 0.0170.205 0.027 * 0.193 0.0170.210 0.019*lactate ( mmol / l)1.48 0.602.84 1.34 * 1.12 0.452.03 0.89*data are presented as mean sd . tym t : tympanic temperature ; ibe : immediately before exercise ; iae : immediately after exercise ; rating scale range , 3 to 4 ; * p<0.05 vs. ibe ; p<0.05 vs. control test . the tympanic temperature iae was significantly elevated compared to the temperature ibe in the control test ( p<0.05 ) , while no significant change was observed in the experimental test . in both tests , the hsp70 and lactate levels measured iae were significantly increased compared to the levels ibe ( p<0.05 ) . the disc score iae was significantly increased compared to the score evaluated ibe in both tests ( p<0.05 ) , and it was significantly higher iae in the control test compared to that in the experimental test ( p<0.05 ) . tym t : tympanic temperature ; ibe : immediately before exercise ; iae : immediately after exercise ; rating scale range , 3 to 4 ; * p<0.05 vs. ibe ; p<0.05 vs. control test organic ge has been reported to promote immune activation and play an important role in regulating antioxidative activities8 , 9 . for this reason , it has been widely utilized for manufacturing not only health functional foods but also bracelets , necklaces , and textile products such as underwear . to verify the effects of wearing ge - coated functional clothing during exercise on thermal stress , in the present study , the results in the control test ( wearing common clothing ) showed a significant increase in tympanic temperature measured iae ; however , the experimental test ( wearing ge - coated clothing ) did not show such an increase . in addition , the measurement of thermal sensation using the disc score showed significantly lower levels iae in the experimental test than that in the control test . the reasons for such results could be that ge - coated clothing alleviated exercise - induced thermal stress by promoting blood circulation during exercise . this interpretation is supported by the observations that increase in skin blood flow during exercise is an effective mechanism for dispersing heat accumulated in the body10 , and that ge - containing clothing had the effect of promoting blood circulation5 , 6 . in both tests in the present study , the serum hsp70 and plasma lactate levels showed a significant increase iae , but without a significant difference between the 2 tests . the result for hsp70 levels supports the results of previous studies that reported a significant increase after acute exercise11 , 12 , and suggests that not only high core temperature but also various other factors contribute to hsp70 expression . this interpretation is consistent with the reports of a positive correlation between hsp70 expression and core temperature13 ; however , hsp70 expression was also affected by various physiological and environmental stress factors such as heat , training status , oxidative stress , ph change in the body , and glucose depletion14 , 15 . conversely , the lactate level is considered independent of thermal stress , probably due to the same level of exercise intensity used for both tests ; this result suggests that wearing ge - coated clothing during exercise exerts no significant effect on exercise - induced fatigue . this interpretation is supported by ishii and nishida , who reported that circulating lactate concentrations were dependent on exercise intensity16 , and also by mohr et al . , who reported that exercise under different environmental conditions ( 21 c vs. 43 c ) showed no difference in plasma lactate concentration in spite of significant differences in core temperature17 . in conclusion , the present study suggests that wearing ge - coated clothing during endurance exercise may have the positive effect of alleviating thermal stress that accumulates during exercise .

[ purpose ] the present study investigated the effects of wearing germanium - coated functional clothing on tympanic temperature , thermal sensation , heat shock protein 70 ( hsp70 ) , and lactate during endurance exercise . [ subjects and methods ] nine healthy and untrained male subjects were enrolled . subjects ran for 60 min on a treadmill ( 75% heart rate reserve ) in the following 2 tests : 1 ) control test ( wearing conventional clothing ) and 2 ) experimental test ( wearing germanium - coated functional clothing ) . during each test , the tympanic temperature and thermal sensation were measured , and blood samples were collected immediately before exercise and immediately after exercise . thermal sensation was measured using a disc score . [ results ] the tympanic temperature immediately after exercise was significantly increased compared to the temperature immediately before exercise in the control test , while no significant change was observed in the experimental test . in both tests , the disc score and hsp70 and lactate levels immediately after exercise were significantly increased compared to those immediately before exercise . in addition , the disc score immediately after exercise was significantly higher in the control test than in the experimental test . [ conclusion ] wearing germanium - coated functional clothing during endurance exercise may have the positive effect of alleviating thermal stress that accumulates in the body during exercise .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

a 70 year old woman first visited our clinic for right hand bradykinesia and gait disturbance unresponsive to levodopa treatment . at age 67 , she started to feel clumsiness in her right hand and to walk with very short stride and festination . her gait disturbance rapidly worsened until she was unable to walk without assistance after two years . at age 69 , she was diagnosed with pd and started to take levodopa . at age 70 , she took 450 mg of levodopa by regular levodopa / carbidopa , 150 mg by controlled release form of levodopa / carbidopa , 6 mg of ropinirole , and 200 mg of amantadine per day , but felt no clinical improvement . her verbal output was markedly reduced , and she could speak only simple words . on neurological examination , she showed masked face and no rest tremor . muscle tone was moderately elevated in her right limbs and mildly in the left limbs . she could not perform hand rolling , finger tapping , and foot tapping movements with her right limbs . deep tendon reflexes were normoactive . the stimulus sensitive jerks and ideomotor apraxia were observed in her right hand . a t2-weighted brain magnetic resonance imaging ( mri ) study showed diffuse cortical atrophy which was prominent in the left fronto - tempo - parietal cortex , and moderate degree of hypersignal intensities in diffuse subcortical white matter . an [ f]-deoxyglucose positron emission tomography study showed severe hypometabolism in left fronto - temporo - parietal cortex , striatum and thalamus ( figure 1 ) . because there was no response to levodopa treatment , we started to taper off anti - parkinsonian medications . three days after abrupt discontinuation of ropinirole , amantadine , and controlled release form of levodopa except regular levodopa / carbidopa , she developed severe dysphagia and dyspnea . serum ck level was 10,632 iu / l and urine myoglobin level was 2,881 ng / ml . the dosage of levodopa was increased to 750 mg per day and antibiotics treatment was started . during the course of following seven days , body temperature and serum ck level slowly returned to the normal level . the pd patients with longer disease duration , higher motor severity and motor fluctuation are likely to develop nms after abrupt reduction of anti - parkinsonian medications.3 abrupt deterioration of dopaminergic transmission is suggested to be a common pathophysiological mechanism of nms.2,4 however , the nms can occur in atypical parkinsonisms , including progressive supranuclear palsy , multiple system atrophy , and cbd , which accompany the pathology in postsynaptic striatal neurons and poor levodopa responsiveness.2,5 these suggest that peripheral mechanism or the alteration of neurotransmitters other than dopamine may also be involved in the pathogenesis of nms.2,4 in this patient , both reduction of levodopa dosage and concurrent infection might precipitate nms . therefore , prevention of infection , a common complication of parkinson - plus syndromes , is necessary to prevent nms . additionally , dose reduction or withdrawal of anti - parkinsonian medications should be carefully performed even in the patients with cbd who are expected to be unresponsive to levodopa treatment .

parkinson s disease is a principal underlying disease of neuroleptic malignant syndrome ( nms ) occurring in parkinsonian disorders , but nms may occur in patients with progressive supranuclear palsy and multiple system atrophy . we report first patient with corticobasal degeneration ( cbd ) who developed nms after abrupt reduction of antiparkinsonian medication and concurrent infection . it should be kept in mind that the prevention of infectious illness , which is common complication in parkinson - plus syndrome , is important , and dose reduction or withdrawal of anti - parkinsonian medications should be carefully performed even in the patients with cbd who are expected to be unresponsive to levodopa treatment .

Case Discussion

abstracting data from electronic medical records , we retrospectively selected a sample of adult inpatients who received insulin at boston medical center , most of whom had a diagnosis of diabetes , admitted between 1 july 2005 and 31 december 2009 . diabetes was defined by any one of the following : 1 ) administration of an oral antidiabetes medication ( metformin , a thiazolidinedione , or a sulfonylurea ) during hospitalization ; 2 ) a diabetes icd-9 code of 250.xx ; or 3 ) an a1c > 6.5% . patients were excluded if they were aged < 18 years , were admitted to an intensive care unit , had a primary diagnosis of hypoglycemia , had hypoglycemia ( any blood glucose < 70 mg / dl ) within 24 h of admission , or had no point - of - care ( poc ) capillary glucose values . the sample was restricted to only the first admission per patient , and data subsequent to the first hypoglycemic event after 24 h were excluded . only poc values were used to measure glucose because of variability among different glucose assays . case subjects were defined by a poc glucose < 70 mg / dl after the first 24 h of admission . the time period examined for each case was 24 h prior to the first hypoglycemic event in order to include all relevant doses of insulin . control subjects , defined by a poc glucose 70 mg / dl , were matched one to one on the basis of the hospital day of hypoglycemia , age ( 1830 , 3164 , or 6580 years ) , sex , and bmi ( < 18.5 , 18.525 , 2530 , or > 30 kg / m ) . matching on hospital day of hypoglycemia provided a control time frame as well as controlled for variation in clinical status during hospitalization . the exposure was defined as the total insulin dose per body weight ( units / kg ) over the 24-h study period . insulin doses were stratified into nonoverlapping ranges , as follows : 0 , < 0.2 , 0.20.4 , 0.40.6 , 0.60.8 , and 0.8 units / kg . the exposure was grouped by insulin regimen type as follows : 1 ) glargine plus any other insulin , 2 ) nph plus any other insulin , 3 ) lispro and/or regular insulin only , and 4 ) no insulin . for 11 patients who received both glargine and nph in 1 day , group assignment was on the basis of whichever insulin dose was greater . for two patients who received the same total 24-h dose of glargine and nph , group assignment was on the basis of the insulin given closest to the hypoglycemic event . the following known predictors of hypoglycemia and potential confounders were examined : weight , race , serum creatinine , albumin , liver function ( aspartate aminotransferase and alanine aminotransferase ) , a1c , white blood cell count , hematocrit , use of sliding - scale insulin ( ssi ) , fasting , length of stay , service ( medical or surgical ) , and disease severity defined by the charlson comorbidity index ( 12 ) . use of ssi was categorized as only ssi , no ssi , or ssi plus scheduled insulin . data were collected during hospitalization , except for missing values that were substituted by the closest value up to 3 months prior to admission . bmi values < 10 or > 100 kg / m , weight < 20 or > 400 kg , and height < 100 or > 300 cm were considered erroneous and were deleted . for these individuals , the average height of the sample for each sex was imputed ( 174.2 9.6 cm for male subjects and 160.8 8.5 cm for female subjects ) . admission glucose was defined as the first poc glucose value within 24 h of admission . summaries of categorical variables included counts and percentages , whereas for continuous variables means and sds were used . comparability between the case and control groups for categorical variables was determined using the test for categorical variables and the two - sample t test for continuous variables . regression was used for length of stay because this continuous , positive variable was not normally distributed . conditional logistic regression was used to examine the unadjusted and the adjusted association of insulin dose with hypoglycemia . the initial adjusted model included all variables that were associated with hypoglycemia , as well an interaction term for dose by regimen to assess effect modification by insulin regimen type on the relationship between insulin dose and hypoglycemia . we then performed backward selection with the level to keep variables in the model set at 0.2 ( 13 ) . all analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . abstracting data from electronic medical records , we retrospectively selected a sample of adult inpatients who received insulin at boston medical center , most of whom had a diagnosis of diabetes , admitted between 1 july 2005 and 31 december 2009 . diabetes was defined by any one of the following : 1 ) administration of an oral antidiabetes medication ( metformin , a thiazolidinedione , or a sulfonylurea ) during hospitalization ; 2 ) a diabetes icd-9 code of 250.xx ; or 3 ) an a1c > 6.5% . patients were excluded if they were aged < 18 years , were admitted to an intensive care unit , had a primary diagnosis of hypoglycemia , had hypoglycemia ( any blood glucose < 70 mg / dl ) within 24 h of admission , or had no point - of - care ( poc ) capillary glucose values . the sample was restricted to only the first admission per patient , and data subsequent to the first hypoglycemic event after 24 h were excluded . only poc values were used to measure glucose because of variability among different glucose assays . case subjects were defined by a poc glucose < 70 mg / dl after the first 24 h of admission . the time period examined for each case was 24 h prior to the first hypoglycemic event in order to include all relevant doses of insulin . control subjects , defined by a poc glucose 70 mg / dl , were matched one to one on the basis of the hospital day of hypoglycemia , age ( 1830 , 3164 , or 6580 years ) , sex , and bmi ( < 18.5 , 18.525 , 2530 , or > 30 kg / m ) . matching on hospital day of hypoglycemia provided a control time frame as well as controlled for variation in clinical status during hospitalization . the exposure was defined as the total insulin dose per body weight ( units / kg ) over the 24-h study period . insulin doses were stratified into nonoverlapping ranges , as follows : 0 , < 0.2 , 0.20.4 , 0.40.6 , 0.60.8 , and 0.8 units / kg . the exposure was grouped by insulin regimen type as follows : 1 ) glargine plus any other insulin , 2 ) nph plus any other insulin , 3 ) lispro and/or regular insulin only , and 4 ) no insulin . for 11 patients who received both glargine and nph in 1 day , group assignment was on the basis of whichever insulin dose was greater . for two patients who received the same total 24-h dose of glargine and nph , group assignment was on the basis of the insulin given closest to the hypoglycemic event . the following known predictors of hypoglycemia and potential confounders were examined : weight , race , serum creatinine , albumin , liver function ( aspartate aminotransferase and alanine aminotransferase ) , a1c , white blood cell count , hematocrit , use of sliding - scale insulin ( ssi ) , fasting , length of stay , service ( medical or surgical ) , and disease severity defined by the charlson comorbidity index ( 12 ) . use of ssi was categorized as only ssi , no ssi , or ssi plus scheduled insulin . data were collected during hospitalization , except for missing values that were substituted by the closest value up to 3 months prior to admission . bmi values < 10 or > 100 kg / m , weight < 20 or > 400 kg , and height < 100 or > 300 cm were considered erroneous and were deleted . for these individuals , the average height of the sample for each sex was imputed ( 174.2 9.6 cm for male subjects and 160.8 8.5 cm for female subjects ) . admission glucose was defined as the first poc glucose value within 24 h of admission . summaries of categorical variables included counts and percentages , whereas for continuous variables means and sds were used . comparability between the case and control groups for categorical variables was determined using the test for categorical variables and the two - sample t test for continuous variables . regression was used for length of stay because this continuous , positive variable was not normally distributed . conditional logistic regression was used to examine the unadjusted and the adjusted association of insulin dose with hypoglycemia . the initial adjusted model included all variables that were associated with hypoglycemia , as well an interaction term for dose by regimen to assess effect modification by insulin regimen type on the relationship between insulin dose and hypoglycemia . we then performed backward selection with the level to keep variables in the model set at 0.2 ( 13 ) . all analyses were performed using sas ( version 9.2 ; sas institute , cary , nc ) . of 6,376 eligible patients abstracted from hospital records , 1,012 ( 15.8% ) had hypoglycemia . after matching , 995 case subjects ( 98.3% ) and 995 nonhypoglycemic control subjects remained ( supplementary table 1 ) . compared with 4,386 unmatched patients , the matched control sample was older ( aged 63.3 vs. 60.6 years , p < 0.001 ) , had lower weights ( 86.1 vs. 91.4 kg , p < 0.001 ) , were less obese ( bmi 31.2 vs. 33 kg / m , p < 0.001 ) , had a higher proportion of african americans ( 39.4 vs. 36.6% , p = 0.04 for race ) , and a had lower proportion of hispanics ( 12.7 vs. 16.1% ) . the matched and unmatched control subjects were similar in terms of sex distribution and mean a1c . the median time from admission to the first hypoglycemic event in case subjects was 2.7 days ( range 1.033.7 ) . during the index 24-h period , glargine was the most frequently administered insulin ( 31.7% ) , followed by short - acting insulin ( 28.4% ) , no insulin ( 24.0% ) , and nph insulin ( 15.9% ) ( table 1 ) . ssi only was less common than ssi plus scheduled insulin and no ssi at all ( 25.3 , 37.0 , and 37.7% ) . approximately 50% of the patients were given metformin , 33% were given a sulfonylurea , and 13% were given a thiazolidinedione . the sample was racially diverse : 40.6% were african american , 34.1% were white , 13.9% were hispanic , and 11.4% were of other race / ethnicity . the mean a1c was 8.1 2.2% , and mean serum creatinine was 1.5 1.7 mg / dl . of the 1,990-patient sample , 1,418 ( 71.3% ) were not on insulin as an outpatient . sample characteristics of unmatched factors by case status data are n ( % ) , percentages , or means sd . * p values for categorical variables were generated by tests . hypoglycemia prevalence among patients who received lispro was similar to that among patients given regular insulin ( 31.8 vs. 36.1% ) . hypoglycemic patients were more likely to be given glargine or nph and less likely to be given only ssi or no insulin than control subjects ( p < 0.001 ) . ssi plus scheduled insulin was more common , whereas ssi without scheduled insulin was less common , among case subjects than control subjects ( p < 0.001 ) . hypoglycemic patients had a lower albumin and hematocrit and higher creatinine and length of stay than nonhypoglycemic patients ( p < 0.001 for each comparison ) . case subjects also had higher comorbidity index scores than control subjects ( p < 0.001 ) . there was no difference in mean a1c ( 8.0 2.1 vs. 8.1 2.3% , p = 0.44 ) or admission glucose ( 199.8 109.5 vs. 193.6 96.9 mg / dl , p = 0.20 ) between case and control subjects . higher insulin doses were associated with progressively higher blood glucose levels ( supplementary table 2 ) . within each dose range , the mean glucose levels of case subjects were lower than the mean glucose levels of control subjects . relative to insulin doses < 0.2 units / kg , the unadjusted odds of hypoglycemia increased with increasing insulin dose ( table 2 ) . adjusted for insulin regimen , ssi use , and albumin , creatinine , and hematocrit levels , the higher odds of hypoglycemia with increasing insulin doses remained ( table 3 and fig . 1 ) . patients who received insulin doses of 0.6 units / kg were at increased odds of hypoglycemia . in contrast , the adjusted odds of hypoglycemia were not higher in patients who received 0.20.6 units / kg . the association of insulin dose with hypoglycemia did not vary by regimen type , as indicated by a statistically nonsignificant interaction term in the multivariate model ( p = 0.524 ) . distribution of insulin dose by case status data are n ( % ) or percentages . predictors of hypoglycemia in the multivariate conditional logistic regression model multivariate - adjusted odds of hypoglycemia at different insulin doses in 995 case subjects vs. 995 matched control subjects . error bars represent 95% cis . or , odds ratio . * p < 0.05 by conditional logistic regression . in addition to insulin dose , there were several significant predictors of hypoglycemia , including creatinine and hematocrit . the odds of hypoglycemia were threefold greater among those who did not receive ssi relative to those who did . patients who received ssi plus scheduled insulin or ssi alone had higher blood glucose levels than patients who did not receive ssi ( 203 64 , 185 52 , and 128 33 mg / dl , p < 0.001 ) . there was a trend toward higher odds of hypoglycemia among patients who received nph compared with patients given glargine or short - acting insulin . to investigate whether the risk of hypoglycemia depends on the ratio of basal insulin ( nph or glargine ) relative to the total daily dose , a post hoc analysis revealed that basal ratios < 0.4 confer lower odds of hypoglycemia than 0.40.6 ( odds ratio 0.50 [ 95% ci 0.250.97 ] , p = 0.040 ) . in contrast , there was no difference in the odds of hypoglycemia between basal ratios > 0.6 or 0 and 0.40.6 . hypoglycemia was not more common among patients given insulin with an oral diabetes medication compared with those on insulin alone ( 49.4 vs. 54.6% , p = 0.13 ) . however , of the 201 patients who did not receive insulin but did receive an oral agent , sulfonylureas were more common among hypoglycemic patients than in control subjects ( 27.4 vs. 16.4% , p < 0.001 ) . patients who received only ssi were more likely to be given an oral agent than those on ssi plus scheduled insulin and those not given ssi ( 54.5 , 28.1 , and 38.3% , p < 0.001 ) . although hypoglycemia was proportionately more common on medical services than on surgical services ( table 1 , p = 0.008 ) , this variable was not significant in the multivariate model . there was no difference in mean blood glucose in medical versus surgical patients ( 173 62 vs. 168 59 , p = 0.12 ) . however , medical patients received more insulin than surgical patients ( 0.35 0.88 vs. 0.26 0.38 units / kg , p = 0.013 ) . in addition , ssi only was less common among medical patients ( 20.9 vs. 36.0% , p < 0.001 ) . the sample included 77 patients ( 3.9% ) without a diagnosis of diabetes who had an inpatient a1c > 6.5% . a sensitivity analysis excluding these patients yielded similar results . this retrospective , matched , case - control study of 1,990 hospital - ward patients with diagnosed or probable diabetes shows that higher weight - based insulin doses are associated with greater odds of hypoglycemia , independent of the types of insulin used . adjusted for insulin regimen , ssi use , and albumin , creatinine , and hematocrit levels , patients given at least 0.8 units / kg within a 24-h period are at threefold - higher odds of hypoglycemia than patients who receive < 0.2 units / kg . however , 0.6 units / kg seems to be a threshold below which the odds of hypoglycemia are relatively low . in addition , patients who do not receive ssi are at threefold - greater odds of hypoglycemia than patients who receive ssi with or without scheduled insulin . because ssi typically is administered in response to a glucose value above a predetermined threshold , patients given ssi are hyperglycemic by design . patients who did not receive ssi may have been given excessive scheduled insulin , resulting in more frequent hypoglycemia . together , these data suggest that the insulin program with the lowest risk of hypoglycemia consists of scheduled insulin plus ssi at total daily doses < 0.6 units / kg . our findings are broadly consistent with other studies of insulin use in non intensive care unit inpatients that show that insulin is a risk factor for hypoglycemia ( 14,15 ) . these studies , however , did not examine how the risk of hypoglycemia varies by insulin dose or type . the lack of a clear difference in the odds of hypoglycemia between glargine and nph is similar to data from a randomized trial comparing weight - based analog insulins with nph and regular insulins that found no difference in hypoglycemia rates between the treatment groups ( 11 ) . also consistent with other studies are our findings that high creatinine and low hematocrit levels are independently associated with hypoglycemia ( 16,17 ) . the frequent use of oral agents was surprising given that our medication guideline for hospitalized patients with diabetes encourages the discontinuation of oral agents . however , we support the judicious use of oral diabetes medications in patients who are eating regularly , who are no longer acutely ill , and/or who preparing for discharge , consistent with some expert opinions ( 1,18 ) . our data provide evidence for the safety of this approach in terms of hypoglycemia . a number of limitations must be acknowledged . also , bias can not be completely accounted for by matching and multivariate analysis of retrospective data . the use of poc glucose values at the exclusion of serum glucose values may underestimate the occurrence of hypoglycemia . this effect , however , is likely to be small because almost all inpatients with diabetes are ordered poc testing four times daily , and a poc glucose is more likely to be checked on a patient with hypoglycemic symptoms than a serum glucose . another limitation is that a case - control design precludes the calculation of hypoglycemia rates ; however , the odds ratio provides a reasonable estimate of relative risk . an additional limitation is our inability to classify the sample by diabetes type because of unavailable data . however , at least 90% of the sample is estimated to have type 2 diabetes , in keeping with national estimates ( 19 ) . finally , the focus of our study was hypoglycemia , and we did not examine the association of insulin dose with hyperglycemia . although lower - dose insulin regimens may confer a lower risk of hypoglycemia , they may be associated with a higher risk of hyperglycemia . these data , however , suggest that 0.40.6 units / kg , a dose range tested in randomized controlled trials and recommended by the american diabetes association ( 5,9,11 ) , may be the highest dose at which the risk of hypoglycemia remains low . to determine a starting dose of insulin within this range for a specific patient , clinicians must balance the presence of hypoglycemia risk factors with the benefits of achieving glycemic control . despite these limitations , strengths of our study include a large sample size spanning 4.5 years . because the case and control subjects had similar admission glucose values and because we adjusted for numerous potential confounders , it is likely that we have isolated the effect of inpatient insulin dose on hypoglycemia . matching case subjects on the basis of hospital day , age , sex , and bmi furthermore , no published study has investigated the association of weight - based insulin with inpatient hypoglycemia across a range of doses and insulin types . one is whether the risk of hypoglycemia depends on the ratio of basal insulin relative to the total daily dose , which is an important component of insulin protocols ( 911 ) . post hoc analysis of our data revealed that basal ratios < 0.4 confer a lower odds of hypoglycemia than basal ratios of 0.40.6 . this may reflect a higher proportion of patients who received ssi , who are by definition hyperglycemic as discussed above . prospective studies are necessary to determine the effect of basal ratio in scheduled insulin doses across heterogeneous inpatient populations . another question is whether higher insulin doses cause hypoglycemia more quickly than lower insulin doses . in conclusion , we found that higher weight - based insulin doses are associated with greater odds of hypoglycemia , and this association does not differ by the type of insulin used . insulin doses < 0.6 units / kg confer a lower odds of hypoglycemia , whereas doses > 0.6 units / kg are associated with higher odds of hypoglycemia . given that insulin is recommended for most hospitalized patients with diabetes ( 4 ) , and hypoglycemia is associated with increased length of stay and mortality ( 15 ) , strategies to reduce hypoglycemia are of critical importance . our findings provide additional information to help clinicians dose insulin more safely for inpatients with diabetes .

objectiveto determine the association of weight - based insulin dose with hypoglycemia in noncritically ill inpatients with diabetes.research design and methodswe performed a retrospective , case - control study of 1,990 diabetic patients admitted to hospital wards . patients with glucose levels < 70 mg / dl ( case subjects ) were matched one to one with nonhypoglycemic control subjects on the basis of the hospital day of hypoglycemia , age , sex , and bmi.resultsrelative to 24-h insulin doses < 0.2 units / kg , the unadjusted odds of hypoglycemia increased with increasing insulin dose . adjusted for insulin type , sliding - scale insulin use , and albumin , creatinine , and hematocrit levels , the higher odds of hypoglycemia with increasing insulin doses remained ( 0.60.8 units / kg : odds ratio 2.10 [ 95% ci 1.084.09 ] , p = 0.028 ; > 0.8 units / kg : 2.95 [ 1.545.65 ] , p = 0.001 ) . the adjusted odds of hypoglycemia were not greater in patients who received 0.20.4 units / kg ( 1.08 [ 0.641.81 ] , p = 0.78 ) or 0.40.6 units / kg ( 1.60 [ 0.902.86 ] , p = 0.11 ) . although the relationship between insulin dose and hypoglycemia did not vary by insulin type , patients who received nph trended toward greater odds of hypoglycemia compared with those given other insulins.conclusionshigher weight - based insulin doses are associated with greater odds of hypoglycemia independent of insulin type . however , 0.6 units / kg seems to be a threshold below which the odds of hypoglycemia are relatively low . these findings may help clinicians use insulin more safely .

RESEARCH DESIGN AND METHODS Study sample Variable definitions Statistical analysis RESULTS CONCLUSIONS Supplementary Material

the receptor apj remained orphan until 1998 , when tatemoto and his coworkers isolated its endogenous ligand from bovine stomach extract . they isolated a 36-amino - acid peptide which was named apelin ( from apj endogenous ligand ) . apelin exists in at least three forms , consisting of 13 , 17 , or 36 amino acids , all originating from a common 77-amino - acid precursor . apelin has been shown to be involved in vessel formation , where it exerts a pro - angiogenic role [ 3 , 4 ] , and in the regulation of cardiovascular function , by reducing arterial blood pressure , via stimulation of nitric oxide - mediated vasorelaxation [ 5 , 6 ] . moreover , apelin has recently been added to the family of adipokines [ 7 , 8 ] , which are adipocytokines mainly derived from adipose tissue as well as endothelial cells ( ecs ) in various parts of the body . hepatitis c virus ( hcv ) has been recognized as a major cause of chronic liver disease(s ) ( cld ) in egypt . the emerging role of apelin in cld is complex , as described in a recent report linking apelin to the initiation and maintenance of the inflammatory and fibrogenic processes occurring in the fibrotic liver , as well as to the vascular and haemodynamic abnormalities in cirrhosis and its complications [ 11 , 12 ] . however , clinical data demonstrating the role of apelin in cld is limited , as depicted by bertolani and marra . although barely detectable in normal liver , production of tnf- from t - cells occurs at the early onset in many types of liver injury and has been related to fibrosis progression . moreover , adipokines as leptin , resistin , visfatin , and adiponectin are recently considered as regulators of liver fibrogenesis and may explain why obesity influences fibrosis progression . hepatic insulin signaling is markedly impaired in hcv patients ; in addition , down regulation of hepatic insulin mediators is associated with enhanced hepatocyte apoptosis and fibrogenesis . currently some evidence supports a relationship between insulin resistance ( ir ) and hepatitis c on one hand , playing role in progression of liver disease [ 1720 ] , and between ir and apelin level on the other hand in cases of obesity . therefore , highlighting the apelin system would present a new therapeutic target for liver disease . thus , this study aims to address if there is relation between apelin serum levels and liver disease progression in a previously characterized cohort of established chc egyptian patients . the second question is whether obesity , inflammation ( tnf- ) , and ir are cofounders for this association . twelve subjects served as healthy controls ( group i ) selected from healthy subjects working in , or attending with their relatives , to the outpatient clinics of ain shams university specialized hospitals ( asush ) . none of the healthy controls took any medication or dietary supplements including vitamin(s ) and/or antioxidant(s ) . seventy three patients with chc were selected from cairo liver center , dokki , cairo , egypt . after protocol approval , all subjects gave written informed consent prior to participation and the study was approved by the committees on medical ethics of the cairo liver center and asush . the study was carried out in accordance with the regulations and recommendations of the declaration of helsinki . patients enrolled in the study were classified into the following groups : asymptomatic chronic hepatitis c carriers ( asc ) ( group ii ) ( n = 20 ) with normal liver function tests ( lfts ) , namely , alanine transaminase ; alt , for at least 6 months . group iii were patients with liver fibrosis ( n = 20 ) and patients with cirrhosis ( group iv ) ( n = 33 ) . histopathological staging was done using metavir scoring system where fibrosis was staged as ( f1f3 ) and ( f4 ) represents established cirrhosis . the following exclusion criteria were used for all subjects : hypertension , cardiopulmonary disease , renal disease , endocrine disorders including diabetes , malignancy , having previous interferon treatment or recently received any antiinflammatory drugs , as well as smokers and alcoholics , and other causes of cld as chronic hepatitis b , autoimmune hepatitis , acute hepatitis , haemochromatosis , hepatocellular carcinoma , and biliary disorders . a detailed drug treatment(s ) history was collected , and physical examination of the patients was carried out with special emphasis on previous surgical procedures and prior parenteral therapy . complete clinical examination was done , on the day of sample withdrawal which included the manifestations of hepatitis such as hepatomegaly , tenderness in the right hypochondrium , splenomegaly , and lower limb edema , or liver cell failure such as jaundice , hepatic encephalopathy , bleeding varices , and ascites . biopsy samples were assessed unless contraindication or established cirrhosis was present ; abdominal ultrasonography and endoscopy were also done side by side with routine laboratory investigations including complete blood picture , liver , and kidney function tests . hcv was diagnosed by anti - hcv assay by a third generation enzyme immunoassay , and hcv rna by real time pcr . medical records indicated that all patients were of hcv genotype 4 which is the most common in egyptian patients . body mass index ( bmi ) was calculated as an index of the weight ( in kilograms ) divided by the square of the height ( in meters ) measured on the same day of sample withdrawal . centers for disease control and prevention ( cdc ) classify the normal range of bmi to be between 18.524.9 kg / m , overweight bmi between 2529.9 kg / m , and the obese bmi > 30 kg / m . since bmi could be influenced by the presence of ascites in patients with cirrhosis , earlier weights taken prior to the diagnosis of ascites were used for the calculation of bmi . all subjects were advised to take no medication on the morning before blood sample collection . fasting blood ( 5 ml ) was obtained from the antecubital vein , after an overnight fasting period ( 1012 hours ) . samples were divided into two parts ; one containing trisodium citrate ( final concentration 1 mg / ml ) for prothrombin time ( pt ) determination . the other part was taken into vacutainer clotted tubes , where sera were separated by centrifugation at 3000 rpm for 10 min for other lab measurements . other sera aliquots were kept frozen at 70c for measurement of serum apelin ( with no need for aprotinin pretreatment step applied to plasma samples ) , tnf- , and insulin . sera were obtained , aliquoted for the measurement of lfts : aspartate transaminase ( ast ) , alt , bilirubin , albumin , fasting blood glucose ( fbg ) , and lipids ( total cholesterol ( tc ) and triacylglycerol ( tag ) ) by using standard enzymatic techniques using appropriate kits and semiautomated photometer 5010 . determination of serum apelin ( phoenix pharmaceuticals , burlingame , calif ) using the apelin-12 microplate enzyme - linked immunosorbant assay ( elisa ) kit , tnf- ( orgenium laboratories elisa ) , and insulin ( monobind inc . ) using enzyme - linked immunosorbant assay ( elisa ) kits , and , following elisa , procedures were carried out according to the manufacturers ' instructions . insulin resistance was determined by the homeostasis model of assessment ( homa ) using the formula : fasting insulin ( iu / ml ) fasting blood glucose ( mg / dl)/405 . data was expressed as mean s.d for quantitative parametric measures , in addition to median and iqr for nonparametric data and percentiles for categorical data . student 's t test was used for comparison of two independent groups for parametric data and wilcoxon rank sum for nonparametric data . however , for comparison between more than 2 patient groups for parametric data , we used analysis of variance ( anova ) . multiple comparisons ( post hoc test : lsd ( least significant difference ) ) were also followed to investigate the possible statistical significance between each 2 groups . moreover , comparison between more than 2 patients ' groups for nonparametric data kruskall wallis test was used . finally , spearman 's ranked correlation test , to study the possible association between each two variables among each group for nonparametric data , using the probability of error at 0.05 was considered significant , while at 0.01 and 0.001 were highly significant . multiple linear regression analysis was done on all measured parameters to allow for adjustment of apelin level . the clinical and demographic characteristics as well as studied parameters of all participants ( control and chc patients ) are shown in table 1 . significant differences in median values of apelin , tnf- , and homa - ir levels existed between control and chc patients as shown in table 1 . chc patients were subdivided into three groups : asc ( 20 patients ) , fibrotic ( 20 patients ) , and cirrhotic ( 33 patients ) . laboratory investigations and studied parameters for each group are detailed in table 2 . regarding ir , it was significantly higher in all groups when compared to control . it was also significantly higher in cirrhotic patients ( 4.1 ) in comparison to asc ( 2.75 ) . tnf- level was also higher in all groups in comparison to controls with median ( 76.6 ) being highest among asc ( 616 ) . hcv - related disease progression was associated with greater median values of apelin compared to nondiseased control patients , while those with cirrhosis had the highest apelin concentrations among those with liver disease , with median value ( 3.9 ) . patients with fibrosis also displayed higher median values of apelin ( 3 ) when compared to either controls ( 1.11 ) or asc ( 2 ) , with nonsignificant differences among different fibrosis metavir scores . these results were consistent when apelin was adjusted for bmi , tag , and tc covariants , whereas elevation in apelin levels disappeared only in case of asc after adjustment as depicted in table 2 . table 3 illustrates apelin levels among different 2 patient subgroups , where cirrhotic patients ( n = 33 ) were further subdivided into : ( a ) patients not suffering from complications including portal hypertension and/or ascites ( 13 subjects ) ; ( b ) patients encountering complications as portal hypertension and/or ascites ( 20 patients ) . upon comparing adjusted - apelin level among the two subgroups , it was found to be significantly higher in those with complications with median ( 2.36 ) in comparison to those free of such complications ( 1.87 ) as shown in ( table 3 ) . in an attempt to test our second aim reclaiming whether obesity is a cofounder for apelin serum levels association with chc disease progression , we analyzed apelin levels in the obese ( 36 patients ) versus lean ( 37 patients ) . it was found to be significantly higher in the latter with median value ( 3.6 ) versus ( 2.96 ) in the former ( table 4 ) . additionally , chc patients were subdivided into ir ( 43 patients ) and non - ir ( 30 patients ) . adjusted - apelin level was significantly higher in ir subjects with median ( 1.9 ) than non - ir ones ( 1.7 ) ( table 4 ) . correlations of adjusted - apelin levels to different parameters showed modest positive relation to ir in all chc patients ( r = 0.2663 , p < 0.05 ) ( figure 1(a ) ) . such relation appeared to be significant in group of asc ( r = 0.665 , p < 0.01 ) but was nonsignificant for fibrotic and cirrhotic groups ( table 5 ) . additionally , adjusted - apelin was found to be negatively correlated to tnf- in all chc patients ( n = 73 ) with high significance ( r = 0.5994 , p < 0.0001 ) ( figure 1(b ) ) . further analysis within patients groups revealed significant negative correlation in the cirrhotic group ( r = 0.488 , p < 0.01 ) ( table 5 ) . recent emerging studies pointed to the possible multiple effects of the apelinergic system in the liver and related it to oxidative stress , inflammation , fibrosis , angiogenesis , as well as haemodynamic and vascular disturbances [ 11 , 12 ] . apelin was evaluated in all groups both before and after adjustment for bmi , tag , and tc that acted as potential covariants . studies assessing apelin-36 and apelin-12 levels in patients with nonalcoholic fatty liver disease ( nafld ) indicated that the elevation of this peptide did not persist after adjustment for potential confounders and rather attributed apelin elevation in these cases to obesity and ir that are closely associated with nafld [ 27 , 28 ] . in contrast to nafld models , the present study shows that serum apelin level is elevated in patients with either fibrosis or cirrhosis due to hcv , with significant differences among the two groups being higher in the latter , even after apelin adjustment . this is in line with previous investigations demonstrating that patients with cirrhosis showed significant increase in apelin circulating levels [ 11 , 29 ] . moreover , recent emerging studies speculated that activated hepatic stellate cells ( hscs ) represent a potential source for apelin in liver and that apelin could be an important mediator of the profibrogenic gene induction that markedly stimulates collagen - i synthesis . in addition , apelin contributes to platelet - derived growth factor - induced proliferation of hsc 's in vitro , all of which are known to contribute largely to fibrosis progression and extracellular matrix deposition [ 30 , 31 ] . in this setting , we also found apelin serum level to be significantly elevated in patients with fibrosis . thus , apelin emerges as a major contributor to the fibrogenic process(es ) occurring in liver disease as well as playing role in disease progression . on the other hand , this elevation in case of asc disappeared after apelin adjustment to cofounders , which points to the interference of these cofactors in elevating apelin levels and may result in the early upset of the system in chc patients . moreover , the current study demonstrated that this peptide is closely associated to ascites and portal hypertension complications . tiani and his coworkers suggested that the expression of endogenous apelin / apj signaling is associated with development of portal hypertension and contributes to the formation of portosystemic collateral blood vessels and splanchnic neovascularization in portal hypertensive rats . in the last few years , several data have accumulated suggesting that obesity also plays role in development and progression of liver disease of well - defined etiology . recently , expression of both apelin and apj has been described in adipocytes and is suggested to stimulate blood vessel growth , due to its proangiogenic activity , thus leading to increased growth of adipose tissue . coinciding with that line , our study also demonstrated that obese subjects with chc had significant increased circulating apelin levels than lean patients , regardless of the stage or grade of liver disease . on the other hand , studies also demonstrated that apelin expression was higher in animal models of obesity associated with hyperinsulinemia , in addition to its role in adipogenesis and steatosis ; all of which contribute largely to fibrosis progression , as well as higher degree of inflammation [ 32 , 34 ] . further insight in our study revealed that ir was significantly higher among all groups of chc in comparison to the control group . moreover , significant difference was also found between asc and cirrhotic groups , being higher in the latter . moreover , when comparing apelin levels in ir and non - ir groups , it was significantly higher in ir group . additionally , there was significant positive correlation between ir and adjusted - apelin in patients with chc . this is in agreement with a previous study conducted by aktas et al . , who depicted that apelin , the novel adipokine , was associated with the components of the metabolic syndrome ( hyperlipidemia , obesity , and ir ) in nafld patients . however , our correlation was quite significant in asc but was unexpectedly nonsignificant in the case of progressive stages of fibrosis and cirrhosis , suggesting that the upset of the apelin system in these stages may follow a unique different pattern irrelevant to ir . moreover , previous investigations had pointed to tnf- as an inductor of apelin synthesis in adipocytes . tnf- was mostly elevated in cases of asc , this was in agreement with zekri and his coworkers but was opposite to what was reported by goyal et al . and toyoda et al . . this could be attributed to the difference in the epitopes of the elisa system used by the different investigating groups or to the difference in genotypes where all our patients were hcv genotype 4 . moreover , the striking elevation of this proinflammatory cytokine in carriers may reflect both insufficiencies of hcv elimination and/or a failure to control the cytokine cascade . in the current study , negative correlation was found between tnf- and adjusted - apelin in patients with chc . further analysis of such correlation in chc groups revealed a positive correlation between tnf- and apelin in cases of asc but was nonsignificant after adjustment for covariants , emphasizing the role of covariants in the induction of apelin in early stages of hepatitis . interestingly , in case of , cirrhosis , there was significant negative correlation , suggesting that apelin induction in cld follows alternative signaling pathways . this is in agreement with melgar - lesmes and his coworkers who pointed to the diminution of apelin expression induced by tnf- in hsc cultures and explained that it could represent a homeostatic protective response toward reducing the overactivated hepatic apelin system in patients with advanced liver disease . moreover , this provides a rationale to investigate new drugs targeting the apelin / apj signaling pathway to reduce fibrosis and to improve hemodynamics in those patients . circulating serum apelin level varies in different stages of chc , which in conjunction with ir and obesity , would contribute to fibrosis progression . additionally , apelin is more elevated in patients with ascites and portal hypertension and may have role in the development of these complications . apelin , being recently added to the list of adiposity signals , in this setting , apelin targeting could be of additional benefit in obese patients with concomitant cld , for alleviating both conditions and possibly resulting in better outcomes .

objective . highlighting the apelin system would present a new therapeutic target for liver disease . apelin ; endogenous ligand for the orphan receptor apj , was recently suggested to be associated with fibrosis progression and cirrhosis in addition to insulin resistance ( ir ) and inflammation . the present study was conducted to evaluate blood apelin level changes among 73 chronic hepatitis c ( chc ) egyptian patients and if associated with body mass index ( bmi ) , ir , and tumor necrosis factor - alpha ( tnf- ) . serum apelin levels were significantly higher in patients with chc with median value ( 3.25 ) when compared with controls ( 1.11 ) , at p < 0.0001 , with significant apelin variations among asymptomatic carriers ( asc ) , fibrosis , and cirrhosis patients , and also among obese and nonobese patients . multiple regression analysis depicted that bmi , triglycerides , and total cholesterol were independent correlation factors to apelin levels , whereas tnf- was found to be significantly negatively correlated to adjusted apelin in chc patients ( r = 0.5944 , p < 0.0001 ) . ir was positively correlated to adjusted apelin in chc patients ( r = 0.2663 , p < 0.05 ) . conclusion . apelin level varies among stages of chc , which may contribute to fibrosis progression . in addition , obesity and ir could act as comorbid factors affecting apelin level in patients with chc .

1. Introduction 2. Subjects and Methods 3. Results 4. Discussion 5. Conclusion Conflict of Interests

artery - ureteral fistula ( auf ) is a relatively rare but life - threatening condition because of the potential of massive hemorrhage and hematuria . risk factors for auf include previous abdominal vascular surgery , previous extensive and radical oncologic treatment in the pelvis and chronic ureteral stenting . because of the increased prevalence of these risk factors , the chances of encountering auf are increasing . various treatment options have been reported to manage an existing fistula . conventionally , auf is treated by open surgery to close the fistula ; however , this is associated with high mortality and morbidity rates because the surgical field often has adhesions from previous treatments such as surgery or radiotherapy . recently , endovascular stent graft placement for auf has been employed as a less invasive and safer therapeutic option with satisfactory outcomes . herein , we present a case of auf after surgery for an abdominal aortic aneurysm , which was successfully treated with an endovascular procedure using a covered stent . he had undergone a y - graft surgery for an abdominal aortic aneurysm 3 years earlier . a computed tomography ( ct ) , 1 year after y - graft surgery revealed that the right ureter had crossed the distal anastomosis of the vascular graft . two years after the surgery , the ureter was observed to be extrinsically compressed by fibrosis at the site of its crossing over the y - graft and a ureteral stent was placed at another hospital , 7 months before presentation to us . the patient was transferred to our hospital after transfusion of four units of red cell concentrates . laboratory data showed anemia with a hemoglobin concentration of 9.7 g / dl ( normal , 13.517.0 g / dl ) , a hematocrit level of 28.9% ( normal , 4050% ) , an elevated serum creatinine level of 1.41 mg / dl ( normal , 0.40.8 mg / dl ) and a slightly elevated c - reactive protein concentration of 0.40 mg / dl ( normal , 0.000.30 mg / dl ) . he was hemodynamically stable and a urine bacterial culture was negative . at the time of consultation at our hospital , contrast - enhanced ct of the abdomen revealed a possible fistula between the right common iliac artery and the ureter , although no flow of contrast medium to the ureter was detected [ figure 1a ] . the ct showed that the process originated at the level of the distal anastomosis of a vascular y - graft [ figure 1b and c ] . an elective endovascular surgery was performed by vascular surgeons following embolization of the right internal iliac artery to prevent endoleak caused by backflow from the right internal iliac artery . arteriography at the operation detected no leak from the artery to the ureter [ figure 2 ] . covington , ga , usa ; diameter 10 mm ; length , 80 mm ) was placed via the right femoral artery . extrinsic obstruction caused by inflammatory fibrotic change was considered to be so dense that it was impossible to treat the ureteral stricture . two weeks after treatment , contrast - enhanced ct showed disappearance of the fistula and improvement of right hydronephrosis . six months after the endovascular surgery , severe right hydronephrosis was observed with an elevated serum creatinine level of 1.42 mg / dl . ( a ) contrast - enhanced computed tomography ( ct ) in arterial phase shows a minimal process ( arrow ) that was a possible fistula between the right common iliac artery and the right ureter . ( b and c ) ct and 3d - ct angiography show that the right ureter with a ureteral stent crosses the distal site of anastomosis of the vascular graft ( arrow ) arteriography shows no leak of contrast medium to the right ureter . urological complications after vascular surgery were previously considered to be rare because a unilateral hydronephrosis may not produce symptoms , mild obstruction may not be identified and no systematic follow - up after vascular surgery was performed . however , it is now known that ureteral obstruction occurs in 1020% of all bypass operations . patients with ureteral obstruction are significantly more likely to have graft complications , including auf , compared with those without ureteral obstruction . in particular , delayed ureteral obstruction , as observed in the present case , appears to be a marker for current or impending graft complications . it is important to recognize the predisposing risk factors for auf , and the urologist should suspect auf in any patient with hematuria and the predisposing risk factors . the pathogenesis of auf is closely related to inflammatory or ischemic changes in the ureter , vessels or both . the predisposing factors from the outside of the ureter that could cause the pathogenesis are previous genitourinary or pelvic oncologic surgery , previous vascular surgery , radiotherapy and an underlying vascular disease . in addition , chronic ureteral stenting is a strong predisposing factor from the inside of the ureter . chronic ureteral stenting acts as a counter - brace to facilitate the transmission of arterial pulsations to a fragile ureter , producing pressure necrosis and resulting in fistula formation . in a previous study assessing a large patient cohort , 85 of 139 auf patients ( 61% ) had chronic ureteral catheterization prior to the fistula formation . auf in a patient who underwent vascular graft surgery is often located at the level of the proximal or distal anastomosis of the vascular graft . this is because anastomosis is most fragile in the artery . in the review of 139 auf cases , a significant portion of the fistulas originated at the level of the anastomosis of the vascular graft . consistent with this , the fistula in the present case was located at the level of the distal anastomosis . the treatment of auf in an arterial lesion is either via an open procedure or via an endovascular procedure . endovascular treatments are less invasive and safer than open procedures because they avoid direct procedures on areas damaged during previous radiation or surgery . fox et al . reported that the overall endovascular success rate of no recurrent hemorrhage was 85% ( 12 of 14 cases ) , including 11 patients with endovascular stents with fewer complications than with open procedures . endovascular treatment is classified into two categories : the first using angiographic vascular occlusion and the other using a covered stent . the former procedure requires extra - anatomical vascular repair to maintain antegrade blood flow to the lower extremity . however , an endovascular procedure using a covered stent does not require revascularization ; therefore , this treatment option can be considered less invasive than open procedures or endovascular treatment using angiographic vascular occlusion . although endovascular treatment using a covered stent has some disadvantages , such as not dealing with fistula formation , severe complications such as stent occlusion or stent graft infection are very rare . additional cases and longer follow - ups are likely to demonstrate the further utility of endovascular procedures with covered stents . in conclusion , in a patient with history of treatment of aortic aneurysm , a ureteral stent should not be placed for a long time , and endovascular treatment of auf using a covered stent could become the preferred treatment modality because of its less invasiveness and safety .

artery ureteral fistula ( auf ) is a rare condition but there is an increase in the number of reported cases . it is frequently difficult to treat . a 63-year - old male who had undergone a dacron y - graft placement for an infrarenal aortic aneurysm 3 years earlier , presented with hematuria . contrast - enhanced computed tomography revealed a fistula located between the right common iliac artery and the right ureter at graft anastomosis . endovascular treatment using a covered stent was performed successfully .

INTRODUCTION CASE REPORT DISCUSSION

1 . perithecia , antheridia and axis of receptacle produced at the second or third layer of receptacle ... euphoriomyces agathidii 1 ' . perithecia , antheridia and axis of receptacle produced on the third layer of receptacle ... 2 2 . thallus with a single perithecium , antheridium and only the primary axes without appendages ... euphoriomyces cybocephali2 ' . thallus with 2~3 perithecia , antheridia formed as coner cells of the distal receptacle and composed of 2~3 axis with a few appendages ... euphoriomyces sugyamae 2 . thallus with a single perithecium , antheridium and only the primary axes without appendages ... euphoriomyces cybocephali 2 ' . thallus with 2~3 perithecia , antheridia formed as coner cells of the distal receptacle and composed of 2~3 axis with a few appendages ... euphoriomyces sugyamae ectinomyces agathidii maire . bull . total length to the top of the perithecium ( up to the upper most perithecium ) 85~117 m . receptacle axis ( up to the third cell bearing perithecia ) 35~38 15~35 m ( included the foot ) ; cell i obtriangular , cell ii about two times broader than length , cell iii up to the three times broader than length ; except for the cell i , most of the cells cut off , unilaterally or bilaterally , isodiametric basal cells of secondary antheridial branchlets or inflated stalk cells of perithecia ; the primary axis is a prolongation of receptacle , usually simply once branched on the fourth or fifth cell , consisting of 5~11 superposed cells ( except for the three cells of receptacle ) , its distal cell bears one or two phialides proliferated into short branchlets ; secondary lateral branchalets short , each with 1~2 terminal phialides which proliferate . perithecia composed of the stalk cell and the perithecium proper , produced unilaterally or bilaterally usually between the second and the fourth cell of the main axes , 1~3 in number , ovate or elongated cylindrical , with narrowed subapical part , the apical protruding cell indistinct ; the perithecium proper 40~65 13~20 m ; the stalk cell nearly isodiametric , 10 10 m . host genera : agathidium and amphicyllis ( leiodidae , coleoptera)host species in korea : agathidium sp.distribution : germany , north africa , spain , and south koreaspecimens examined : youngsil , mt . halla , jeju island , 13 july , 1997 , l - y-1445 - 1 , 1445 - 2 and 1445 - 3 host genera : agathidium and amphicyllis ( leiodidae , coleoptera ) host species in korea : agathidium sp . distribution : germany , north africa , spain , and south korea specimens examined : youngsil , mt . halla , jeju island , 13 july , 1997 , l - y-1445 - 1 , 1445 - 2 and 1445 - 3 this species is very similar to e. rossii , from which it is distinguished by the larger thalli and primary axis composed of some more cells than the latter . thalli were collected from elytra , metasternum , abdomen and legs of host insects living on russula densifolia ( russulaceae , agaricales ) in the deciduous broad - leaved trees forest . receptacle ( primary axis ) consisting of the basal and distal portions ; the basal portion composed of three superposed cells , 25 13 m ; cell i obtriangular , narrower towards the basal portion , broader towards the distal portion ; cell ii flattened , isodiametric ; cell iii the largest and broadest to the others ; the distal portion of the receptacle ( primary axis ) consisting of usually 5~9 superposed cells , divided unilaterally into two cells at the lower portion ( usually from second to fifth layer except for the basal portion of receptacle ) ; the secondary axis bearing unilaterally on the third cell of the receptacle , composed of the stalk cell and the antheridium proper ; the antheridium proper nearly flask - shaped , 20 4 m ; the stalk cell 3 5 m . perithecium composed of the stalk cell and the perithecium proper ; perithecium proper elongated cylindrical or ovate , only one in number , 38~40 10 m ; the stalk cell 10 7 m . all coleoptera.host species in korea : pseudocolenis hilleri reitter ( known newly from korea).distribution : south korea and sri lanka.specimens examined : mt . l - y-1401 - 1 , 1401 - 2 , 1401 - 3 , 1401 - 4 and 1401 - 5 . l - y-1401 - 1 , 1401 - 2 , 1401 - 3 , 1401 - 4 and 1401 - 5 . the present species is closely related to e. cioideus , but it differs from following features ; in e. cybocephali the primary axis are composed of 5~9 superposed cells and not branched , whereas in e. cioideus they are composed of 8~10 superposed cells and one or two appendages are branched at the distal portion . thaxter 's materials ( 1931 ) showed two kinds of specimens having the primary ( thaxter , 1931 , fig . korean materials are nearly agreed with his representatives , although the length of primary axis are longer than those of thaxter . they are identified to e. cybocephali , because of the structures of cells and the figures of thalli are closely related to the latter . host insects were caught in a mountain forest with quercus mongolica ( fagaceae ) on suilus grevillei ( polyporaceae ) . this species have been found only on the genus cybocephalus as far , but korean materials have been collected newly from the genus pseudocolenis . the axis of the receptacle and primary appendages consist of 9~13 superposed cells that are slightly elongated or ( except for the basal cell ) flattened ; the receptacle gradually broadens to the third cell ; the higher situated cells are narrower and of nearly the same breadth . the fourth cell of the receptacle divides , laterally separating on one or both side the stalk cell of the perithecium and on the other the basal cell of the secondary appendage ; antheridia directed upwards , arranged in one row , are separated in the upper corner of 3~5 distal cells of the receptacle . a few long branchalets ( up to 56 m long ) arise from the distal cell of the receptacle . the secondary appendage resembles the paralled distal part of the receptacle , but is usually made up of somewhat more narrow cells . the stalk cell of the perithecium slightly elongated , basal cells obvious . the perithecia almost cylindrical elongate , 1~3 in number , 53~68 14~23 m ; the apex narrows fairly abruptly to a narrow rounded top ; one side of the subapex with a projecting part . host genera : pseudocolenis ( coleoptera , leiodidae ) and scaphisoma ( coleoptera , scaphidiidae ) , ( known newly from korea)host species in korea : scaphisoma rufum acharddistribution : japan and south koreaspecimens examined : near temple hwaeom , mt . jiri guryegun , jeonnam province , 20 july , 1998 , l - y-2000 , 2001 , 2002 , 2003 , 2004 , 2005 , 2006 , 2007 , 2008 , 2009 , 2010 , 2011 and 2013 host genera : pseudocolenis ( coleoptera , leiodidae ) and scaphisoma ( coleoptera , scaphidiidae ) , ( known newly from korea ) host species in korea : scaphisoma rufum achard distribution : japan and south korea specimens examined : near temple hwaeom , mt . jiri guryegun , jeonnam province , 20 july , 1998 , l - y-2000 , 2001 , 2002 , 2003 , 2004 , 2005 , 2006 , 2007 , 2008 , 2009 , 2010 , 2011 and 2013 this species is very unique in having the antheridia formed as corner cells with lateral necks and the receptacle composed of the unicells from the basal cell to the third cell . although the present species should be included in the genus carpophoromyces according to personal information from dr . i. tavares ( majewski , 1994 ) , in this paper it is described to the genus euphoriomyces . the specimens of this species were found newly on scaphisoma rufum in a vally forest with quercus variabilis ( fagaceae ) . 1 . perithecia , antheridia and axis of receptacle produced at the second or third layer of receptacle ... euphoriomyces agathidii 1 ' . perithecia , antheridia and axis of receptacle produced on the third layer of receptacle ... 2 2 . thallus with a single perithecium , antheridium and only the primary axes without appendages ... euphoriomyces cybocephali2 ' . thallus with 2~3 perithecia , antheridia formed as coner cells of the distal receptacle and composed of 2~3 axis with a few appendages ... euphoriomyces sugyamae 2 . thallus with a single perithecium , antheridium and only the primary axes without appendages ... euphoriomyces cybocephali 2 ' . thallus with 2~3 perithecia , antheridia formed as coner cells of the distal receptacle and composed of 2~3 axis with a few appendages ... euphoriomyces sugyamae total length to the top of the perithecium ( up to the upper most perithecium ) 85~117 m . receptacle axis ( up to the third cell bearing perithecia ) 35~38 15~35 m ( included the foot ) ; cell i obtriangular , cell ii about two times broader than length , cell iii up to the three times broader than length ; except for the cell i , most of the cells cut off , unilaterally or bilaterally , isodiametric basal cells of secondary antheridial branchlets or inflated stalk cells of perithecia ; the primary axis is a prolongation of receptacle , usually simply once branched on the fourth or fifth cell , consisting of 5~11 superposed cells ( except for the three cells of receptacle ) , its distal cell bears one or two phialides proliferated into short branchlets ; secondary lateral branchalets short , each with 1~2 terminal phialides which proliferate . perithecia composed of the stalk cell and the perithecium proper , produced unilaterally or bilaterally usually between the second and the fourth cell of the main axes , 1~3 in number , ovate or elongated cylindrical , with narrowed subapical part , the apical protruding cell indistinct ; the perithecium proper 40~65 13~20 m ; the stalk cell nearly isodiametric , 10 10 m . host genera : agathidium and amphicyllis ( leiodidae , coleoptera)host species in korea : agathidium sp.distribution : germany , north africa , spain , and south koreaspecimens examined : youngsil , mt . halla , jeju island , 13 july , 1997 , l - y-1445 - 1 , 1445 - 2 and 1445 - 3 host genera : agathidium and amphicyllis ( leiodidae , coleoptera ) host species in korea : agathidium sp . distribution : germany , north africa , spain , and south korea specimens examined : youngsil , mt . halla , jeju island , 13 july , 1997 , l - y-1445 - 1 , 1445 - 2 and 1445 - 3 this species is very similar to e. rossii , from which it is distinguished by the larger thalli and primary axis composed of some more cells than the latter . thalli were collected from elytra , metasternum , abdomen and legs of host insects living on russula densifolia ( russulaceae , agaricales ) in the deciduous broad - leaved trees forest . receptacle ( primary axis ) consisting of the basal and distal portions ; the basal portion composed of three superposed cells , 25 13 m ; cell i obtriangular , narrower towards the basal portion , broader towards the distal portion ; cell ii flattened , isodiametric ; cell iii the largest and broadest to the others ; the distal portion of the receptacle ( primary axis ) consisting of usually 5~9 superposed cells , divided unilaterally into two cells at the lower portion ( usually from second to fifth layer except for the basal portion of receptacle ) ; the secondary axis bearing unilaterally on the third cell of the receptacle , composed of the stalk cell and the antheridium proper ; the antheridium proper nearly flask - shaped , 20 4 m ; the stalk cell 3 5 m . perithecium composed of the stalk cell and the perithecium proper ; perithecium proper elongated cylindrical or ovate , only one in number , 38~40 10 m ; the stalk cell 10 7 m . all coleoptera.host species in korea : pseudocolenis hilleri reitter ( known newly from korea).distribution : south korea and sri lanka.specimens examined : mt . l - y-1401 - 1 , 1401 - 2 , 1401 - 3 , 1401 - 4 and 1401 - 5 . l - y-1401 - 1 , 1401 - 2 , 1401 - 3 , 1401 - 4 and 1401 - 5 . the present species is closely related to e. cioideus , but it differs from following features ; in e. cybocephali the primary axis are composed of 5~9 superposed cells and not branched , whereas in e. cioideus they are composed of 8~10 superposed cells and one or two appendages are branched at the distal portion . thaxter 's materials ( 1931 ) showed two kinds of specimens having the primary ( thaxter , 1931 , fig . korean materials are nearly agreed with his representatives , although the length of primary axis are longer than those of thaxter . they are identified to e. cybocephali , because of the structures of cells and the figures of thalli are closely related to the latter . host insects were caught in a mountain forest with quercus mongolica ( fagaceae ) on suilus grevillei ( polyporaceae ) . this species have been found only on the genus cybocephalus as far , but korean materials have been collected newly from the genus pseudocolenis . thallus hyaline yellowish . total length to the top of the highest perithecium 110~118 m . the axis of the receptacle and primary appendages consist of 9~13 superposed cells that are slightly elongated or ( except for the basal cell ) flattened ; the receptacle gradually broadens to the third cell ; the higher situated cells are narrower and of nearly the same breadth . the fourth cell of the receptacle divides , laterally separating on one or both side the stalk cell of the perithecium and on the other the basal cell of the secondary appendage ; antheridia directed upwards , arranged in one row , are separated in the upper corner of 3~5 distal cells of the receptacle . a few long branchalets ( up to 56 m long ) arise from the distal cell of the receptacle . the secondary appendage resembles the paralled distal part of the receptacle , but is usually made up of somewhat more narrow cells . the perithecia almost cylindrical elongate , 1~3 in number , 53~68 14~23 m ; the apex narrows fairly abruptly to a narrow rounded top ; one side of the subapex with a projecting part . host genera : pseudocolenis ( coleoptera , leiodidae ) and scaphisoma ( coleoptera , scaphidiidae ) , ( known newly from korea)host species in korea : scaphisoma rufum acharddistribution : japan and south koreaspecimens examined : near temple hwaeom , mt . jiri guryegun , jeonnam province , 20 july , 1998 , l - y-2000 , 2001 , 2002 , 2003 , 2004 , 2005 , 2006 , 2007 , 2008 , 2009 , 2010 , 2011 and 2013 host genera : pseudocolenis ( coleoptera , leiodidae ) and scaphisoma ( coleoptera , scaphidiidae ) , ( known newly from korea ) host species in korea : scaphisoma rufum achard distribution : japan and south korea specimens examined : near temple hwaeom , mt . l - y-2000 , 2001 , 2002 , 2003 , 2004 , 2005 , 2006 , 2007 , 2008 , 2009 , 2010 , 2011 and 2013 this species is very unique in having the antheridia formed as corner cells with lateral necks and the receptacle composed of the unicells from the basal cell to the third cell . although the present species should be included in the genus carpophoromyces according to personal information from dr . i. tavares ( majewski , 1994 ) , in this paper it is described to the genus euphoriomyces . the specimens of this species were found newly on scaphisoma rufum in a vally forest with quercus variabilis ( fagaceae ) .

three species of the genus euphoriomyces ( laboulbeniales ) were found newly from korea . e. agathidii ( maire ) tavales was collected from agathidium sp . ( leiodidae , coleoptera ) . the characteristic traits of this species are the distal portion of the primary axis composed of 5~11 superposed layers and bearing more or less the elongated appendages . e. cybocephali ( thaxter ) thaxter was collected from pseudocolenis hilleri reitter ( leiodidae , coleoptera ) . the primary axis of this species composed of 5~9 superposed layers is simple , not branched and a single antheridium occurs on the apex of the secondary axis . e. sugiyamae majewski was collected from scaphisoma rufum achard ( scaphidiidae , coleoptera ) . this species is very unique in having the antheridia formed as coner cells with lateral necks .

Description of Species Key to the species of genus 1. 2. 3.

oral squamous cell carcinoma ( oscc ) is the most common type of tumor in the oral cavity . the majority of the tumors are locally advanced and have relatively poor prognosis with 5 years survivals < 50 - 60% . at present , the standard of care for resectable locally advanced oscc is the surgical treatment of the primary tumor and neck followed by postoperative radiotherapy or chemoradiotherapy , depending on the presence of intermediate- or high - risk features . extensive procedures are required in these locally advanced cancers which are associated with a substantial amount of cosmetic deformity and functional morbidity . the recent advances in reconstruction techniques have enabled the possibility of wider resections with limited morbidity . however , resectability of the tumor must be a fine balance between achieving negative surgical margins with acceptable functional and cosmetic deformities . neoadjuvant chemotherapy ( nact ) in head and neck cancers has been investigated for long with an aim of reducing surgical margins , distant metastasis rates , and improving outcomes . the meta - analysis of chemotherapy on head and neck cancer meta - analysis of 31 trials involving more than 5000 patients failed to demonstrate significant survival benefit following induction chemotherapy . however , the trials that used 5- fluorouracil ( 5-fu ) and cisplatin as a part of nact regime showed significant overall survival ( os ) benefit compared to other combination regimens and single agent nact . the trials included in the meta - analysis did not specifically address oral cavity cancers and had less number of oscc patient . most of these trials were from the pretaxane era , and impact of taxanes in the neoadjuvant setting is not addressed with this meta - analysis . the interest in nact has been rekindled by recent studies such as tax 323 and 324 which included taxane ( docetaxel ) along with fluorouracil and cisplatin ( tpf ) containing regimen . the tpf induction regimen showed improved survival in advanced head and neck squamous cell carcinoma ( hnscc ) as compared to patients receiving an induction regimen with cisplatin and fluorouracil ( pf ) alone . however , these trials included various head and neck subsites and also were not exclusively designed for oscc . the aim of this paper is to critically review the current evidence for nact in locally advanced oscc and suggest an algorithmic approach to the patient population who might benefit from nact for oscc . published a retrospective single institution review of nact in patients with 141 operable oral cavity cancers . the nact consisting of two cycles of cisplatin , vincristine , and peplomycin , with or without mitomycin c showed overall response rate was 51.5% , and there was a documented decrease in the rates of distant metastases . there was no decrease in overall or disease - free survival when all 141 patients were compared . the type of surgery performed , the response to chemotherapy according to the t stage , the impact of effective chemotherapy and the adequacy of surgical margins achieved were not reported . the drawbacks of the trial were that this was a single institution retrospective study with a combination of drugs which were not tested rigorously in head and neck cancer . with regards to the use of nact prior to surgery in resectable oral cavity cancers , at least two well - structured and adequately powered randomized trials have been published . licitra et al . in their article , published the results of a randomized , multicenter trial for 195 patients with advanced resectable , stage t2-t4 ( > 3 cm ) , n0-n2 , m0 untreated squamous cell carcinoma ( scc ) of the oral cavity . patients were randomly assigned to three cycles of cisplatin and fluorouracil followed by surgery ( chemotherapy arm ) or surgery alone ( control arm ) . complete or partial clinical response of the primary tumor was observed in 28 ( 33% ) and 42 ( 49% ) patients , respectively , adding up to a total of 82% objective response rate . however , stage iv tumors the response rate was only 18% . segmental mandibulectomy was performed in the chemotherapy arm ( 31% vs. 52% in the control group ) there was no increase in the number of positive surgical margins . there was no difference in terms of locoregional recurrence , distant metastasis and os between the two arms . however , it was observed that patients achieving pathologically complete response had statistically significant higher 5 years survival rates . , with a median follow - up of 11.5 years showed no difference in the incidence of locoregional relapse between chemotherapy and control group ( p = 0.6337 ) , nor in distant metastasis development ( p = 0.1527 ) , or os ( p = 0.3402 ) . patients with a pathological complete response ( pcr ) to the nact had a higher probability of survival than those without ( 10-year os : 76.2% vs. 41.3% , p = 0.0004 ) . published the results of their prospective open - label phase iii trial of 256 untreated stages iii or iva locally advanced resectable oscc . patients were randomized into two groups who received two cycles of nact ( tpf ) ( docetaxel 75 mg / m on day 1 , cisplatin 75 mg / m on day 1 , and fluorouracil 750 mg / m on days 1 - 5 ) followed by radical surgery and postoperative radiotherapy ( 54 - 66 gy ) versus up - front radical surgery and postoperative radiotherapy . the primary endpoint was os , and the secondary endpoints included local control and safety . the pathological response rate was 13.4% . a statistically insignificant lower rate of distant metastasis in the nact arm was observed ( 5.5% vs. 8.7% ) . at a median follow - up of 30 months , there was no difference in the disease - free survival and the os between the two arms ( 68.8% vs. 68.2% ) . patients in the nact arm with a clinical response or favorable pathologic response ( < 10% viable tumor cells ) had superior os and locoregional and distant control . the long - term results of this study confirmed that at a median follow - up of 70 months there were no significant differences in survival rates between experimental and control groups . however , patients with favorable pathologic responses had improved outcomes compared to those with unfavorable pathologic responses and also to those in the control group in terms of os , distant metastasis free survival ( dmfs ) , local recurrence free survival which were statistically not significant . subgroup analysis demonstrated that cn2 patients receiving tpf nact had a better outcome than those not receiving tpf nact , especially os and dmfs . in addition , primary tongue carcinomas had a trend toward better os and dmfs compared to other oral cavity subsites . although , this is a post - hoc analysis is interesting and hypothesis generating , it must be interpreted with caution . although neoadjuvant strategy provided no significant advantage , the chemotherapy sensitivity of oral cancers has been demonstrated in both these randomized trials . favorable pathologic response was noted in 27.7% with pcr of 13.4% by zhong et al . the extent of surgery after nact was different in the trial reported by licitra et al . with significantly while the lower rates of segmental mandibulectomy are associated with better quality of life , the impact of reduced surgical effort to achieve margin negativity in the chemotherapy group remains unclear . a meta - analysis of induction chemotherapy between 1965 and 2011 in resectable hnscc of all subsites , which included 14 randomized control trials with 2099 patients showed a 8% reduction in rate of distant metastasis in patients receiving induction chemotherapy . in summary , multiple studies have failed to demonstrate improvement in locoregional control , os in resectable oral cavity cancer . however , the effect of nact on reducing distant metastasis , mandibular preservation in tumors without gross invasion of mandible and disease control in patients with higher nodal stage seems interesting and warrants further investigation . paccagnella et al . showed in their study that patients with either stage iii or iv scc tumors of the head and neck without distant metastasis who were not candidates for surgery had a 21% os rate at 5 years when they underwent 4 cycles of neoadjuvant cisplatin and 5-fu followed by radiotherapy as opposed to an 8% os rate in similar patients who received radiotherapy alone . the participants in the study who were deemed resectable after treatment underwent surgical resection . these patients did not have any survival benefit with the addition of chemotherapy , however , showed significantly lower rates of distant metastasis . hitt et al . published the first randomized trial comparing pf to pf plus paclitaxel . the trial showed significant improvements in terms of response rates and time to treatment failure in favor of tpf regimen . tax 323 trial , compared tpf versus pf followed by radiotherapy ( 4 cycles ) in 358 patients with locally advanced hnscc ( lahnscc ) . tax 324 trials , a randomized , open - label phase 3 trial consisting of 501 patients with lahnscc compared three cycles of tpf induction chemotherapy with three cycles of pf . however , oral cavity patients consisted only 13% in tpf and 15% in pf group . the long - term follow - up of 5 years of tax 324 study showed that the os was significantly better with tpf versus pf ( hazard ratio [ hr ] = 0.74 , 95% confidence interval [ ci ] : 0.58 - 0.94 ) , with an estimated 5-year survival rate of 0.52 and 0.42 in the tpf and pf arms , respectively . median survival time was 70.6 months ( 95% ci : 49.0 - 89.0 months ) with tpf versus 34.8 months ( the 95% ci : 22.6 - 48.0 months ) in the pf group ( p = 0.014 ) . pfs was also significantly better with tpf ( 38.1 months ; 95% ci : 19.3 - 66.1 months vs. 13.2 months , 95% ci : 10.6 - 20.7 months ; hr = 0.75 , 95% ci : 0.60 - 0.94 ) . none of these trials was designed exclusively for oral cavity cancers and only had a small proportion of patients with oscc . the outcomes were also not compared with the outcomes with the standard of care which is surgery . these studies clearly demonstrate the advantage of using three drugs over two drug regime for induction . however , feasibility of using tpf chemotherapy in this setting remains low in the indian context . ajcc staging 2010 defines very advanced , local disease , or unresectable t4b oscc as tumor invading the masticator space , pterygoid plates , and skull base , or encasing the internal carotid artery . the term technically unresectable has been also been used to include tumors which are not staged as t4b but are known to carry poor prognosis and high morbidity after surgical resection . a rationale of proposing nact in these cases is to improve the overall outcome by reducing tumor burden prior to radiation or facilitate possible resection following tumor shrinkage . it has been seen that the results of chemoradiation or radical radiation alone in t4b cancers are not satisfactory with a disease - free survival of 1 year . primary surgical resection using compartment techniques seems to have good loco - regional control in oscc patients with infratemporal fossa involvement . although a small series , a study by trivedi et al . showed good locoregional control rate using compartmental resections for tumors involving masticator space . liao et al . in a single institution study reported encouraging results on upfront surgery in t4b oral cavity cancers below the mandibular notch . patil et al . published a retrospective study of 123 patients with technically unresectable locally advanced oral cavity cancers . unresectibility in these cases was defined as disease reaching up to the zygoma and/or soft tissue swelling up to the zygoma , extensive soft tissue involvement reaching up to the hyoid cartilage , extensive skin infiltration , and the involvement of the infratemporal fossa . the response rate with the three drug and two drug regimens was 32.00% and 27.37% , respectively . resectability was achieved in 17 patients with 3 drug regimen ( 68.00% ) and 36 patients with 2 drug regimen ( 37.89% ) . the estimated median os in these patients was 8 months ( p = 0.0001 ) . they demonstrated the effectiveness of nact in down - staging tumors and enabling radical surgery with comparable 2 years survival to primary surgery . in a subsequent article by the same group , joshi et al . they assessed the efficacy and impact of nact in t4b tumors in a retrospective study of 110 patients . 20% of patients received 3 drug regimens while the rest received various combinations of 2 drug regimen . partial response was achieved in 28 patients , stable disease in 49 patients and progression was noted in 23 patients . the estimated median os in patients who underwent surgery was 18.0 months ( 95% ci : 13.6 - 22.46 months ) and for those treated with nonsurgical treatment was 6.5 months ( 95% ci : 5.6 - 7.4 months ) ( p = 0.0001 ) . it is noteworthy to know that none of the patients had a complete response to nact . however , resectability was achieved in 30.9% of the tumors considered unresectable prior to chemotherapy thereby improving the outcome . in a follow - up article by patil et al . , retrospectively analyzed 721 patients with t4a and t4b oscc deemed as technically unresectable who received nact . the locoregional control rate was 20.6% for the overall cohort . for patients undergoing surgery , the lrc was 32% and 15% for the nonsurgical group . in addition , the pattern of pathological shrinkage in advanced oscc following nact has not been well documented . the pathological and radiological studies in breast cancer and other cancers have demonstrated a variable pattern of tumor shrinkage following nact . the tumors may not shrink concentrically and may have a satellite residual tumor beyond the visible tumor . the clinical and radiological findings in advanced oscc suggest a similar variable tumor shrinkage pattern [ figures 1 and 2 ] . further studies are warranted to clearly establish the pathological pattern of tumor shrinkage following nact . postneoadjuvant chemotherapy clinical specimen showing nonconcentric tumor shrinkage in separate areas with intervening tissue pre- and post - neoadjuvant chemotherapy positron emission tomography - computed tomography scan showing nonconcentric shrinkage of tumor these patients have been shown to have better locoregional control , disease - free survival , and os . the identification of tumor response predictors may allow a more rational selection of therapeutic strategies , sparing unnecessary toxicities to patients who would not benefit from nact . it could further lead to the development of new drug regimens to overcome primary resistance to nact . tsuji et al . retrospectively studied 70 patients with oscc for anti - tumor effects of cisplatin - based nact in relation to biological markers of tumor cell proliferation activity : tumor grade , cellular dna content , mitotic index , apoptotic index , ki-67 positive rate , and p53 and bax expression . it was observed that tumor grade , bax expression , apoptotic index , and cellular dna content correlated significantly with the anti - tumor effects of nact in univariate analysis . by using multiple logistic analyses , tumor grade , bax expression , and apoptotic index analyzed 53 pretreatment biopsies of oral cavity scc patients receiving primary cisplatin and fluorouracil chemotherapy followed by surgery for the predictive value of tp53 mutations and their functional status on the basis of the transactivation activity of p53 mutant proteins . 28% patients achieved a pathologic complete remission ( pcr ) at both t and n sites , and 38 patients had residual tumor cells . among 53 pretreatment biopsies , 24 ( 45% ) tp53 mutation predicted pcr in 4 ( 17% ) of 24 patients and a nonfunctional mutation in only 2 ( 9% ) of 22 patients . they concluded that the loss of function ( transactivation activities ) of p53 mutant proteins might predict a significant low pcr rate and a suboptimal response to cisplatin - based nact in patients with oscc . retrospectively analyzed 89 patients who underwent radical surgery for tongue cancer and examined the effect of nact on tumor local recurrence . expression of cd44v6 and abcg2 was significantly associated with regional lymph node metastasis , the pattern of invasion , depth of invasion , perineural invasion , and local recurrence , respectively . they suggested that local recurrence in nact treated cases was associated with cancer stem - like cells and nact may lead to the selection and/or residue of more aggressive cancer stem - like cells . there is an unmet need to develop a biomarker panel specific for predicting response to nact . current evidence suggests that there is a limited role for nact in locally advanced resectable oral cancer and should not be used outside of a well - designed clinical trial . the addition of nact has failed to demonstrate an improvement in local control rates and os in resectable oscc . the improvement in the distant metastasis rate among a select group of patients requires further validation . there is a need to standardize the definition of resectable advanced oscc for both clinical practices and for inclusion in future clinical trials . patients with unresectable oral cavity cancers may have responses to the tune of 70% with aggressive three - drug regime ( tpf ) , and a third of these patients may benefit for subsequent surgical treatment . the patient with good response and subsequent resections has a better outcome . in view of limited benefit with nact , newer strategies need to be conceived in order to improved outcome in patients with advanced oscc .

the standard of care treatment for oral squamous cell carcinoma ( oscc ) at present , consist of surgical resection followed by adjuvant radiotherapy and chemotherapy as indicated . despite recent advances the overall prognosis remains guarded . role of neoadjuvant chemotherapy is being explored with premise of reducing extent of surgical resection , improving loco - regional control and decreasing distant metastasis , thereby improving treatment outcomes by decreasing mortality and morbidity . however , indications of neoadjuvant chemotherapy in oral cancers are not clearly defined . majority of studies have failed to demonstrate a significant benefit of neoadjuvant chemotherapy in terms of loco regional control and overall survival in resectable oscc . in a select subset of patients with locally very advanced and unresectable oscc , neoadjuvant chemotherapy has been shown to cause tumor shrinkage and improve resectability . these hypothesis generating findings of reduction in distant metastasis , improved resectability and functional outcome , however need further validation . in summary , the role of neoadjuvant chemotherapy for oscc remains investigational and has a limited role outside clinical trial .

INTRODUCTION NEOADJUVANT CHEMOTHERAPY FOR RESECTABLE ORAL SQUAMOUS CELL CARCINOMA NEOADJUVANT CHEMOTHERAPY IN LOCALLY ADVANCED CANCER NEOADJUVANT CHEMOTHERAPY IN LOCALLY VERY ADVANCED/UNRESECTABLE ORAL CAVITY CANCER POTENTIAL PREDICTIVE BIOMARKERS FOR NEOADJUVANT CHEMOTHERAPY IN ORAL CANCERS SUMMARY Financial support and sponsorship Conflicts of interest

many reports have demonstrated that gene polymorphisms of the renin - angiotensin system ( ras ) are associated with hypertension . there have been some reliable reports about susceptible genes for hypertension including the results from the millennium genome project for hypertension in japan ( 2000~2005 ) [ 1 , 2 ] ; however , no convincing gene has yet been detected . some of the genes regulating blood pressure might also be related to the response to antihypertensive medication [ 3 , 4 ] . indeed , we and other collaborators have investigated several susceptible genes related to hypertension [ 515 ] , including genes of not only the renin - angiotensin system and sodium handling but also insulin resistance , oxidative stress , and sympathetic nervous system ( described in the section 2 ) ; however , the genes involved in the response to antihypertensive medication have not yet been identified . in addition , exhaustive gene expression analysis ( transcriptome analysis ) for lifestyle - related diseases has not been performed thus far . we performed a large collaboration with the study group led by professor katayama at saitama medical university to perform a randomized controlled trial called azelnidipine ( a calcium ( ca ) channel blocker ) and temocapril ( an ace inhibitor ) in hypertensive patients with type 2 diabetes study ( attest ) , which included genetic analysis to evaluate the therapeutic effects of azelnidipine and temocapril . the goals of this study were , first , to assess the association between polymorphisms of susceptible genes for hypertension ( 18 genes ) and each treatment or phenotype and , second , to assess the association between expression in peripheral blood of susceptible genes for hypertension and each treatment or phenotype . attest study , a multicenter , randomized , open - label study , was originally performed to investigate the efficacy and safety of combination therapy using the calcium channel blocker ( ccb ) azelnidipine and angiotensin - converting enzyme ( ace ) inhibitor temocapril in hypertensive diabetics , led by professor katayama of saitama medical university . all of the subjects fulfilled the following inclusion criteria : ( 1 ) age : 30~70 years , outpatients , ( 2 ) mean systolic bp ( bp ) : 140~180 mmhg and/or mean diastolic bp : 90~110 mmhg in washout period ( four weeks ) and stable bp without fluctuation in systolic bp of more than 30 mmhg or diastolic bp of more than 15 mmhg , and ( 3 ) fasting blood glucose : more than 126 mg / dl or hba1c : more than 6.5% at 6 months before entry . these mild or moderate hypertensive subjects with diabetes were treated with the following medication : ccb : azelnidipine and ace inhibitor : temocapril in accordance with the protocol ( figure 1 ) . each medication was started at a dose of 8 mg azelnidipine or 2 mg temocapril and increased to 16 mg azelnidipine or 4 mg temocapril until bp of less than 130/80 mmhg was achieved . all subjects were measured for body height , body weight , systolic bp , diastolic bp , fasting blood glucose level , triglyceride level , low density lipoprotein ( ldl ) cholesterol level , and serum creatinine ( cr ) level . informed consent for genetic analysis was obtained from all subjects , and finally a total of 44 subjects were recruited in this study . the genotypes of the 22 polymorphisms of the 18 susceptible genes ( ace ins / del , add1 gly460trp ( rs4961 ) , adipoq ile164thr , adrb1 ser49gly ( rs1801252 ) and arg389gly ( rs1801253 ) , adrb2 gly16arg ( rs1042713 ) and glu27gln ( rs1042714 ) , adrb3 trp64arg ( rs4994 ) , agt met235trp ( rs699 ) , agtr1 a1166c ( rs17231380 ) , aldh2 glu487lys ( rs671 ) , arhgap8 arg338gly ( rs6007334 ) , bdkrb2 t-58c , fasl g-670a , grk4 ala486val ( rs1801058 ) and arg65leu ( rs2960306 ) , hogg1 ser326cys ( rs1052133 ) , mthfr c677 t ( rs1801133 ) , rgs2 a to g in promoter ( rs3767489 ) , rgs2 a-638 g ( rs2746071 ) , slc12a3 arg904gln ( rs11643718 ) , and tgfb1 leu10pro ( rs1982073 ) ) for which positive associations with hypertension were previously reported were successfully determined using taqman pcr method ( applied biosystems inc . , foster city , ca , usa ) . according to quantitative rt - pcr analysis using cdna extracted from peripheral blood , 11 genes were expressed in peripheral blood : ace , add1 , adrb1 , adrb2 , arhgap8 , fasl , grk4 , mthfr , rgs2 , slc12a3 , and tgfb1 . rna samples were extracted from the peripheral blood of the subjects , and cdna was made from rna using reverse transcriptase . quantitative real - time pcr ( rt - pcr ) for all the genes was performed using taqman pcr method to determine their expression levels . the associations between polymorphisms and clinical variables were analyzed using one - way analysis of variance ( anova ) . odd ratios were calculated as an index of the association of each genotype with the prevalence of hypertension . to assess the contribution of confounding factors , we performed multiple logistic regression analysis using the computer software application , jmp 7.0 ( sas institute inc . , cary , nc , u.s.a . ) . we focused on testing for snp medication and gene - expression medication interactions ( i.e. , whether the effects of snps on systolic bp or diastolic bp differed between hypertensives on azelnidipine and temocapril ) . multivariate analysis of variance ( manova ) was performed to determine whether bp change showed a significant interaction of each antihypertensive medication with the genotypes of the susceptible genes . baseline characteristics of the subjects treated with azelnidipine and temocapril are shown in table 2 . there was no difference in sex , age , body mass index ( bmi ) , affected period of hypertension , systolic bp , triglyceride level , ldl cholesterol level , and serum creatinine level between the azelnidipine and temocapril groups ; however , fasting glucose level was higher in the azelnidipine group than in the temocapril group at the baseline . in the current study , the same level of bp reduction was observed in both the azelnidipine and temocapril groups , and both groups achieved a mean bp of less than 130/80 mmhg at 52 weeks . there was no difference in the dose of each treatment at 52 weeks ( group started with azelnidipine : bp at 52 weeks 121.9 11.4/74.7 7.1 mmhg , final dose of azelnidipine 15.0 2.7 mg , final dose of temocapril 3.8 0.6 mg ; group started with temocapril : bp at 52 weeks 125.6 10.3/74.7 6.1 mmhg , final dose of azelnidipine 14.9 2.8 mg , final dose of temocapril 3.7 0.7 mg , figure 2 ) . the genotypes of 22 polymorphisms of the 18 genes were successfully determined at each time point ( 0 , 8 , 16 , and 52 weeks ) . only the ile164thr polymorphism of the adiponectin gene has no mutant genotype , and the other genotype frequencies were not significantly different from the values of hardy - weinberg 's expectation ( figure 3 ) . according to the analysis of the association between bp , changes of bp , and all genotypes at each time point ( 0 , 8 , 16 , and 52 weeks ) , only the a-638 g polymorphism of the regulator of g protein signaling-2 gene ( rgs2 ) showed a significant association with changes in bp ( bp : bp at 8 weeks bp at 0 week ) . as shown in figure 3 , there was a significant relationship between the a-638 g polymorphism of the rgs2 gene and the changes in bp between 0 and 8 weeks in subjects with azelnidipine ( systolic bp : aa 28.0 10.1 mmhg , ag 15.5 12.6 mmhg , gg + 7.0 12.2 mmhg , p = .0013 ; diastolic bp : aa 17.2 9.8 mmhg , ag 8.1 9.0 mmhg , gg 4.0 11.0 mmhg , p = .067 ) , but not in subjects with temocapril ( systolic bp : aa 5.3 11.8 mmhg , ag 8.4 13.0 mmhg , gg 9.9 10.0 mmhg , p = .81 ; diastolic bp : aa 0.5 7.2 mmhg , ag 4.4 3.5 mmhg , gg 10.0 4.5 mmhg , p = .34 ) . at 8 weeks , changes in bp showed a significant interaction between a-638 g and treatment with azelnidipine and temocapril ( systolic bp , diastolic bp : p = .0014 , p = .036 , resp . , by manova , after adjustment for age , sex , and bmi ) ( table 3 ) . there was no gene whose expression was associated with bp phenotypes or the polymorphisms of each gene through analysis of gene expression in peripheral blood . in terms of the rgs2 gene , the a-638 g polymorphism was not related to the change of rgs2 expression between 0 and 8 weeks either in subjects with azelnidipine ( rgs2/18srna with azelnidipine : aa 1.06 2.1 , ag + 0.31 0.85 , gg + 0.57 0.44 , p = .13 , figure 4 ) or in subjects with temocapril ( rgs2/18srna with temocapril : aa + 0.75 1.3 , ag 0.10 0.62 , gg + 0.38 0.44 , p = .24 , figure 4 ) . this study demonstrated the importance of pharmacogenetic research . previously , lynch et al . reported that the nppa t2238c variant was associated with modification of antihypertensive medication effects on cardiovascular disease and bp , and tt allele carriers had more favorable outcomes when randomized to receive a ccb ( amlodipine ) . beitelshees et al . also demonstrated that the kcnmb1 genotype influenced responsiveness to verapamil sr and risk of adverse cardiovascular outcomes . these reports could support the possibility of the existence of genes influencing drug efficacy . signaling by g - protein - coupled neurotransmitter receptors in the autonomic nervous system and vasoregulatory factor receptors in the periphery governs both blood pressure , by controlling the constriction and dilatation of resistance arterioles , and electrolyte and fluid balance by the kidney [ 19 , 20 ] . the recently identified regulator of g - protein signaling ( rgs ) proteins is important in regulating signaling cascades initiated by g - protein - coupled receptors ( gpcrs ) activation . rgs proteins facilitate the intrinsic inactivating guanosine triphosphatase reaction of g - protein a - subunits , and thereby serve as effector channel blockers . rgs2 is unique among the rgs proteins in its apparent selectivity towards gpa , which mediates the action of mouse physiological vasoconstrictors , including norepinephrine , angiotensin ii , endothelin-1 , and thrombin . rgs2 can also attenuate gi- and gs - mediated pathways [ 22 , 23 ] , which can also affect blood pressure via other physiologically important agonists such as serotonin , dopamine , and bradykinin . it was recently reported that mice lacking rgs2 exhibit a strong hypertensive phenotype ( increase in sbp of 50 mmhg ) and resistance vasculature [ 24 , 25 ] . both heterozygous and homozygous rgs2-null mice exhibited a similar level of marked hypertension , suggesting that a naturally occurring mutation that affects the level of rgs2 protein may have a significant impact on blood pressure regulation . recently there have been two reports that genetic changes in rgs2 are associated with a hypertensive phenotype [ 26 , 27 ] . a-638 g , t1026a , and 1891 - 1892 del tc polymorphisms were extracted on the condition the allele frequencies of these polymorphisms were > 0.1 , and the t1026a and 1891 - 1892 del tc polymorphisms of this gene were associated with hypertension in women . these findings suggest that some functional variants of the rgs2 gene might be involved in regulating blood pressure in humans . this attest gene study revealed that one possible gene related to the effect of antihypertensive agents . in the current study , the a-638 g polymorphism of the rgs2 gene was a predictive factor for therapeutic performance of a ccb , azelnidipine . other classical candidate genes including genes associated with the renin - angiotensin system , sodium handling , vasodilatation and vasoconstriction , and the sympathetic nervous system did not show significant association between their polymorphisms and the effect of a ca channel blocker or ace inhibitor . in addition , there was no significant relationship between rgs2 polymorphisms and bp - related phenotypes in this study , unlike previous reports . it has not yet been reported that the promoter variant a-638 g of the rgs2 gene may change rgs2 function ; however , this polymorphism has the possibility to change rgs2 protein production because this polymorphism is in the promoter region . there has been no evidence supporting the idea that rgs2 could be involved in the effect of ccbs . in several previous reports , genes without a drug - metabolizing effect , such as ace or agtr1 , showed a significant association between their polymorphisms and response to antihypertensive medication ; however , the mechanisms have not been clarified in any of these reports . in terms of the present study , rgs2 is suggested to function as a switch to turn on or off the g protein - associated pathway , and rgs2 can regulate blood pressure through smooth muscle cells . from this viewpoint , rgs2 might have the possibility of changing the effect of ccbs , because those mainly act through smc ; however , the detailed mechanism merits further investigation in the future . peripheral blood mainly contains white blood cells , red blood cells , platelets , and other circulating hormones , so the distribution of genes expressed in peripheral blood has to be investigated to clarify the significance of disease susceptibility gene expression in peripheral blood . in the present study , there was no significant relationship between gene expression including the rgs2 gene and the effect of antihypertensives and phenotypes . from this viewpoint , rgs2 expression in peripheral blood does not seem to do anything and might not be a marker for bp regulation . however , further study is needed on its clinical application as a marker of drug efficacy . as the limitation of this study , we could not exclude the possibility of the false positive association ( type i error ) due to the use of small number of subjects . however , this study was carried out under the strict protocol of a clinical trial so that the reliability of the results obtained seems to be high . to confirm the effect of rgs2 gene in the tailored medicine of hypertension , further study using another panel of hypertensive subjects should be required . in conclusion , the a-638 g polymorphism of the rgs2 gene could be a predictive factor for therapeutic effectiveness of ccbs such as azelnidipine . further research is needed to determine the optimal approach for personalizing antihypertensive medication treatment regimens according to genotype information and for achieving the best blood pressure control .

we performed a prospective study to examine the genetic effect on the response to a calcium ( ca ) channel blocker , azelnidipine and an ace inhibitor , temocapril treatment in patients with hypertension , as a part of the prior clinical trial , the azelnidipine and temocapril in hypertensive patients with type 2 diabetes study ( attest ) . methods and results . all subjects who gave informed consent for genetic research were divided into two groups : the subjects treated with azelnidipine or temocapril , for 52 weeks . we selected 18 susceptible genes for hypertension and determined their genotypes using taqman pcr method . rna samples were extracted from peripheral blood , and quantitative real time pcr for all genes was performed using taqman method . one of the polymorphisms of the rgs2 gene was extracted as being able to influence the effect of these treatments to reduce bp . at eight weeks , bp change showed a significant interaction between the a-638 g polymorphism of regulator of g protein signaling-2 ( rgs2 ) gene and treatment with azelnidipine or temocapril . there was no gene whose expression was associated with bp phenotypes or the polymorphisms of each gene . conclusions . a-638 g polymorphism of the rgs-2 gene could be a predictive factor for therapeutic performance of ca channel blockers .

1. Introduction 2. Methods 3. Results 4. Discussion

factors adversely affecting short term survival include impaired consciousness , leg weakness and increasing age . on the other hand , long term survival the pattern of survival is changing with time in many populations with gradual improvement of survival and decrease in mortality.3 review of the literature suggests that immediate mortality after stroke approaches 30% in the first month.4 five year survival rates range between 35 - 45% . furlan reported a 5 year survival rate in stroke patients of 77% compared to 85% in matched normal controls.4 al rajah , in a study from saudi arabia showed that ischemic strokes accounted for 76.2% of all strokes . strokes caused by intro - cerebral and sub - arachnoid hemorrhage constituted 21.4% and 2.4% respectively.5 radiological changes in brain computed tomography ( ct ) correlates with survival . hankey reported that lesions greater than 50 mm in diameter have a higher mortality than lesions less than 50 mm8 . poor prognosis is also noted in ct findings of intracerebral hemorrhage , mass effect and deep intracerebral lesions.7 in this study , the prognostic effects of age , sex , hypertension , diabetes mellitus and presentation in coma on the survival pattern of stroke patients presenting to a referral hospital , are reviewed . all patients hospitalized with definite stroke at king fahd specialist hospital ( kfsh ) , buraidah , saudi arabia for the period between june 1986 and june 1991 were reviewed to determine the effect of age , sex , hypertension , diabetes mellitus , heart disease , atrial fibrillation , transient ischemic attacks , and the pathological type of the stroke on survival . the medical records of patients were reviewed retrospectively according to pre - established diagnostic and inclusion criteria . stroke is defined as acute focal or global loss of cerebral functions with symptoms lasting more than 24 hours , without an apparent cause other than the vascular origin . patients were included if they were saudi adults of 18 years of age or more , with a definite first stroke . the clinical diagnosis was made by the admitting physician and reviewed independently by two members of the study group ( em and mn ) . the ct brain scans were reviewed by two observers , a radiologist ( ka ) and a physician ( mn ) to determine the pathological type of the stroke . the reliability of the method was confirmed by assessing a random 10% sample of the radiographs without knowledge of prior reports . survival duration was estimated from the date of onset of stroke to the date of death or last contact . kaplan - meier analysis was used with log - rank procedure to compare unadjusted survival . cox proportional hazard analysis was used to investigate the effect of each prognostic factor on survival . in all data analysis , the bmdp statistical software package was used ( university of california press , berkeley , 1991 ) . there were 106 males and 68 females with a male to female ratio of 1.56:1 . hypertension , diabetes mellitus and heart disease were found in 54 ( 31% ) , 49 ( 33% ) and 57 ( 28% ) of patients respectively . at the time of analysis , 126 patients , 72.4% of all patients , were alive . over the follow up period , 19 patients ( 10.9% ) died within 2 weeks , 32 patients ( 18.4% ) died within 4 weeks and 38 patients ( 21.8% ) died within 24 weeks . while the median survival has not been reached , the lower 95% confidence limit for the median survival was estimated at 103.14 weeks . kaplan - meier survival curve for all patients in the study is shown in figure 1 . figures 2a , 2b and 2c show the survival curves of the patients who presented with or without coma , heart disease and age below and above 60 years consecutively . unadjusted analysis of survival as a function of various variables is shown in table 1 . a logistic regression analysis to examine the adjusted effect of these variables to estimate their independent influence on survival overall survival of patients with stroke survival of patients with heart disease compared to patients with no heart disease ( bold line ) . survival of patients presenting in coma compared to patients who were conscious on presentation ( bold line ) survival of patients below age 60 years ( bold line ) compared to patients of age above 60 years survival estimates ( unadjusted analysis ) logistic regression analysis of factors influencing survival adversely ( cox proportional hazards ) mortality risk was statistically significant for patients above age 60 ( p < 0.04 ) , patients presenting in coma ( p<0.003 ) and patients with heart disease ( p<0.0009 ) . there was no significant statistical difference in mortality risk due to gender , hypertension or diabetes mellitus . cerebral infarction was the most frequently observed pathological type of stroke seen in 142 patients ( 82% ) . there were 17 patients with intracerebral hemorrhage with a mean survival of 105 weeks , and 6 patients with sub - arachnoid hemorrhage with a mean survival of 29 weeks . in common with other reports , the single most important determinant of overall survival in the saudi stroke patients , was the state of consciousness on presentation , with the presence or absence of coma1237 . age over 60 years and the presence of heart disease also adversely affected survival . ischemic , valvular and hypertensive disease were the most frequent cardiac culprits . . the poor survival may be the result of the added mortality risk from heart disease or the associated high risk of cerebral vascular disease in patients with coronary artery disease . it was not possible to study other factors such as body mass index , previous stroke , hemianopia , and blood glucose due to the limitations of the retrospective study . several multi - variate models have been designed to predict the effects of different variables on the outcome of stroke . the prediction value for the belfast model is 75%,8 guy 's hospital 65%9 and for uppsala 50%.10 the state of consciousness and urinary incontinence predicted good and poor outcome with accuracy comparable to that of multi - variate models . these multi - variate methods may have possible values in defining prognostic factors for designing clinical studies . yet in prospective studies , these models did not show advantage over simple clinical variables . in this study , data from a regional referral hospital was analyzed to define the prognostic factors useful in predicting the outcome and mortality of stroke in the saudi population . these factors can be used to evaluate the effectiveness of interventions in the treatment of stroke . factors affecting survival after stroke in saudi patients , has been found to be more or less similar to those commonly observed in other populations.11

background : prognosis of stroke has been studied in various population . factors adversely affecting short term survival include impaired consciousness , leg weakness and increasing age.aim of the study : in this study , the prognostic effects of age , sex , hypertension , diabetes mellitus and presentation in coma on the survival pattern of stroke patients presenting to a referral hospital , are reviewed.methodology:the medical records of all patients hospitalized with definite stroke at king fahd specialist hospital , buraidah , for the period between june 1986 and june 1991 , were reviewed . the cranial ct scans were also reviewed.results:one hundred and seventy four patients , 106 males and 68 females , with a mean age of 64 years who had definite stroke were studied to estimate overall survival and the various risk factors influencing it . the case records and cranial ct scans were reviewed.conclusion : the factors adversely affecting survival in this study , were age above 60 ( p<0.04 ) , presentation in coma ( p<0.003 ) and pre - existing heart disease ( p<0.0009 ) . there was no significant effect on survival due to sex , hypertension and/or diabetes mellitus .

INTRODUCTION PATIENT POPULATION STATISTICAL METHOD RESULTS DISCUSSION

the elderly appear to be one of the fastest growing population groups worldwide . never before in human history has our planet included such a large percentage of old people . only 5.4% of the population . in the year 2000 , people older than 65 years represented 12.4% of the population and this number is expected to rise to 19% by 2030 , particularly in developed countries.1 cognitive skills play an important role in older people s daily functioning , such as remembering appointments and driving . unfortunately , some of these cognitive skills decline during the process of aging . in spite of the fact that cognitive aging varies among different individuals , there are some common features.2 research studies show that changes in the cognitive domain are based on crystallized and fluid intelligence.35 crystallized intelligence is the ability to exploit acquired skills and knowledge such as formulation of general information , use of vocabulary , reading comprehension , analogs , and numerical reasoning . in fact , it can be even enhanced with advancing age at a rate of 0.020.003 sds per year at the age of 60 years and 70 years.6 in contrast , fluid intelligence , which usually starts in middle age , significantly influences the cognitive functions because this intelligence requires speed processing , problem - solving skills , working memory , abstract reasoning , and learning.7 this ability usually declines at a rate of 0.02 per year and considerably affects older people s performance of daily activities and independent living.6,8 in addition , there are a number of other aspects that can negatively influence cognitive functions in aging . these include sensory factors ( eg , impaired vision or hearing ) , health - related factors ( eg , pain or arthritis ) , changes in mood ( eg , depression or anxiety ) , and drugs that can have side effects ( eg , drowsiness).5 thus , there is currently a general trend to delay these negative changes of cognitive decline by effective nonpharmacological approaches.9 additionally , information and communication technologies ( ict ) can be one of the noninvasive solutions to the prevention of cognitive decline in the process of normal aging . the use of technologies is also supported by three major current trends : an increase in the number of older people worldwide;ict as a tool for providing older people with the promise of greater independence;the generation of baby boomers approaching retirement being relatively comfortable using ict ; they will bring many technology - related skills into their retirement years.10 an increase in the number of older people worldwide ; ict as a tool for providing older people with the promise of greater independence ; the generation of baby boomers approaching retirement being relatively comfortable using ict ; they will bring many technology - related skills into their retirement years.10 in fact , the older generation is now much more digitally literate in the use of ict than they were 2 decades ago.11,12 heart and kalderon13 stated that 80% of older people have fairly easy access to computers . recent research studies also reveal that there is a significant increase in the use of ict , especially the internet , for health purposes.1418 this concept of using the internet to enable health care and improve health in the population is called e - health.19 at present , e - health shows big potential for older people because it can enable easier access to better and more effective health care . de veer et al20 reported that older people start to be open - minded toward the use of e - health applications . furthermore , bujnowska - fedak15 stated that although the main users of the internet are young people , the key users of online health services are older adults . this is also confirmed by the italian study17 and the german study18 in which more than a half of the seniors admitted using the internet in search for health - related information . the kaiser family foundation21 survey presented that older people use selected e - health services that include , for example , obtaining information on their health , receiving reminders for scheduled visits , medication instructions , and consulting a doctor at a distance . furthermore , they use internet for researching health information about proper nutrition , exercise or weight issues , diseases , such as cancer , heart disease , and arthritis , high cholesterol , and health providers . bujnowska - fedak15 also added accessing and managing personal health records , participating in forums on self - help groups focusing on health and illness , and ordering medicine and other medicinal products . in addition , the internet use may promote health literacy skills through providing opportunities for health knowledge acquisition , improving other cognitive functions such as reasoning skills , providing social benefits through social media and networking sites , and training the specific navigational and digital skills required to use the internet.22 thus , the purpose of this study is to discuss the role of the internet in the prevention of cognitive decline in normal aging . the author examines clinical studies that exploit the use of the internet , including online training programs , in the prevention of cognitive decline in healthy older individuals . the methodology of this review study is based on the study of moher et al23 and the study of kurz and baelen.24 the main method was a systematic review whose aim was to find the research studies on the basis of the keywords in five databases : web of science , sciencedirect , springer , scopus , and medline . this review was conducted in the period from 2010 to 2015 for the following keywords : use of the internet and cognitive decline in normal aging . altogether , 2,037 studies were found via the database search and 154 studies via other sources , which included conference proceedings and books outside the scope of the databases described earlier . after a thorough review of the titles , duplication , and abstracts of the selected studies , only 42 studies remained for the full - text analysis . the study was included if it matched the corresponding period , ie , from 2010 to 2015 . the selection period starts with the year of 2010 since this is the year when the clinical trials conducted among older adults started to appear due to older individuals being more digitally literate and able to use the internet on a daily basis.11 thus , the study was included if it matched the corresponding period , ie , from 2010 to 2015 ; if it involved healthy older people and focused on the use of the internet , especially online training programs . the remaining selected theoretical articles , review articles , and book chapters were exploited in the discussion and in the introduction for the exploration of the research topic and comparison of findings . figure 1 illustrates the selection procedure of the final number of studies , which was done in the following four steps : identification ( identification of the relevant studies on the basis of the keywords and , consequently , on the basis of the titles);duplication check ( the same articles were included only once);assessment of relevancy ( verification on the basis of abstracts whether the selected study corresponds to the set goal);after the exclusion of the irrelevant studies , 42 sources were analyzed and 34 eventually excluded . identification ( identification of the relevant studies on the basis of the keywords and , consequently , on the basis of the titles ) ; duplication check ( the same articles were included only once ) ; assessment of relevancy ( verification on the basis of abstracts whether the selected study corresponds to the set goal ) ; after the exclusion of the irrelevant studies , 42 sources were analyzed and 34 eventually excluded . the studies were included if the internet / online intervention activities were involved and if participants were healthy older individuals ( usually > 60 years ) although in two cases the intervention group also comprised subjects with mild cognitive impairment . four of the clinical studies were randomized controlled trials , which means that the efficacy of the treatment of the intervention group could have been compared with the control group ; two were longitudinal cohort studies , one was a randomized clinical study , and one was a pilot study . the efficacy of the intervention treatment in the studies was predominantly measured with available validated cognitive assessment tools such as a computerized cognitive assessment battery . one of the most reliable tools is , for example , brain aging monitor - cognitive assessment battery.25 as far as the intervention tool is concerned , five of the clinical studies focused on online cognitive training programs , one on the internet - based conversation , one on the use of the internet / email , and one on the use of computers . the findings of the clinical studies described earlier show that the use of the internet , mediating access to email , online activities such as email and cognitive training programs , with one exception , has a positive effect on the improvement of cognitive functions in healthy older adults . the findings of the study by xavier et al26 indicated that the internet enhances brain and cognitive reserve or leads to the engagement of more efficient cognitive networks , which may delay cognitive decline . this statement has been confirmed by other studies explored in table 1 , ie , the study of almeida et al27 and that of dodge et al.28 almeida et al,27 for example , argued that the use of computers , including emails , the internet , word processors , and games , may serve as an appropriate intervention tool against the development of dementia . as this study proved , the risk of incident of dementia was ~30%40% lower among elderly computer users than nonusers . this is in compliance with another research study by tun and lachman,29 who reported that the use of computers has been connected with enhanced cognitive functioning in adulthood and old age . in addition , the reviewed study by xavier et al26 claimed that the use of the internet , ie , digital literacy , may have a positive impact on healthy behaviors such as physical activities , higher consumption of fruits and vegetable , and less smoking among older individuals . moreover , the intervention of online cognitive training programs presented in table 1 , which can be done , thanks to the internet access and digital literacy of older adults , may also contribute to the improvement of certain cognitive functions . the findings demonstrate the enhancement especially in the area of reasoning skills and short - term memory with transfer effects on nonpracticed tasks.3032 however , the findings of the most of the reviewed clinical studies in table 1 illustrate that the samples of the subjects using the internet , especially doing online based activities , are relatively small and the intervention period is short . therefore , larger sample longitudinal randomized controlled clinical trials aimed at the use of the internet in the prevention of cognitive decline among healthy older adults are needed . this conclusion is also supported by other reviewed studies such as those of bozoki et al,33 haesner et al,34 and lampit et al,35 who believe that longer and more intense interventions might significantly enhance cognitive functions . moreover , research studies conducted in the area of the prevention of cognitive decline include other noninvasive intervention approaches such as physical activities , adherence to the mediterranean diet , chinese herbal medicine , playing a musical instrument , and learning a new foreign language.3644 most preferably , these activities , including the use of the computer , ie , the internet , should be done simultaneously . anderson - hanley et al45 stated that there is a much bigger potential for the delay of cognitive decline if , for example , cognitive and physical exercises are performed at the same time . they can also have positive effects on psychic problems such as mood improvement , removal of agitation or anxiety , and strengthening of overall fitness.46,47 the findings of the reviewed research studies suggest that the use of the internet , especially online cognitive training programs , might have a positive effect on the improvement of cognitive functions in healthy older adults . in addition , the use of the internet , including online cognitive training programs , seems to be beneficial in several ways : it is a noninvasive , in many cases , personalized intervention , and cost - effective in the sense of widespread dissemination and reduction of administration and treatment costs.4850 nevertheless , more randomized controlled clinical trials should be conducted to establish conclusive efficacy of these intervention approaches such as the use of the internet and online cognitive training programs in the prevention and delay of cognitive decline among healthy older individuals . the limitations of this review study consist in the lack of available clinical studies on the research issue and different methodologies of the included publications . this might result in the overestimated effects of the use of the internet in the prevention of cognitive decline , which can consequently have a negative impact on the validity of these reviewed studies.51,52

recent demographic trends indicate that older people appear to be one of the fastest growing population groups worldwide . in the year 2000 , people older than 65 years represented 12.4% of the population . this number is expected to rise to 19% by 2030 , particularly in developed countries . therefore , there is sustained effort at both national and international levels to prolong the active life of these people as long as possible . since the present older generation at the age of 55 years is already digitally literate , the use of technologies is one of the solutions . the purpose of this study is to discuss the role of the internet in the prevention of cognitive decline in normal aging . the author examines clinical studies that exploit the use of the internet , including online training programs , in the prevention of cognitive decline in healthy older individuals . the findings of the clinical studies indicate that the use of the internet , especially online cognitive training programs , may have a positive effect on the improvement of cognitive functions in healthy older adults . nevertheless , larger sample longitudinal randomized controlled clinical trials aimed at the prevention of cognitive decline among healthy older adults are needed .

Introduction Methods Findings Discussion Conclusion

diabetes is the seventh leading cause of death in the u.s . and is widely considered a forthcoming global epidemic ( 1 ) . it is more prevalent in certain vulnerable subgroups , particularly in african americans and latinos and those of lower socioeconomic status ( ses ) . additionally , there are significant racial / ethnic and ses disparities in diabetes care , management , and health outcomes ( 2 ) . african americans and latinos , for example , have two to four times the rate of related renal disease , blindness , amputation , and amputation - related mortality compared with non - hispanic whites ( 3 ) . disparities in diabetes outcomes are the product of multiple interacting factors , including individual , family , neighborhood , and community factors . disparities in outcomes are also likely due to poorer - quality diabetes care received by vulnerable populations . as evidence of this , the agency for healthcare research and quality has reported disparities of up to 20% in the receipt of preventive services for diabetes across race / ethnicity , income , and education groups ( 4 ) . other studies have found that differences in diabetes care vary according to characteristics of primary care practices ( 5,6 ) . individuals with diabetes receive the majority of their care in a primary care setting ( 7 ) . thus it is imperative to understand health care factors that influence care in this setting , especially with the advent of changes to the primary care environment . a medical home reflects an accessible , ongoing source of primary care that delivers or coordinates the majority of a patient s care . medical - home definitions vary considerably across organizations , but there is consensus on at least four key features : first - contact care , continuity , comprehensiveness , and coordination ( 8,9 ) . other features sometimes include aspects of community - oriented care , family - centered care , and cultural competence . the evidence cited to promote the medical home is derived almost wholly from the literature on primary care delivery ( 10 ) . there is a substantive literature demonstrating the benefits of primary care to equity and efficiency and in health care ( 11 ) , and primary care is a cornerstone of health system efforts to reduce disparities ( 12 ) . most of these studies assess the general population in need of preventive care , not those from vulnerable populations who have chronic conditions , such as diabetes . thus we undertook this study to evaluate whether patient - reported indicators of care that are consistent with the medical - home model are associated with patient - reported receipt of recommended preventive diabetes services for adults considered vulnerable . we hypothesized that patients with physicians who are reported to function more like a medical home will be more likely to report better preventive diabetes care ( including both recommended screening and patient education ) . the data for this analysis are from a cross - sectional survey of patients aged 1963 years with type 2 diabetes and medicaid ( medi - cal ) health insurance in los angeles county . first , primary care physicians ( i.e. , family medicine , internal medicine , and general practice ) were identified through publicly available network data for one of the largest medicaid plans in the county . from a list of 471 eligible physicians , they were randomly drawn and recruited until we reached our target sample size of 100 ( n = 104 ; 75% response rate ) . random selection of physicians was accomplished on a rolling basis through a random cell selection algorithm in our spreadsheet software . second , all physicians were asked to refer a minimum of 10 patients with type 2 diabetes who met the study criteria aged 18 to 63 years and having medi - cal health insurance using one of two methods : retrospective or prospective referrals . the retrospective referral method involved referring all eligible patients who had visited the physician , working from most recent up to the past 6 months . before referral , the offices called the patients using a study - provided script that offered a chance to opt out of being referred . physicians referred all eligible patients visiting the office for up to the next 6 months or until a minimum of 10 referrals were made . in this case , a flier was provided to each patient about the study by the office staff , allowing the patient an opportunity to opt out of being referred . both methods received an institutional review board approved waiver of health insurance portability and accountability act authorization by the university of southern california office for the protection of research subjects . in both approaches , the potential for physicians to hand - select patients is reduced by the requirement that all patients meeting the criteria be referred . a total of 1.8% of patients opted out of being referred , accounted for in the study response rate . from the referrals , we then randomly selected patients ( again on a rolling basis using a random cell selection algorithm in our spreadsheet software ) until we reached a goal of 5 patients per physician . there were no significant differences in opt - out rates , response rates , or demographics between referral methods . we used the primary care assessment tools ( pcat ) adult expanded to assess patient - reported indicators of medical - home quality ( 13,14 ) . the pcat has good reliability and validity and consists of 96 questions that evaluate 7 features : 1 ) first - contact care , 2 ) continuity of care , 3 ) comprehensiveness , 4 ) coordination , 5 ) community - oriented care , 6 ) family - centered care , and 7 ) cultural competence . each question is scored using a likert - type response scale as follows : definitely not ( 1 point ) , probably not ( 2 points ) , probably ( 3 points ) , and definitely ( 4 points ) . missing values in any of the pcat items were assigned the average score of 2.5 . a total medical - home score averages the responses across all of the features . the first four features have two subcomponents each : one based on the structure indicating the capacity to practice as a medical home and one based on the process indicating actual care delivery . first - contact care refers to the concept that care is available and first sought from the medical home when a new health or medical need arises , reflecting that services are accessible ( structure ) and utilization occurs when a need arises ( process ) . continuity of care refers to the use of a regular source of care over time , including tracking a defined population ( structure ) and the perceived ongoing patient - provider relationship ( process ) . comprehensiveness refers to the range of services offered ( structure ) and the recognition of problems and their appropriate delivery ( process ) . coordination refers to arranging for and following up on specialist health services , including effective information systems ( structure ) and use of that information as it bears on current needs for integration of all patient care ( process ) . these include community - oriented care that refers to the concept that all primary care is delivered in the context of the community , such that providers recognize the common health needs of the community and strive to be aware of , and oriented to , providing services to address those needs . family - centered care refers to the recognition of the family as a major participant in the diagnosis , treatment , and recovery of patients . providers must be aware of the family context , learn about the family history of illness and health risks , and work to incorporate the family as needed into care . cultural competence refers to provision of care that respects the language , beliefs , and attitudes of people as they influence health . providers should be prepared to address language barriers and account for cultural beliefs and practices in working to promote health . we asked patients when they had their last hba1c test ( when was your last hba1c test ? this is a test that measures your average blood sugar level over the past 2 or 3 months . ) and dilated eye exam ( when was the last time that you had an eye exam during which the doctor put drops in your eyes that made your pupils large ? you may have been unable to see enough to drive or had to wear dark glasses afterward . ) . responses were dichotomized at 6 months for last hba1c test and at 1 year for last eye exam . patients were asked if their physician gave them a plan to manage their own care at home , if they have had diabetes education outside of their usual doctor or nurse visits , and whether they had met with a dietitian ( defined for them as a person who teaches others what foods to eat to help you manage diabetes ) . patients were asked if in the past 6 months their physician had discussed any of nine topics with them : when and how to take insulin or diabetes pills , when and how to check blood glucose , how to time their meals , what to eat , how to check and care for their feet , how to increase their physical activity , how to make changes in medications , how to deal with the emotional demands of diabetes , and where to find community resources to help with diabetes . responses to each item were dichotomous ( yes / no ) and are reported in this study as the total number of items discussed ( 0 to 9 ) . descriptive statistics were first obtained for demographic characteristics , including race , education , employment , marital status , self - reported health status , medical - home scores for each feature and total score , as well as diabetes care and diabetes education received . next , bivariate analysis was conducted to examine the differences in medical - home scores and demographics among different diabetes care and diabetes education received . to test the significance of differences , t tests or anova finally , logistic regression of total medical - home score on diabetes care and linear regression of total medical - home score on diabetes education received were conducted , controlling for demographics . seven people had one or more missing values in the study covariates and were dropped in the regression analyses . the data for this analysis are from a cross - sectional survey of patients aged 1963 years with type 2 diabetes and medicaid ( medi - cal ) health insurance in los angeles county . first , primary care physicians ( i.e. , family medicine , internal medicine , and general practice ) were identified through publicly available network data for one of the largest medicaid plans in the county . from a list of 471 eligible physicians , they were randomly drawn and recruited until we reached our target sample size of 100 ( n = 104 ; 75% response rate ) . random selection of physicians was accomplished on a rolling basis through a random cell selection algorithm in our spreadsheet software . second , all physicians were asked to refer a minimum of 10 patients with type 2 diabetes who met the study criteria aged 18 to 63 years and having medi - cal health insurance using one of two methods : retrospective or prospective referrals . the retrospective referral method involved referring all eligible patients who had visited the physician , working from most recent up to the past 6 months . before referral , the offices called the patients using a study - provided script that offered a chance to opt out of being referred . physicians referred all eligible patients visiting the office for up to the next 6 months or until a minimum of 10 referrals were made . in this case , a flier was provided to each patient about the study by the office staff , allowing the patient an opportunity to opt out of being referred . both methods received an institutional review board approved waiver of health insurance portability and accountability act authorization by the university of southern california office for the protection of research subjects . in both approaches , the potential for physicians to hand - select patients is reduced by the requirement that all patients meeting the criteria be referred . a total of 1.8% of patients opted out of being referred , accounted for in the study response rate . from the referrals , we then randomly selected patients ( again on a rolling basis using a random cell selection algorithm in our spreadsheet software ) until we reached a goal of 5 patients per physician . there were no significant differences in opt - out rates , response rates , or demographics between referral methods . we used the primary care assessment tools ( pcat ) adult expanded to assess patient - reported indicators of medical - home quality ( 13,14 ) . the pcat has good reliability and validity and consists of 96 questions that evaluate 7 features : 1 ) first - contact care , 2 ) continuity of care , 3 ) comprehensiveness , 4 ) coordination , 5 ) community - oriented care , 6 ) family - centered care , and 7 ) cultural competence . each question is scored using a likert - type response scale as follows : definitely not ( 1 point ) , probably not ( 2 points ) , probably ( 3 points ) , and definitely ( 4 points ) . missing values in any of the pcat items were assigned the average score of 2.5 . a total medical - home score averages the responses across all of the features . the first four features have two subcomponents each : one based on the structure indicating the capacity to practice as a medical home and one based on the process indicating actual care delivery . first - contact care refers to the concept that care is available and first sought from the medical home when a new health or medical need arises , reflecting that services are accessible ( structure ) and utilization occurs when a need arises ( process ) . continuity of care refers to the use of a regular source of care over time , including tracking a defined population ( structure ) and the perceived ongoing patient - provider relationship ( process ) . comprehensiveness refers to the range of services offered ( structure ) and the recognition of problems and their appropriate delivery ( process ) . coordination refers to arranging for and following up on specialist health services , including effective information systems ( structure ) and use of that information as it bears on current needs for integration of all patient care ( process ) . these include community - oriented care that refers to the concept that all primary care is delivered in the context of the community , such that providers recognize the common health needs of the community and strive to be aware of , and oriented to , providing services to address those needs . family - centered care refers to the recognition of the family as a major participant in the diagnosis , treatment , and recovery of patients . providers must be aware of the family context , learn about the family history of illness and health risks , and work to incorporate the family as needed into care . cultural competence refers to provision of care that respects the language , beliefs , and attitudes of people as they influence health . providers should be prepared to address language barriers and account for cultural beliefs and practices in working to promote health . we asked patients when they had their last hba1c test ( when was your last hba1c test ? this is a test that measures your average blood sugar level over the past 2 or 3 months . ) and dilated eye exam ( when was the last time that you had an eye exam during which the doctor put drops in your eyes that made your pupils large ? you may have been unable to see enough to drive or had to wear dark glasses afterward . responses were dichotomized at 6 months for last hba1c test and at 1 year for last eye exam . patients were asked if their physician gave them a plan to manage their own care at home , if they have had diabetes education outside of their usual doctor or nurse visits , and whether they had met with a dietitian ( defined for them as a person who teaches others what foods to eat to help you manage diabetes ) . patients were asked if in the past 6 months their physician had discussed any of nine topics with them : when and how to take insulin or diabetes pills , when and how to check blood glucose , how to time their meals , what to eat , how to check and care for their feet , how to increase their physical activity , how to make changes in medications , how to deal with the emotional demands of diabetes , and where to find community resources to help with diabetes . responses to each item were dichotomous ( yes / no ) and are reported in this study as the total number of items discussed ( 0 to 9 ) . we used the primary care assessment tools ( pcat ) adult expanded to assess patient - reported indicators of medical - home quality ( 13,14 ) . the pcat has good reliability and validity and consists of 96 questions that evaluate 7 features : 1 ) first - contact care , 2 ) continuity of care , 3 ) comprehensiveness , 4 ) coordination , 5 ) community - oriented care , 6 ) family - centered care , and 7 ) cultural competence . each question is scored using a likert - type response scale as follows : definitely not ( 1 point ) , probably not ( 2 points ) , probably ( 3 points ) , and definitely ( 4 points ) . missing values in any of the pcat items were assigned the average score of 2.5 . a total medical - home score averages the responses across all of the features . the first four features have two subcomponents each : one based on the structure indicating the capacity to practice as a medical home and one based on the process indicating actual care delivery . first - contact care refers to the concept that care is available and first sought from the medical home when a new health or medical need arises , reflecting that services are accessible ( structure ) and utilization occurs when a need arises ( process ) . continuity of care refers to the use of a regular source of care over time , including tracking a defined population ( structure ) and the perceived ongoing patient - provider relationship ( process ) . comprehensiveness refers to the range of services offered ( structure ) and the recognition of problems and their appropriate delivery ( process ) . coordination refers to arranging for and following up on specialist health services , including effective information systems ( structure ) and use of that information as it bears on current needs for integration of all patient care ( process ) . these include community - oriented care that refers to the concept that all primary care is delivered in the context of the community , such that providers recognize the common health needs of the community and strive to be aware of , and oriented to , providing services to address those needs . family - centered care refers to the recognition of the family as a major participant in the diagnosis , treatment , and recovery of patients . providers must be aware of the family context , learn about the family history of illness and health risks , and work to incorporate the family as needed into care . cultural competence refers to provision of care that respects the language , beliefs , and attitudes of people as they influence health . providers should be prepared to address language barriers and account for cultural beliefs and practices in working to promote health . we asked patients when they had their last hba1c test ( when was your last hba1c test ? this is a test that measures your average blood sugar level over the past 2 or 3 months . ) and dilated eye exam ( when was the last time that you had an eye exam during which the doctor put drops in your eyes that made your pupils large ? you may have been unable to see enough to drive or had to wear dark glasses afterward . ) . responses were dichotomized at 6 months for last hba1c test and at 1 year for last eye exam . patients were asked if their physician gave them a plan to manage their own care at home , if they have had diabetes education outside of their usual doctor or nurse visits , and whether they had met with a dietitian ( defined for them as a person who teaches others what foods to eat to help you manage diabetes ) . patients were asked if in the past 6 months their physician had discussed any of nine topics with them : when and how to take insulin or diabetes pills , when and how to check blood glucose , how to time their meals , what to eat , how to check and care for their feet , how to increase their physical activity , how to make changes in medications , how to deal with the emotional demands of diabetes , and where to find community resources to help with diabetes . responses to each item were dichotomous ( yes / no ) and are reported in this study as the total number of items discussed ( 0 to 9 ) . descriptive statistics were first obtained for demographic characteristics , including race , education , employment , marital status , self - reported health status , medical - home scores for each feature and total score , as well as diabetes care and diabetes education received . next , bivariate analysis was conducted to examine the differences in medical - home scores and demographics among different diabetes care and diabetes education received . to test the significance of differences , t tests or anova finally , logistic regression of total medical - home score on diabetes care and linear regression of total medical - home score on diabetes education received were conducted , controlling for demographics . seven people had one or more missing values in the study covariates and were dropped in the regression analyses . a total of 540 patient interviews were completed by telephone in spanish ( 55.3% ) , english ( 43.7% ) , and mandarin or armenian ( 1% ) . the most conservative response rate ( 56.9% ) is calculated as the total completed patient interviews ( n = 540 ) out of all patients sampled ( n = 949 ) . approximately one - fifth of all patients sampled had an incorrect phone number and address ( n = 165 ) and were not considered usable . the response rate among just the usable listings ( n = 784 ) was 68.9% . among patients that we reached and spoke with ( n = 635 ) , the response rate was 85.0% . table 1 provides a listing of the descriptive details of the 540 patients surveyed . of note , 77% were hispanic , 56% had less than a high school education , and 72% were unemployed . medical - home performance measurements yielded an average score of 2.85 0.29 ( based on a 14 scale , with 4 representing the highest level of care ) . the lowest score was for community orientation ( 2.10 0.61 ) and the highest for first - contact utilization ( 3.81 0.40 ) . with regards to diabetes care , a majority ( 83% ) reported having their hba1c level measured within the past 6 months , while only 58% recollected having had a dilated ophthalmologic examination within the past year . descriptive statistics ( n = 540 ) for medical - home measures , all 88 , 99 , and missing values were coded as 2.5 . for last hba1c test , 31 observations of do nt know were coded as 6 months . for last eye exam , 14 observations of do nt know were coded as 1 year . for diabetes education , education score equals the number saying yes to the nine education items with minimum of 0 and maximum of 9 . e / vg / g , excellent / very good / good . table 2 provides a bivariate comparison between measures of diabetes care and medical - home performance . higher medical - home performance was reported among patients who had received more timely and thorough diabetes care . for example , the first - contact access feature score was higher among patients who had an hba1c test in the past 6 months compared with those who had the test longer ago ( 2.38 vs. 2.25 ; p < 0.05 ) and an eye exam in the past year compared with an eye exam longer ago ( 2.39 vs. 2.31 ; p < 0.05 ) . similar patterns were found for all measures of medical - home performance except the comprehensiveness - services available feature . interestingly , none of the sociodemographic variables , except health status , were found to be associated with any of the diabetes care measures . bivariate analysis of medical - home measures , covariates , and diabetes care mean medical - home score ranges from 14 , with 4 = best . . table 3 shows the relationship between overall medical - home performance and the diabetes care measures after adjustment for sociodemographics and health status . we show the total medical - home performance score that summarizes all of the features here ( rather than the individual features ) because they were nearly all associated with the diabetes care measures in table 2 . this table shows that total medical - home score ( on a scale from 1 to 4 ) is associated with improvements in each of the diabetes care measures . for example , a 1-point increase in total medical - home performance is associated with 4.53 higher odds of reporting they had an hba1c test in the past 6 months and 1.88 higher odds of having an eye exam in the past year . also , a 1-point increase in total medical - home performance was associated with a 5.66-point increase in our tally of diabetes education items received ( on a scale of 19 items ) . regressions of diabetes care on medical - home total score controlling for demographics data are odds ratio / coefficient ( 95% ci ) . the concept of a patient - centered medical home is becoming an accepted model for the delivery and coordination of care for patients in primary care settings . this study is one of the first to specifically target diabetes in relationship to medical - home performance . diabetes is one of our greatest emerging health care problems , and it is particularly suited for this sort of analysis given the availability of clearly defined preventive care guidelines . in addition , the treatment of diabetes involves engagement of patients regarding lifestyle behaviors , which may benefit from the ongoing patient - provider relationship and other features in the primary care medical home . indeed , this study found that among individuals with medicaid insurance , there was a relationship between patient - reported indicators of the medical - home model and better patient - reported diabetes care . in bivariate analysis , medical - home features nearly across the board were associated with better diabetes care . when summarized as a total score in multivariate analysis , medical - home performance was more predictive of diabetes care than any sociodemographic measures in our analysis . this is somewhat expected given that the study is limited to medicaid patients ( limiting heterogeneity ) , but this also provides evidence that medical - home performance may lead to improvements in patient - reported diabetes care , even in this lower ses population . the overall pcat score reported by this population was slightly lower than generally found for privately insured patients in hmo settings ( 15 ) . many projects have been developed to enhance primary care practices , and some may be adaptable to practices predominantly serving medicaid or otherwise low - ses patients . one review identified more than 40 medical - home transformation projects nationwide , with 8 reporting measures of diabetes care or outcomes ( 16 ) . absent randomized controlled trials , before and after data in these pilots suggested the potential for improvements in diabetes care , outcomes , and costs . if medical - home performance as shown in this study is indeed important for patients with diabetes , then the challenge will be to enable more practices to adopt at least some of these features ( 17,18 ) . second , the data were collected from an important and growing , but unique , population ( mostly hispanic medicaid - insured patients in los angeles ) , and so the results may not be fully generalized to other vulnerable groups . third , features in the pcat to assess medical - home quality align closely , but not perfectly , with the other measures of medical home ( such as those by the national committee for quality assurance ) . the national committee for quality assurance measures are different in that they are reported by physicians and include elements of reimbursement not included in the pcat fourth , an element of endogeneity may exist in the results because the independent and dependent measures were reported by the same individual . for example , an individual who has a positive outlook on their medical care in general may report positive responses on both medical - home quality and diabetes care . while we can not rule out this potential endogeneity , it is reduced by the use of more reporting measures ( e.g. , do you have to wait more than 30 min before you are seen by a doctor or nurse ? ) rather than rating measures that tend to be based on more evaluative or subjective satisfaction measures ( e.g. , how satisfied are you with the wait time to be seen ? ) . lastly , the diabetes quality measures were obtained by patient self - report and thus may be neither accurate measures of either medical - home performance nor complete and objective measures of clinical care . previous work suggests the level of concordance between patient self - report and medical record or administrative data are only fair ( 5565% ) , with patients tending to overreport ( rather than underreport ) diabetes services ( 1921 ) . these data reflect the patient s understanding of their condition and their perceived health care needs and thus likely influence care - seeking behaviors ( 22 ) . the findings should , however , be confirmed with medical record or administrative data , but even those sources are not considered fully accurate and unbiased ( 20,23 ) . despite these limitations , this study adds important evidence to the value of the primary care medical home for patients with chronic disease . the study also adds to the literature on how patients perceive the medical - home model and its impact on perceptions of chronic disease care . in our analysis , features consistent with higher medical - home performance were related to improvements in patient - reported diabetes care measures . this adds to the literature on how patients perceive the medical - home model and suggest that the results may be particularly important , given that this study was conducted among a medicaid ( low - ses ) population where the prevalence of diabetes is higher , the potential for poor outcomes is greater , and resources dedicated to primary care tend to be fewer .

objectivethe patient - centered medical home has gained much traction . little is known about the relationship between the model and specific health care processes for chronic diseases such as diabetes . this study assesses the impact of features of a medical home on diabetes care.research design and methodsa cross - sectional survey of 540 patients with medicaid ( medi - cal ) health insurance and type 2 diabetes in los angeles county was performed . the primary care assessment tools was used to measure seven features of medical - home performance.resultsthe response rate of the patient survey was 68.9% . patient - reported medical - home performance averaged a score of 2.85 0.29 ( on a 14 scale , with 4 equaling the best care ) . patients who received more timely and thorough diabetes care reported higher medical - home performance in every feature except for the comprehensiveness - services available . for example , the first - contact access feature score was higher among patients who had an hba1c test in the past 6 months versus those who did not ( 2.38 vs. 2.25 ; p < 0.05 ) . before and after adjusting for sociodemographics and health status , total medical - home performance was positively associated with each diabetes care measure . a 1-point increase in total medical - home score was associated with 4.53 higher odds of an hba1c test in the past 6 months and 1.88 higher odds of an eye exam in the past year.conclusionsfeatures consistent with higher medical - home performance are associated with improvements in patient - reported diabetes care process measures , even in this low socioeconomic status setting . the patient - centered medical - home model may help in caring for people with type 2 diabetes .

Introduction Research Design and Methods Study Sample Measures Medical-Home Total Score and Its Features Diabetes Care Received Diabetes Education Received Analysis Results Conclusions

non - alcoholic fatty liver disease ( nafld ) is one of the main causes of the liver disease in the world . the disease comprises a range of damages , from mild abnormal hepatic triglyceride accumulation , simple steatosis , to non - alcoholic steatohepatitis ( nash ) , which may lead to brosis and even irreversible cirrhosis ( 1 - 3 ) . the worldwide prevalence of nafld has been roughly estimated to affect 20% - 30% of the general population ( 3 , 4 ) . studies show that this disorder is directly associated with obesity , insulin resistance , and diabetes mellitus . thus , it is expected that the incidence of nafld is rapidly increasing likely due to the rising of these relevant conditions ( 5 , 6 ) . recent data have provided evidence that mitochondria and oxidative stress play a key role in the pathogenesis of nafld ( 7 , 8) . mitochondria have several functions in fat and energy homeostasis in hepatic cells . some of the mitochondria active roles include beta - oxidation of free fatty acids , electron transfer and production of atp and reactive oxygen species ( ros ) ( 9 , 10 ) . mitochondrial dysfunction will lead to the lipid deposits accumulation of non - metabolized fatty acids in the cytosol by disruption of fatty acid beta - oxidation . as a response to the higher circulating levels of fatty acids , an increase in fatty acid beta - oxidation occurs . therefore , increased demand of mitochondrial oxidation could damage normal function of this organelle through the induction of ros production ( 1 , 7 , 11 ) . the pathogenesis of nafld results from a multiplex interaction of genetic variations with environmental factors such as sedentary lifestyle and high calorific food intake ( 12 ) . there is increasing evidence that genetic components have an important influence in the development of nafld ( 13 , 14 ) . human mitochondrial dna ( mtdna ) comprises 16,569 bp and encodes 13 essential subunits of the oxidative phosphorylation system and also 2 rrnas and 22 trnas ( 15 ) . genetic mutations in mtdna , including deletions , could exhibit broad effects on mitochondrial function and may contribute to the disease susceptibility . the 4,977 bp deletion is located between nucleotides 8,469 and 13,447 and includes several essential oxidative phosphorylation genes encoding atpase6/8 , cytochrome oxidase iii , nadh dehydrogenase subunit 3 ( nd3 ) , nd4 , and nd5 . this large deletion is the most common deletion in the mitochondrial genome that has been detected in several types of human diseases ( 16 , 17 ) . moreover , it has been suggested that variations in mtdna copy numbers may also be associated with some disorders . therefore , the possible relationship between mitochondrial copy number and diseases susceptibility has been investigated in several studies ( 18 , 19 ) . however , a few studies have examined the relationship between these mutations and nafld susceptibility . chiappini et al . reported that the mean ratio of mitochondrial dna to nuclear dna ( ndna ) content is higher in liver steatosis compared to normal liver biopsies ( 20 ) whereas two other studies showed that mtdna copy number is reduced in nafld ( 21 , 22 ) . to date , a few studies have investigated the association of mtdna copy number with nafld on the liver tissue samples . in addition , to the best of our knowledge , there is no previous report on the association between the mtdna 4,977-bp deletion and nafld . accordingly , the current study aimed to evaluate the possible association of the mtdna copy number and 4,977-bp large deletion levels with susceptibility to nafld in a sample of iranian population . this case - control study included 43 nafld patients and 20 obese control subjects undergoing sleeve gastrectomy who were recruited during the period of may 2014 to november 2015 in the khatamol anbia hospital , tehran , iran . tissue samples were taken by using liver needle biopsy from imaging or histologically - diagnosed patients with nafld by a pathologist according to the nash clinical research network criteria ( nash crn ) . based on the presence of hepatic steatosis or hepatocellular injury , nafld patients were divided into two groups : nonalcoholic fatty liver ( nafl ) and nash . nafl was dened as the presence of hepatic steatosis with no evidence of hepatocellular injury and nash was dened as the presence of hepatic steatosis and inflammation with hepatocellular injury ( 23 ) . for the control group , 20 normal liver samples were obtained by using biopsy from obese , nafld free subjects whose histological findings were completely normal . subjects were excluded if they met any of the following criteria : other liver disorders , secondary causes of steatosis including alcohol consumption , hepatitis b and c virus infection , use of drugs that may cause steatosis , and glucocorticoids therapy . subjects who had been diagnosed with any cancer as well as diabetic patients were also excluded . the study was approved by the ethics committee of hamadan university of medical sciences and written informed consent was obtained from all subjects . body mass index ( bmi ) was calculated as the body weight divided by the square of the body height ( weight ( kg)/height ( m ) ) . alanine transaminase ( alt ) , aspartate transaminase ( ast ) , cholesterol , triglyceride , hdl - cholesterol , and fasting blood glucose levels were measured according to the enzymatic methods using an automatic biochemical analyzer . genomic dna was extracted from fresh liver tissue samples by using a dna isolation kit ( allprep dna / rna micro kit , qiagen ) according to manufacturer s instructions . the relative level of mtdna copy number was measured by quantitative real - time pcr ( q - pcr ) ( 24 ) . in brief , q - pcr was performed using the onp 86 , onp 89 primers and -actin as an internal control . amplification was carried out using the sybr master mix ( real qpcr 2x mix , ampliqon ) according to the protocol of the manufacturer . the pcr was done in 40 cycles at 95c for 15 minutes , followed by 40 cycles at 95c for 30 seconds , and 58c for 1 minute . the level of mtdna copy number was calculated using the delta ct ( ct ) of average ct of mtdna and ndna ( ct = ctmtdna -ct-actin ) . the relative level of mtdna copy number was calculated using the 2method . for detection of mtdna 4,977-bp deletion , a multiplex polymerase chain reaction ( pcr ) protocol was performed using the onp 86 , onp 89 , onp 25 , and onp 74 primers ( 23 ) . the specificity of primers was checked using primer - blast from ncbi ( http://www.ncbi.nlm.nih.gov/ ) . the cycling conditions were as follows : 94c for 3 minutes followed by 35 cycles at 95c for 40 seconds , annealing time at 57.5c for 40 seconds , and extension at 72c for 40 seconds with a nal extension time of 7 minutes at 72c . onp 86 and onp 89 primers amplified a normal internal mtdna fragment ( 279 bp ) that served as a control for the pcr preparation and analysis . the appearance of a 497-bp band ( using the onp 74 and onp 25 primers ) on a 1.5% agarose gel indicated an expression of the 4,977-bp deletion . the accuracy of amplifications was verified by direct sequencing analysis . to determine 4,977-bp deletion levels , the mtdna content was measured in individuals who carried this mutation using a real - time pcr and normalized by -actin genes . q - pcr was carried out using specific primer pairs ( onp 86/onp 89 and onp 12/onp 13 ) in a 20 l reaction volume containing sybr master mix ( real qpcr 2x mix , ampliqon ) according to the protocol of the manufacturer . onp 12 ( 5-tcgtagtaacagccattctc-3 ) and onp 13 ( 5-gaggttagcgaggcttgcta-3 ) primers were located within the common deletion region and amplified a specific product only in wild type mtdna . in addition , whole mtdna pcr product was amplified using the onp 86 and onp 89 primers . the pcr conditions were 95c for 15 minutes , followed by 35 cycles at 95c for 30 seconds , and 58c for 60 seconds . the threshold cycle number ( ct ) values of the mitochondrial genes and -actin were determined . the percentage of mtdna deletion levels was quantified by calculating the common deletion region relative to total mitochondrial dna . the quantitative data were expressed as mean sd ( standard deviation ) or median ( interquartile range ) . depending on whether the data were normally distributed or not , parametric or non - parametric tests were employed . parametric statistical tests including student s t - test and one way anova were used to compare the normally distributed quantitative variables between the groups . mann - whitney u test was used to compare non - normally distributed variables between the groups . logistic regression analysis was performed in order to adjust for the confounders of age , bmi , and lipid levels . pearson correlation analysis was performed to determine the relationship of the level of mtdna copy number with other variables . this case - control study included 43 nafld patients and 20 obese control subjects undergoing sleeve gastrectomy who were recruited during the period of may 2014 to november 2015 in the khatamol anbia hospital , tehran , iran . tissue samples were taken by using liver needle biopsy from imaging or histologically - diagnosed patients with nafld by a pathologist according to the nash clinical research network criteria ( nash crn ) . based on the presence of hepatic steatosis or hepatocellular injury , nafld patients were divided into two groups : nonalcoholic fatty liver ( nafl ) and nash . nafl was dened as the presence of hepatic steatosis with no evidence of hepatocellular injury and nash was dened as the presence of hepatic steatosis and inflammation with hepatocellular injury ( 23 ) . for the control group , 20 normal liver samples were obtained by using biopsy from obese , nafld free subjects whose histological findings were completely normal . subjects were excluded if they met any of the following criteria : other liver disorders , secondary causes of steatosis including alcohol consumption , hepatitis b and c virus infection , use of drugs that may cause steatosis , and glucocorticoids therapy . subjects who had been diagnosed with any cancer as well as diabetic patients were also excluded . the study was approved by the ethics committee of hamadan university of medical sciences and written informed consent was obtained from all subjects . body mass index ( bmi ) was calculated as the body weight divided by the square of the body height ( weight ( kg)/height ( m ) ) . alanine transaminase ( alt ) , aspartate transaminase ( ast ) , cholesterol , triglyceride , hdl - cholesterol , and fasting blood glucose levels were measured according to the enzymatic methods using an automatic biochemical analyzer . genomic dna was extracted from fresh liver tissue samples by using a dna isolation kit ( allprep dna / rna micro kit , qiagen ) according to manufacturer s instructions . the relative level of mtdna copy number was measured by quantitative real - time pcr ( q - pcr ) ( 24 ) . in brief , q - pcr was performed using the onp 86 , onp 89 primers and -actin as an internal control . amplification was carried out using the sybr master mix ( real qpcr 2x mix , ampliqon ) according to the protocol of the manufacturer . the pcr was done in 40 cycles at 95c for 15 minutes , followed by 40 cycles at 95c for 30 seconds , and 58c for 1 minute . the level of mtdna copy number was calculated using the delta ct ( ct ) of average ct of mtdna and ndna ( ct = ctmtdna -ct-actin ) . the relative level of mtdna copy number was calculated using the 2method . for detection of mtdna 4,977-bp deletion , a multiplex polymerase chain reaction ( pcr ) protocol was performed using the onp 86 , onp 89 , onp 25 , and onp 74 primers ( 23 ) . the specificity of primers was checked using primer - blast from ncbi ( http://www.ncbi.nlm.nih.gov/ ) . the cycling conditions were as follows : 94c for 3 minutes followed by 35 cycles at 95c for 40 seconds , annealing time at 57.5c for 40 seconds , and extension at 72c for 40 seconds with a nal extension time of 7 minutes at 72c . onp 86 and onp 89 primers amplified a normal internal mtdna fragment ( 279 bp ) that served as a control for the pcr preparation and analysis . the appearance of a 497-bp band ( using the onp 74 and onp 25 primers ) on a 1.5% agarose gel indicated an expression of the 4,977-bp deletion . the accuracy of amplifications was verified by direct sequencing analysis . to determine 4,977-bp deletion levels , the mtdna content was measured in individuals who carried this mutation using a real - time pcr and normalized by -actin genes . q - pcr was carried out using specific primer pairs ( onp 86/onp 89 and onp 12/onp 13 ) in a 20 l reaction volume containing sybr master mix ( real qpcr 2x mix , ampliqon ) according to the protocol of the manufacturer . onp 12 ( 5-tcgtagtaacagccattctc-3 ) and onp 13 ( 5-gaggttagcgaggcttgcta-3 ) primers were located within the common deletion region and amplified a specific product only in wild type mtdna . in addition , whole mtdna pcr product was amplified using the onp 86 and onp 89 primers . the pcr conditions were 95c for 15 minutes , followed by 35 cycles at 95c for 30 seconds , and 58c for 60 seconds . the threshold cycle number ( ct ) values of the mitochondrial genes and -actin were determined . the percentage of mtdna deletion levels was quantified by calculating the common deletion region relative to total mitochondrial dna . the quantitative data were expressed as mean sd ( standard deviation ) or median ( interquartile range ) . depending on whether the data were normally distributed or not , parametric or non - parametric tests were employed . parametric statistical tests including student s t - test and one way anova were used to compare the normally distributed quantitative variables between the groups . mann - whitney u test was used to compare non - normally distributed variables between the groups . logistic regression analysis was performed in order to adjust for the confounders of age , bmi , and lipid levels . pearson correlation analysis was performed to determine the relationship of the level of mtdna copy number with other variables . the characteristics of the study population including 43 nafld patients ( 19 with nafl and 24 with nash ) and 20 control subjects are provided in table 1 . in addition , table 2 represents the clinical and biochemical characteristics of the nafl and nash patients . there was no difference in age , systolic and diastolic blood pressure , and lipid levels between the nafld patients and control subjects . however , nafld patients had significantly higher values of bmi ( p < 0.0001 ) . in addition , nafl and nash patients were significantly different in alt level ( p = 0.03 ) . we did not find signicant differences between these groups in terms of other biochemical parameters ( p > 0.05 ) . abbreviations : fbs : fasting blood glucose ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein . p values were computed by test , t - test , and mann - whitney u test . statistically signicant . abbreviations : fbs : fasting blood glucose ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein . p values were computed by test , t - test , and mann - whitney u test . statistically signicant . the comparison of mtdna copy number between nafld cases and healthy controls is shown in figure 1 . the relative expression of mtdna copy number was 3.7 fold higher in nafld patients than healthy controls ( p < 0.0001 ) . the results remained significant after adjustment for age , bmi , gender , and lipid profile ( p = 0.02 ) . moreover , the relative expression of mtdna copy number was 4.3 ( p < 0.0001 ) and 3.2-fold ( p < 0.0001 ) higher in nafl and nash patients than healthy controls , respectively . no significant differences were found in the expression of mtdna copy number between the nafl and nash patients ( p = 0.499 ) . a , comparison of the relative expression of mtdna copy number in nafld and control subjects ; b , comparison of the relative mtdna copy number in liver tissue samples of patients with nafl , nash , and control subjects . p values were computed by test and t - test and data are shown as mean sd . no significant differences were also observed in the expression of mtdna copy number between male and female subjects ( p = 0.494 ) . furthermore , our results showed that there was not a significant difference in mtdna copy number level between older ( > 42 years old ) and younger participants ( < 42 years old ) ( p = 0.118 ) . finally , there was not a signicant correlation between mtdna copy number and biochemical and anthropometrical parameters such as lipid levels , blood pressure , and bmi after adjusting for age and sex ( p > 0.05 ) . based on the results , the 4,977-bp deletion was detected in none of liver tissue samples obtained from the 20 control subjects . whereas , 8 out of 43 nafld patients ( 18.6% ) showed the 4,977 -bp deletion in their liver tissues ( p = 0.039 ) ( figure 2 ) . then , the deletion levels were measured in the nafld patients carrying the deletion by q - pcr . our finding showed that the mtdna deletion levels varied from 14.31% to 28.68% of the total mitochondrial dna in tissues carrying this deletion . of total 8 nafld cases carrying the 4,977-bp deletion , 7 cases ( 87.5% ) belonged to the nash patients . a , electropherograms of multiplex pcr products on a 1.5% agarose gel along with a 100 bp dna ladder . the appearance of a 497-bp band was an expression of the 4,977-bp deletion ; b , schematic presentation of mtdna 4,977-bp deletion . sequence analysis performed using the finch tv 1.4.0 software ( geospiza , inc . , seattle , wa ) . deficiencies observed in mtdna genome and functions along with oxidative stress could play an important role in the pathogenesis of nafld . this case - control study indicated an association between the level of mtdna copy number in the liver tissue and risk of subsequent development of nafld . actually , our results revealed that the mtdna copy number in liver biopsy samples was signicantly higher among 43 patients with nafld than control subjects ( n = 20 ) . in agreement with our results , chiappini et al . reported that the mean ratio of mitochondrial dna to ndna content was higher in liver steatosis compared to normal liver biopsies ( 20 ) however , the findings of the current investigation are in contrast with the results of two studies that showed decreased mtdna copy number was associated with increased risk of nafld . silvia sookoian et al . evaluated the mtdna copy number in liver biopsy samples obtained from 63 patients with nafld and 11 control subjects . they observed that the mtdna / ndna ratio was signicantly lower in the liver of nafld patients than the liver of control subjects ( 21 ) . in another study , carlos et al . showed that patients with nafld ( n = 90 ) have a significantly lower mtdna copy number in fresh liver samples compared to control subjects ( n = 23 ) ( 22 ) . one possible reason for this contradictory result may be due to differences in sample size and ethnic diversity of participants . moreover , several factors are likely to determine mtdna copy number that could be a reason for these negative findings . , several studies have demonstrated a possible association between mtdna copy number and susceptibility to various disorders especially different cancer types . both high and low levels of mtdna copy number have been reported to be associated with an increased risk of malignancies . elevated copy number was observed in tumor tissues of the breast , head , and neck cancers , lung cancer , papillary thyroid carcinoma , ovarian cancer , and prostate cancer ( 19 , 25 - 29 ) . the precise mechanism of increased risk of cancers and other pathologic situations with increased relative mtdna copy number is not completely understood . however , multiple studies provided insights into the potential regulatory mechanism . accumulating evidence suggests that elevated levels of mtdna copy number could possibly be elucidated by an elevation in the level of oxidative stress markers ( 30 , 31 ) . oxidative stress seems to be one of the major pathogenic mechanisms for progression of nafld ( 32 ) . it has been shown that oxidant levels are significantly higher in nafld patients than healthy controls ( 33 , 34 ) . mitochondria are the main target of attack by free radicals , and excessive ros production may cause oxidative modications of lipids and mtdna damages ( 35 ) . several studies suggest that since mtdna does not have protective histones and repair mechanisms ; it is more prone to oxidative damage than ndna ( 36 ) . therefore , increased mtdna copy number could be resulted from a compensatory response to the cumulative exposures to oxidative damage that has been associated with nafld ( 31 ) . the results are in agreement with those of studies which failed to find a significant correlation between age and mtdna copy number ( 37 ) . however , some studies have found that mtdna content is age - dependent and reported both positive and negative associations in this regard ( 38 , 39 ) . to the best of our knowledge , there is no previous report on the association of the mtdna 4,977-bp deletion with nafld . our results showed that the 4977bp deletion was observed in 18.6% ( 8/43 ) of nafld patients , whereas this deletion was not detected in any of liver tissue samples obtained from the 20 control subjects . several studies have found the mtdna 4,977-bp deletion in various types of diseases ( 16 , 17 ) . although , several lines of evidence indicated that oxidative stress inside mitochondria could be attributable to mtdna large deletions ( 40 ) . further studies are needed to confirm this association and also evaluate oxidative stress - related factors that have an influence on mtdna variations . moreover , our results showed that the 4,977-bp deletion frequency was higher in nash cases compared to nafl patients . in addition , although the differences were not statistically significant , the research findings revealed that mtdna content was higher in nafl patients than nash subjects . the decreases in mtdna have been reported in the liver tissues of patients with nash , whereas mtdna content was shown to be signicantly higher in patients with simple fatty liver ( 20 ) . the reduced mtdna level in nash patients could induce progressive inflammation and fibrosis . in conclusion , this case - control study indicated an association between mtdna content in liver tissues and risk of subsequent development of nafld in a sample of iranian population that might be a biological adaptation in the response to oxidative stress .

backgroundthere is growing evidence that deficiencies observed in the mitochondrial dna ( mtdna ) functions could play an important role in the pathogenesis of non - alcoholic fatty liver disease ( nafld ) . we hypothesized that genetic variations in mtdna could affect the mitochondrial function and contribute to the nafld susceptibility.objectivesin this study , the possible association of the mtdna copy number and 4,977-bp deletion levels with nafld susceptibility in a sample of iranian population was evaluated.methodsthis case - control study included 43 nafld patients and 20 control subjects . genomic dna was extracted from fresh liver tissue samples by using a dna isolation kit . the mtdna copy number and mtdna deletion levels were measured by quantitative real - time pcr and multiplex pcr.resultsthe relative expression of mtdna copy number was 3.7 fold higher in nafld patients than healthy controls ( p < 0.0001 ) . the results remained significant after adjustment for age , bmi , and gender ( p = 0.02 ) . in addition , the mtdna copy number was 4.3 ( p < 0.0001 ) and 3.2-fold ( p < 0.0001 ) higher in nonalcoholic fatty liver ( nafl ) and non - alcoholic steatohepatitis ( nash ) patients than healthy controls , respectively . finally , the results showed that the 4,977-bp deletion is not detected in any of liver tissue samples obtained from the 20 control subjects whereas 8 out of 43 nafld patients ( 18.6% ) showed the 4,977 -bp deletion in their liver tissues ( p = 0.039).conclusionsthis study indicated an association between mtdna content in the liver tissue and nafld susceptibility that may be a consequence of compensatory response to the cumulative exposures to oxidative damage .

1. Background 2. Objectives 3. Methods 3.1. Study Population 3.2. Anthropometric and Biochemical Analyses 3.3. DNA Extraction 3.4. mtDNA copy Number Assay 3.5. Detection and Determination of mtDNA 4,977-bp Deletion 3.6. Statistical Analysis 4. Results 5. Discussion

oral health is an integral part of general health . with the life expectancy increasing to 66.8 years1 among the indian population , the role oral health plays in improving the quality of a person s life can not be overstated . over the last five decades , there has been a steady increase both in the prevalence and in the severity of dental caries in india . it is evident from the literature that 30% of children suffer from mal - aligned teeth and jaws diminishing the potential of dentofacial apparatus to function properly.2 oral cancer constitutes 35 - 40% of total body cancers which could be attributed to the wide usage of tobacco , betel nuts , quid , pan masala etc.3 the health care delivery system in india embodies a public sector , a private sector , and an unofficial nexus of health care providers operating within an unchecked environment , with no supervision on the services provided with respect to the provider , way of provision of services , costs involved , and there is no standardization in measuring the quality of care . this impromptu health care system has created a wide disparity in access to dental care , with the frivolous and lackadaisical public health system rubbing salt into the wound . for most indians , a visit to a dental office is considered an extraordinary and an unexpected event . according to the concept given by bhore committee in 1946 , a primary health center ( phc ) is supposed to be a basic health unit that provides curative and preventive health care services in an integrated manner to the rural population emphasizing on the preventive and promotive genres of health care , situated as close to the people as possible . the national health plan ( 1983 ) recommended reconstitution of phcs at a ratio of one phc for every 30,000 rural population in the plains . this ratio is 1:20,000 in hilly , tribal , and backward areas to render comprehensive coverage.4 though the national oral health policy had recommended dentists to be recruited in all phcs , it is unfortunate that this policy has not been implemented till date and there are no dentists appointed in phcs in some states . there are nine clusters under the phc with a population coverage of 41,650.5 the main objective of any oral health care system was to maintain and improve oral health care , which depends on the willingness of the individuals to seek care . in many developing countries , utilization is measured as the number of visits per year or the number of people with at least one visit during the previous year , and utilization studies serve as an important tool in designing and modifying oral health policies.6 with this background a study is planned to know the dental services utilization among the rural population under the phc , with the following objectives : to know the normative needs among the rural population.to know the factors determining utilization of dental services . to know the normative needs among the rural population . to know the factors determining utilization of dental services . the study was carried out in clusters under the primary health center situated 30 km from guntur , andhra pradesh , india . a cross - sectional house to house survey was conducted in the field practice area of a phc in southern india . three clusters were randomly selected for data collection from a total of nine clusters that are covered under the phc . the list of houses was obtained from the respective rural administrative authorities . from each cluster , questionnaires were distributed to adults residing in the selected houses to assess their dental care utilization , followed by screening for existing oral diseases . demographic profile includes details regarding name , age , sex , education , occupation , income ( kuppuswamy s socio - economic status [ ses ] scale - updating for 2007).7 each respondent was asked a series of questions on dental health attitudes , last dental visit , treatments underwent and factors influencing utilization . responses to the questionnaire were analyzed using chi - square test to assess the association between demographic characteristics . a significant relationship was assumed to exist between the groups if the p value was found to be < 0.05 . ethical clearance was obtained from the ethical committee of sibar institute of dental sciences and prior permission was taken from local authorities . subjects who were available on the day of examination and permanent residents of the study area . a cross - sectional house to house survey was conducted in the field practice area of a phc in southern india . three clusters were randomly selected for data collection from a total of nine clusters that are covered under the phc . the list of houses was obtained from the respective rural administrative authorities . from each cluster , questionnaires were distributed to adults residing in the selected houses to assess their dental care utilization , followed by screening for existing oral diseases . demographic profile includes details regarding name , age , sex , education , occupation , income ( kuppuswamy s socio - economic status [ ses ] scale - updating for 2007).7 each respondent was asked a series of questions on dental health attitudes , last dental visit , treatments underwent and factors influencing utilization . responses to the questionnaire were analyzed using chi - square test to assess the association between demographic characteristics . a significant relationship was assumed to exist between the groups if the p value was found to be < 0.05 . ethical clearance was obtained from the ethical committee of sibar institute of dental sciences and prior permission was taken from local authorities . subjects who were available on the day of examination and permanent residents of the study area . of the 385 study subjects , 190 ( 49.4% ) were male and 195 ( 50.6% ) were female with majority of them belonging to the age group of 35 - 44 years ( 22.08% ) . principal number ( 46.23% ) of the study subjects belonged to upper lower ( ul ) ses and very few subjects ( 1.31% ) belonged to upper ses . 4.41% study subjects belonged to lower ses , 40% of study subjects belonged to lower middle ( lm ) ses , and 8.05% study subjects belonged to upper middle ( um ) ses . table 1 shows the distribution of study subjects according to the felt need . major dental problem experienced by the study subjects was toothache which constituted around 68.85% , of which 51.37% study subjects belonged to lm socio economic group . difference in proportions is statistically significant as analyzed by chi - square test ( p = 0.00 ) . majority of study subjects have undergone an extraction which constituted about 50.27% of which 37.17% study subjects belonged to lm socio economic group . the percentage of subjects who underwent restorations and prosthetic treatments constitute 5.46% and 3.28% , respectively . influenced by various factors 31.15% of the subjects with one - third of them belonging to lower ses , availed no treatment despite having dental problems . difference in proportions is statistically significant as analyzed by chi - square test ( p = 0.00 ) . most of the study subjects ( 68.25% ) preferred private dental clinics over government dental hospital ( 75.08% ) in pursuing their dental needs . difference in proportions is statistically significant as analyzed by chi - square test ( p = 0.00 ) . table 4 shows the distribution of study subjects according to factors influencing for not utilization of dental services . impenitent attitude of the patient stands out as prime reason ( 50.88% ) for not visiting the dentist and is followed by the reasons like lack of awareness ( 14.03% ) and notions like dental treatments are expensive ( 14.03% ) . upper ses group reported reason for not visiting a dentist was lack of time . distribution of study subjects according to factors influencing for not utilization of dental services even though they have perceived the problem . table 5 shows the distribution of study subjects according to the reason for taking treatment in a particular center . much of the study subjects ( 45.24% ) stated proximity of dental clinics as influencing factor in choosing a center to avail dental treatments and only 13.49% of the subjects underwent treatment in a particular center as they provide better quality treatment . difference in proportions is statistically significant as analyzed by chi - square test ( p = 0.00 ) . table 6 shows the distribution of normative needs according to gender . majority ( 49.78% ) study subjects were observed with the presence of multiple problems , 27.1% were observed with calculus and 8.05% were observed with decayed teeth . among the study subjects , 9.68% of the um socio economic group presented with decayed teeth . 5.9% of the lower socio economic group had missing teeth . however , the difference observed was statistically not significant ( p = 0.407 ) . utilization is not just the willingness of people to seek care , but the actual attendance at the site of delivery of health care services to receive care . the present study was conducted with a sample of 385 subjects , who were the permanent residents of a phc area . the sample comprised of 190 males ( 49.35% ) and 195 females ( 50.65% ) . in the present study , maximum ( 22.27% ) number of subjects belonged to the 35 - 44 years age group and a minimum of 5.44% subjects belonged to the age group of 0 - 14 years . in our study , it was observed that toothache was the ubiquitous problem , which being an emergency condition should logically have forced them to visit a dentist . but nearly 30% of people having toothache had not visited a dentist , whereas a study conducted by poudyal et al . it was identified from the current study that not giving much importance to dental care was the major factor negatively influencing the utilization of dental care accounting for 50.88% of the total influencing factors . ( 2010),8 where a majority of people opined that they had no dental problems , recognized that the mismatch between normative need and perceived need was the major factor affecting utilization of dental care . in our study , it was observed that fear was not a major reason for non - utilization of dental care , constituting for a mere 3.5% of all the reasons given for non - utilization . nonetheless , these results are not in accordance with a study done by kakatkar et al . ( 2011)9 and tong and tong ( 2005)10 where fear was recognized as the major reason for not utilizing the dental care . this study did not show significant difference between the dental visit history among males and females ; and similar results were observed in a study done by poudyal et al . ( 2010).8 dissimilar results were observed in a study done by liddell and locker ( 1997),11 kakatkar et al . ( 2011).9 most preferred treatment centers by the study subjects were private dental hospitals ( 24.7% ) , and the reason identified was accessibility which constituted ( 15.1% ) of all the reasons given . utilization of oral healthcare services has long been used as an indicator of oral health related behavior . the iterated reason for not visiting the dentist was did not think it was important , which shows the laxity of indian population towards oral health . the only health service available for the rural population is phc , but there is no dentist in it to provide dental care . the need of the hour is to improve the oral health knowledge and awareness among people . motivating them towards availing oral health care is a prime requisite to provide them a socially and economically productive life . recruitment of dentists in primary health centers and providing the necessary infrastructure is the only possible way to abolish the malady of apathy toward procuring oral health care .

background : the oral disease burden in india is showing a steady increase in the recent years . utilization of dental care being the major factor affecting the oral health status of the population is used as an important tool in oral health policy decision - making and is measured in terms of the number of dental visits per annum.materials and methods : a cross - sectional house to house questionnaire survey was conducted in three rural clusters which were randomly selected from a total of eight clusters served by a primary health center . simple random sampling was used to select 100 houses from each cluster . screening was done to examine the existing oral diseases . a total of 385 completed questionnaires were collected from 300 houses.results:of 385 study subjects , 183 have experienced previous dental problems . major dental problem experienced by the study subjects was toothache ( 68.85% ) and the treatment underwent was extraction ( 50.27% ) . most preferred treatment centers by the study subjects were private dental hospital ( 68.25% ) and reason identified was accessibility which constituted ( 45.24% ) of all the reasons given . negative attitude toward dental care is one of the important barriers ; 50.8% of the non - utilizers felt dental treatment is not much important.conclusion:persons attitude , lack of awareness , and affordability remain the barriers for utilization of dental services . effective methods have to be exercised to breach such barriers .

Introduction Materials and Methods Study population and sampling procedure Questionnaire Ethical clearance Inclusion criteria Exclusion criteria Results Discussion Conclusion

the increased attractiveness to infectious mosquitoes coupled with transient depression in cell - mediated immunity increases pregnant women 's susceptibility to malaria and its adverse birth - related outcomes . each year , more than 30 million pregnancies at risk of malaria occur in sub - saharan africa . the adverse effects of mip vary geographically by malaria transmission intensities . in high transmission settings where repeated exposure to infectious mosquito bites is common , most people acquire partial protective immunity by early adulthood . however , pregnant women in these settings remain comparatively more susceptible to malaria than their nonpregnant counterparts as both cellular and humoral immune responses to plasmodium antigens are depressed [ 2 , 4 ] . in such settings , mip is mainly characterised by asymptomatic parasitemia often with sequestration of parasites in the placenta with maternal anaemia and low birth weight ( lbw ) as the principal adverse outcomes [ 5 , 6 ] . in areas of low transmission , , mip is associated with severe clinical illness often with increased risks of miscarriages , still births , lbw , or even maternal death . the pathophysiological processes preceding the adverse foetal consequences of mip are mainly initiated by accumulation of parasitized red blood cells ( prbcs ) in placental intervillous spaces . these prbcs express parasite - encoded surface antigens ( e.g. , pf emp-1 ) that enable their sequestration to host receptors such as chondroitin sulphate a and hyaluronic acid . this is followed by infiltration of inflammatory cells and release of proinflammatory mediators and finally by syncytiotrophoblastic necrosis and the deposition of fibrinoid material . placental malaria ( pm ) is therefore a major manifestation of mip and often occurs without peripheral symptoms . previous studies estimating the burden of pm in sub - saharan africa have yielded variable findings . in one review , prevalence estimates were in the range 9.537% using placental blood smears , 4143% using rapid antigen tests , 5159% using pcr , and 5575% using placental histology . while this may reflect true underlying differences in disease burden by study sites , these differences have been partly attributed to sensitivity differences in the tools used . however , despite the fact that placental blood smear was the least sensitive method , most previous studies have used it , probably due to its ease of application . it therefore appears that , more often than not , previous studies have underestimated the true burden of pm and thus malaria burden in pregnancy . nonetheless , it was estimated in 2007 that at least 25% of african pregnancies were afflicted by malaria at the time of delivery . in kampala district , an area of moderate malaria transmission , data from mulago hospital collected ten years earlier showed that 14% of deliveries had evidence of malaria infection . the country has since witnessed a sustained malaria control campaign implemented by the government in collaboration with development partners . key elements of this campaign have included the introduction of artemisinin - based combination therapies ( acts ) for effective case management , promotion and mass distribution of insecticide - treated bed nets ( itns ) , and increased public education to improve treatment - seeking and prevention practices . whereas these interventions have targeted the population at large , intermittent preventive treatment with sulfadoxine - pyrimethamine ( sp - iptp ) has been particularly implemented as an additional measure to prevent mip . yet , recent times have seen increasing concerns about the effectiveness of sp - iptp both locally [ 1315 ] and globally [ 1618 ] . these concerns have been largely driven by the rising prevalence of higher - level sp resistance mutations . to this end , recent studies from areas with high prevalence of resistance mutations have reported reduced efficacy of sp - iptp [ 14 , 15 , 19 , 20 ] . however , such findings in most cases are hampered by the exclusive reliance on self - reports and antenatal records to ascertain sp use . because unsupervised sp - iptp administration is a common occurrence in many resource - limited settings , we have previously argued that self - reports and anc records may not necessarily translate into sp intake compliance [ 21 , 22 ] . moreover , where intake was ascertainable , studies have continued to show effectiveness of sp - iptp [ 13 , 2329 ] . consequently , some authors have questioned the usefulness of molecular markers in monitoring sp - iptp effectiveness [ 25 , 27 , 30 ] . more recently however , the pfdhps a581 g mutation ( a proxy for higher - level resistance ) has emerged as a better marker for sp - iptp effectiveness [ 31 , 32 ] . prevalence levels exceeding 10% have been found to correlate with sp - iptp failure . in kampala district where prevalence of the quintuple mutation is high but that of the a581 g mutation is thought to be low , this study aimed to assess the relevance of continued sp - iptp use . placental histology , a robust and sensitive method , was used to examine malaria burden among pregnant women who received or did not receive intermittent preventive treatment . this was a descriptive cohort study conducted among pregnant women at mulago hospital between september and november 2014 . the exposure arm of this cohort was part of a larger population pharmacokinetic study in which 87 pregnant women received two supervised doses of sp - iptp and were followed up until delivery . for the control arm , pregnant women presenting in early labour , with no history of sp use , were consecutively identified and invited to participate in the study . these women were required to donate venous blood samples for a retrospective assessment of sp exposure covering the previous 2 - 3 months . mulago hospital is uganda 's national referral facility located in kampala , the capital and commercial centre . in addition to a wide range of specialized inpatient care , the hospital runs a range of outpatient clinics on week days . most outpatients come from the urban and periurban communities of kampala and the neighbouring districts of mukono and wakiso . these areas are mainly inhabited by the ethnic bantu , particularly the baganda . over time , however , other ugandan ethnic communities have settled here , constituting a significant minority . most outpatients tend to be low- and middle - income earners who prefer to utilize the hospital 's free services . malaria is endemic in 95% of uganda 's geographic area with the remaining 5% ( mainly highland areas with altitudes greater than 1,600 m ) subject to low and seasonal transmission . kampala is situated close to the equator at altitudes ranging from 1,300 to 1,500 m above the sea level and experiences a tropical climate with two rainy seasons ( march to may and september to november ) . the last twenty years have witnessed a steady increase in kampala 's urbanization , a change that has possibly contributed to a reduction in malaria burden . recent figures on transmission intensity are hard to come by , but prevalence of malaria among children aged 059 months in the greater kampala region has recently been estimated at 5.5% , down from 12.1% in 2009 . while the prevalence of the dhfr / dhps quintuple mutations is generally high for kampala ( > 80% ) , there are no reliable estimates for the prevalence of the pfdhps a581 g mutation . however , recent estimates from the neighbouring mukono district placed the prevalence at 3.9% among pregnant women presenting for anc . hospital records show that approximately 1,000 outpatients are seen daily , with 25% being pregnant women seeking obstetric care at the two anc clinics located at old and new mulago . all pregnant women seeking care initially present to the general anc clinic run by a team of experienced midwives who offer a standard package of routine anc services . women who require the opinion of an obstetrician or those considered to have , regular attendees of the general clinic , especially those deemed to have undergone a normal pregnancy , are directed for delivery in ward 14 , a labour unit located within the old mulago complex and also run by a team of experienced midwives . women with high risk pregnancies as well as those presenting randomly for the first time while in labour are referred for delivery in ward 5c located within new mulago complex . here , unlike the setting at old mulago , additional services such as caesarean sections , blood transfusions , and special care facilities for premature babies are readily available . sp - iptp users were enrolled from the general anc clinic at old mulago while in the second trimester of gestation ( between 16 and 20 weeks ) . the women received two doses of sp ( one in each of the second and third trimesters ) administered under the direct supervision of a midwife in accordance with national guidelines for sp - iptp at that time . for ethical reasons , sp nonusers were only recruited in early labour from among pregnant women presenting to ward 5c for delivery . to be eligible for enrollment , a woman had to have reported nonuse of sp - iptp during the present pregnancy . as such claims may lack credibility for several reasons , each woman was required to provide a venous blood sample ( 4 ml ) for detection of sulphadoxine ( sdx ) . this requirement allowed for a retrospective assessment of sp exposure over the previous 2 - 3 months . upon delivery , the exclusion criteria included women with known chronic conditions such as hiv or cardiovascular , renal , metabolic , or sickle cell diseases . women who developed pregnancy - related complications such as preeclampsia or antepartum haemorrhage were also excluded . the study protocol received ethical approval from the hospital 's research ethics committee ( reference number mrec 397 ) as well as the uganda national council for science and technology ( reference number hs 1277 ) . all study procedures were performed in accordance with ethical guidelines of the helsinki declaration ( 1964 ) and its amendments as well as the international conference on harmonization ( ich ) guidelines on good clinical practice . within minutes of delivery , the placenta was placed in a labelled container fully immersed in 10% neutral - buffered formalin before transportation to the pathology laboratory for storage and further processing . whole placentas were initially stored for 24 hours in order to firm up the tissue in preparation for biopsy and definitive storage . for each placenta , a 4 4 cm biopsy was cut paracentrically involving the entire thickness of the placenta . this biopsy was placed in another container with 10% neutral - buffered formalin and fixed for 24 hours before being transferred to another container with 70% ethanol where it was stored for 2 - 3 months awaiting histological preparation and assessment . within hours of delivery , the attending midwife collected brief demographic and obstetric data using a standardized questionnaire . items covered included maternal and gestational age , birth weight , sex of baby , history of bed - net use , and physical address of residence . all the histological investigation was done at the department of pathology , college of health sciences , makerere university . paraffin sections , 5 m thick , were stained with hematoxylin and eosin ( h&e ) and giemsa 's stain . each slide was scored for footprints of placental malaria based on the classification criteria proposed by bulmer et al . . this classification is based on the presence or absence of parasites or malaria pigment and has the ability to provide temporal information on malaria history for more than half the pregnancy . briefly , the classification may be summarized as follows : category 0 ( placenta not infected ; here , there is no evidence of parasites or malaria pigment observed ) ; category 1 ( active infection ; parasites seen in maternal erythrocytes in the intervillous space or pigment in erythrocytes and monocytes in the intervillous space ; there is no pigment in fibrin or cells within fibrin ) ; category 2 ( active - chronic infection ; parasites in maternal erythrocytes in intervillous space or pigment present in erythrocytes and circulating monocytes within the intervillous space ; in addition , there is pigment in fibrin or cells within fibrin and/or chorionic villous syncytiotrophoblast or stroma ) ; category 3 ( past - chronic infection ; parasites not present , pigment confined to fibrin or cells within fibrin ) . all bioanalytical procedures for sdx were performed at the department of pharmacology and therapeutics , college of health sciences , makerere university . venous blood samples collected from participants reporting nonuse of sp were centrifuged at 3000 revolutions per minute for 10 minutes . two millilitres of plasma was transferred into cryotubes and stored at 70 degrees centigrade until analysis . plasma was assayed for the presence of sdx using a high performance liquid chromatography - uv method as previously described by bergqvist et al . . therefore , any positive finding served to discredit the claim of sp nonuse while a negative finding corroborated such claims . with a limit of detection ( lod ) as low as 7 mol / l , it was calculated that sdx would be detectable in plasma for at least 10 weeks from the time of intake ( assuming an average cmax of 300 mol / l and a half - life of 1315 days ) . as accurate quantification of plasma sdx levels was not of paramount interest , all assay procedures were performed once for each participant sample . of the 87 women for whom supervised administration of sp - iptp was done , 38 returned for delivery at ward 14 and , from these , 33 placentas were successfully collected and processed for histological examination . to match the number of placentas in the exposure with the control arm , 33 women were also enrolled from the control group presenting to ward 5c . as shown in table 1 , participants in both groups appeared comparable on most baseline characteristics such as age , history of bed - net use , gestational age at delivery , birth weight , and baby 's sex distribution . while 94% of the sp users resided within kampala and the neighbouring districts of mukono and wakiso , 79% of the control group were from these areas . the burden of placental malaria was lower among sp users ( 4/33 ) compared to sp nonusers ( 16/33 ) . among sp nonusers with a positive malaria footprint , most ( 8/16 ) had evidence of active infection at time of delivery . in this limited sample of deliveries at mulago hospital in kampala , women who do not use sp - iptp seem to experience a higher burden of placental malaria compared to users . this finding suggests that sp may still be beneficial in preventing placental malaria and its associated adverse outcomes . this finding is consistent with clinical outcomes from several recent studies that assessed the efficacy of sp - iptp in settings with a high prevalence of sp resistance mutations [ 13 , 23 , 24 , 2628 ] . sp - iptp works by the clearance of asymptomatic parasitemia followed by posttreatment prophylaxis ( ptp ) . the latter effect occurs over a variable length of time depending on drug dose , pharmacokinetic properties , and level of parasite resistance . as the first two factors are relatively constant , it has been suggested that increasing prevalence of higher - level resistance would correlate with reduction in the duration of ptp . notwithstanding such changes , the present results suggest that sp retains significant antimalarial activity in the context of iptp . this finding is consistent with previous reviews that have shown considerable effectiveness of sp - iptp even in areas with > 90% presence of the pfdhps k540e mutation ( a proxy marker for the presence of quintuple mutation ) [ 30 , 39 ] . these observations suggest that additional factors may be critical in determining overall effectiveness of sp - iptp . have previously shown that acquired antimalarial immunity was a better predictor of malaria treatment outcomes when compared with molecular markers of sp resistance . it therefore appears that a similar phenomenon occurs with sp - iptp where the drug complements naturally acquired antimalarial immunity in order to register a beneficial clinical outcome . while various schools of thought have argued for and against the continued use of sp - iptp [ 17 , 41 , 42 ] , the present findings lend support for its continued use . moreover , confidence in this position may be further assured when sp is used according to the revised guidelines advocating for more frequent dosing of sp . in practice , this would offset any anticipated reductions in ptp associated with increasing levels of resistance . whereas the burden of pm appears to have significantly reduced in kampala over the years , no change in profile is apparent from this study . histopathological data collected ten years earlier showed prevalence of 64% , 4% , and 32% for active , chronic , and past infection , respectively . the trend in these proportions is comparable to that observed among sp nonusers in this study , that is , 50% , 12.5% , and 37.5% for active , chronic , and past infections , respectively . in addition , despite clear differences in malaria burden between sp users and nonusers , no appreciable differences in birth weights were apparent between the two groups . this may be explained by the fact that most placental infections were of category 1 ( active infection ) , reflecting infections acquired in late pregnancy . therefore , the pathological sequelae associated with pm may not have been present long enough to adversely impact foetal nutrition and hence birth weight . it has been suggested that the greatest impact of pm on birth weight occurs when infections are acquired earlier on in pregnancy , especially in the first and second trimesters of gestation [ 4345 ] . significant lbw would therefore have been anticipated in pm infections classified under category 2 or category 3 . the use of placental histology for assessment of the burden of mip may be considered a major strength of this study . this is a robust and the most sensitive measure for mip when compared with others such as peripheral , placental , or cord smear microscopy [ 10 , 46 ] . in addition , placental histology provides a temporal history of malaria over more than half of the gestational period . the requirement for participants in the control arm to provide a spot blood sample for residual drug assay the fact that none of the samples turned out positive suggests that participant selection in the control arm was done reasonably well . this scenario contrasts with a previous study in which up to 25% of pregnant women reporting nonuse of sp were found to have residual drug when tested . many previous studies reporting ineffectiveness of sp - iptp have relied on self - reports and anc records as proof of sp use [ 14 , 15 , 19 ] . where no effort is made to ascertain actual user compliance , such findings remain questionable , especially coming from resource - constrained settings where unsupervised administration of sp is the norm , contrary to guidelines . as argued elsewhere , the presence of such records does not necessarily translate into intake compliance [ 21 , 22 ] . our findings are however limited by the small sample sizes used that may not be adequately powered to detect true differences between the groups . furthermore , while only 6% of women in the sp group resided outside kampala and its neighbouring districts , this was not true for the control group , where 21% came from rural upcountry districts . persons from such areas may be predisposed to higher risks of malaria owing to the rural - urban differences in malaria burden . having failed to anticipate and control for this demographic pattern , an element of selection bias in these results can not be excluded . lastly , it is important to note that while 87 women were enrolled in the exposure arm , only 38 ( 43.7% ) returned to deliver at the hospital . going by previous studies , the phenomenon of high anc attendance coupled with low skilled attendance at delivery is not new or unique to this study [ 4751 ] . from previous studies , statistics on this phenomenon reasons advanced for this practice have included a preference for traditional birth attendants , lack of transport ( especially for nighttime onset of labour ) , and financial constraints as well as the need to perform traditional birth rituals . in some cases , rapidly progressing labour may have compelled women to deliver at home or at an alternative health facility [ 50 , 51 ] . compared to nonuse , histological evidence suggests that two doses of sp - iptp were better at preventing placental malaria among pregnant women delivering at mulago hospital . in view of the limited sample size , these findings call for a larger study that is sufficiently powered in order to ascertain these results . meanwhile , frequent doses of sp - iptp as per the revised who iptp policy may offer continued benefit in limiting malaria - related adverse pregnancy outcomes in endemic settings .

intermittent preventive treatment of malaria in pregnancy with sulphadoxine - pyrimethamine ( sp - iptp ) is widely used to reduce the incidence of adverse pregnancy outcomes . as a monitor for continued effectiveness of this intervention amidst sp resistance , we aimed to assess malaria burden among pregnant women who use or do not use sp - iptp . in a descriptive cohort study at mulago hospital , kampala , 87 women who received two supervised doses of sp - iptp were followed up until delivery . controls were pregnant women presenting in early labour without history of sp - iptp . histopathological investigation for placental malaria ( pm ) was performed using the bulmer classification criterion . thirty - eight of the 87 women returned for delivery and 33 placentas were successfully collected and processed along with 33 placentas from sp nonusers . overall , 12% ( 4/33 ) of the users had evidence of pm compared to 48% ( 16/33 ) of nonusers . among nonusers , 17/33 , 8/33 , 2/33 , and 6/33 had no placental infection , active infection , active - chronic infection , and past - chronic infection , respectively . among users , respective proportions were 29/33 , 2/33 , 0/33 , and 2/33 . no difference in birth weights was apparent between the two groups , probably due to a higher proportion of infections occurring later in pregnancy . histological evidence here suggests that sp continues to offer substantial benefit as iptp .

1. Background 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

first - year , undergraduate women living on campus ( n = 5,978 ) were eligible to participate in the body project program through their on - campus residential hall . a total of 430 women signed up to participate in the program across fall and spring semesters of one academic year . during the implementation of the body project , an online study of health behaviors and social networks was simultaneously occurring . this study conducted online assessments of first - year undergraduate women at the end of each semester and included questions regarding participation in the body project program . data from the women residing at the targeted residence halls were abstracted from the dataset . a total of 588 ( mage = 18.69 years , age range : 1832 years ) women endorsed living on campus and not participating in the body project . these participants were asked questions related to why they did not participate in the program . the online study had institutional review board approval , and participants provided online consent prior to completing the online questionnaires . the body project is an empirically supported body acceptance program based on cognitive dissonance theory . extensive research supports the program s efficacy , effectiveness , and dissemination among college populations ( becker et al . , 2008 ; perez , becker , & ramirez , 2010 ) . all body project workshops were run through the university s student development and health and wellness programming at targeted residence halls . information regarding student development , health , and mental health programs occurring within the residential community is advertised through email sent by the community director of the respective residence hall . residents received one email per week advertising the program and offering an incentive to participate . email advertising was randomized so that emails received fall semester vaguely described the program , strategically not stating that this was a body acceptance or eating disorder prevention program . the email provided a colorful picture of the face of a woman , underneath the email was composed of four sections labelled what we are about , past participants have said , incentives to participate , and link to sign up . the what we are about section highlighted that this was a program for undergraduate women by undergraduate women , that it was fun , and focused on personal growth and women s issues . spring semester , residents received emails that disclosed the program was a body acceptance program . the emails distributed were structured like the fall semester emails , with the exception that under the what we are about section , the following quote was added : do nt change your body to get respect from society . the email highlighted that this was a body acceptance program for undergraduate women by undergraduate women , that it was fun , and that it focused on personal growth and women s issues . a series of focus groups was conducted with university staff ( directors of health and wellness programming , directors of greek life , and community directors who oversee residential life programming ) and undergraduate women ( panhellenic sorority presidents , officers of three women s issue student organizations , and 25 women from diverse student organizations ) to identify incentives . the university staff suggested incentives such as t - shirts , which were financially unsustainable , a free meal ( i.e. , pizza ) , or a free dessert ( i.e. , ice cream ) . undergraduate women suggested pairing with other student organizations , offering cash for participation , or a free service , such as a manicure - pedicure treatment . based on these results , six incentives were implemented and evaluated : ( a ) fashion style training one student organization based on making fashion fit your body and reflect your personality , offered free personal styling and in return , this organization received free marketing and a constant supply of new members ; ( b ) $ 100 lottery individuals would have their named entered into a lottery , with three opportunities to win $ 100 ; ( c ) ice cream party the largest group of friends ( defined loosely as residential hall floor , sorority , or group of friends ) to attend a session would receive a free ice cream party ; ( d ) free pizza provided during the workshop ; ( e ) referral a snowball sampling strategy was employed where the participant earned $ 10 for each friend they brought to the workshop . a snowball sampling strategy is a commonly used chain referral sampling technique where participants refer peers to participate and was originally designed to recruit hard - to - reach populations ( paquette , bryant , & de wit , 2012 ; sadler , lee , lim , & fullerton , 2010 ; sedgwick , 2013 ) as well as a method for reducing attrition between follow up sessions ( sedgwick , 2013 ) . , 2012 ) ; ( f ) manicure / pedicure gift certificate local nail salons surrounding the university were asked to donate gift certificates in return for free advertising . in addition , we negotiated with one salon that for every two gift certificates purchased , they would provide a third gift certificate for free ( i.e. , buy two , get one free ) . the online questionnaire asked women if they participated in the body project with a yes or no category response . those who selected no received a second question inquiring why they did not participate and were given the following options : lack of knowledge , lack of interest , inconvenient times , inconvenient locations , stigma associated with this type of program , friends could not attend , i had no one to go with , or other . first - year , undergraduate women living on campus ( n = 5,978 ) were eligible to participate in the body project program through their on - campus residential hall . a total of 430 women signed up to participate in the program across fall and spring semesters of one academic year . during the implementation of the body project , an online study of health behaviors and social networks was simultaneously occurring . this study conducted online assessments of first - year undergraduate women at the end of each semester and included questions regarding participation in the body project program . data from the women residing at the targeted residence halls were abstracted from the dataset . a total of 588 ( mage = 18.69 years , age range : 1832 years ) women endorsed living on campus and not participating in the body project . these participants were asked questions related to why they did not participate in the program . the online study had institutional review board approval , and participants provided online consent prior to completing the online questionnaires . the body project is an empirically supported body acceptance program based on cognitive dissonance theory . extensive research supports the program s efficacy , effectiveness , and dissemination among college populations ( becker et al . , 2008 ; perez , becker , & ramirez , 2010 ) . all body project workshops were run through the university s student development and health and wellness programming at targeted residence halls . information regarding student development , health , and mental health programs occurring within the residential community is advertised through email sent by the community director of the respective residence hall . given the existing structure , residents received one email per week advertising the program and offering an incentive to participate . email advertising was randomized so that emails received fall semester vaguely described the program , strategically not stating that this was a body acceptance or eating disorder prevention program . the email provided a colorful picture of the face of a woman , underneath the email was composed of four sections labelled past participants have said , incentives to participate , and link to sign up . the what we are about section highlighted that this was a program for undergraduate women by undergraduate women , that it was fun , and focused on personal growth and women s issues . spring semester , residents received emails that disclosed the program was a body acceptance program . the emails distributed were structured like the fall semester emails , with the exception that under the what we are about section , the following quote was added : do nt change your body to get respect from society . the email highlighted that this was a body acceptance program for undergraduate women by undergraduate women , that it was fun , and that it focused on personal growth and women s issues . a series of focus groups was conducted with university staff ( directors of health and wellness programming , directors of greek life , and community directors who oversee residential life programming ) and undergraduate women ( panhellenic sorority presidents , officers of three women s issue student organizations , and 25 women from diverse student organizations ) to identify incentives . the university staff suggested incentives such as t - shirts , which were financially unsustainable , a free meal ( i.e. , pizza ) , or a free dessert ( i.e. , ice cream ) . undergraduate women suggested pairing with other student organizations , offering cash for participation , or a free service , such as a manicure - pedicure treatment . based on these results , six incentives were implemented and evaluated : ( a ) fashion style training one student organization based on making fashion fit your body and reflect your personality , offered free personal styling and in return , this organization received free marketing and a constant supply of new members ; ( b ) $ 100 lottery individuals would have their named entered into a lottery , with three opportunities to win $ 100 ; ( c ) ice cream party the largest group of friends ( defined loosely as residential hall floor , sorority , or group of friends ) to attend a session would receive a free ice cream party ; ( d ) free pizza provided during the workshop ; ( e ) referral a snowball sampling strategy was employed where the participant earned $ 10 for each friend they brought to the workshop . a snowball sampling strategy is a commonly used chain referral sampling technique where participants refer peers to participate and was originally designed to recruit hard - to - reach populations ( paquette , bryant , & de wit , 2012 ; sadler , lee , lim , & fullerton , 2010 ; sedgwick , 2013 ) as well as a method for reducing attrition between follow up sessions ( sedgwick , 2013 ) . 2012 ) ; ( f ) manicure / pedicure gift certificate local nail salons surrounding the university were asked to donate gift certificates in return for free advertising . in addition , we negotiated with one salon that for every two gift certificates purchased , they would provide a third gift certificate for free ( i.e. , buy two , get one free ) . the online questionnaire asked women if they participated in the body project with a yes or no category response . those who selected no received a second question inquiring why they did not participate and were given the following options : lack of knowledge , lack of interest , inconvenient times , inconvenient locations , stigma associated with this type of program , friends could not attend , i had no one to go with , or other . the statistical analyses for the current study were predominantly descriptive in nature . to compare the two email advertisements based on participation numbers and number of targeted women , a 22 table was created , and a chi square analysis was used . there were 4,297 female residents who received emails advertising a body acceptance program , of which 200 ( 5.82% ) participated in the program . there were a total of 1,681 female residents who received emails advertising the program as a personal growth program , of which 263 ( 24% ) participated in the program . this differential participation rate was statistically significant , ( 1 ) = 167.78 , p < .01 . a cost analysis was conducted where the total amount of money spent on each incentive was divided by the total number of participants that signed up online while that incentive was offered . table 1 presents participation rates by incentive strategy and the cost per person for each incentive . during baseline , the two most popular incentives were gift certificates for free manicure or pedicure and free personal fashion style training gift certificates , which accounted for 70% of the total participation . moreover , personal fashion style training was the most cost - effective of these popular incentives.table 1 . participation rates as a function of incentive offered and cost.incentivepercentage of participantscost per personmanicure / pedicure gift certificate37$1.88fashion style training33freereferral11$8.47$100 lottery8$8.82no incentive offered4$0free pizza3$28.47free ice cream4$3.71 note . the total amount of money spent on each incentive was divided by the total number of participants that signed up online while that incentive was offered . the total amount of money spent on each incentive was divided by the total number of participants that signed up online while that incentive was offered . a frequency analysis was conducted to examine the reasons provided for not participating in the program . nineteen individuals did not report a reason for not participating in the body project and , therefore , were deleted from the analyses . table 2 displays the percentage of endorsement per item given the total sample of the 569 women who completed the question . though more than half ( 56% ) did not know about the program , nearly a third ( 28% ) reported their friends could not attend , which prevented their participation.table 2 . reasons for not participating in the program.reasonpercentage of endorsementfriends could not go28no one to go with8did not know about the program56stigma3inconvenient time20inconvenient location3lack of interest14 note . the objective of this study was to evaluate two types of email recruitment techniques and six incentive strategies for engaging women in a body acceptance program . in particular , partnerships with local vendors and student organizations can lead to unique , cost effective and sustainable incentives that promote prevention programs as well as supporting businesses and organizations . potentially due to the stigma of mental health programs , advertisements specific to the mental health directive of the prevention program were not as successful as the utilization of a vague goal ( i.e. , personal growth ) . this finding may have important implications for improving recruitment strategies currently employed by health - related programs . in the business world , there is a widespread belief that consumers increasingly desire their prizes ( or incentives ) as soon as they ve won them ; this is potentially due to the popularity of the immediate gratification provided in online gaming ( speeding the reward , 1998 ) . it is possible that the two best incentives ( accounting for 70% of the participants ) were so successful because they fit into this immediate gratification model ; participants received the gift certificates for manicures / pedicures or free fashion advice immediately following the session . conversely , this model may explain why some incentives were less effective ; the referral , lottery , and free ice cream party were received up to a month after the session was completed . though the free pizza incentive fits the instant gratification model , it was the least effective incentive provided in this comparison . this may be due to the negative stereotypes that apply to those who eat excessively ( herman , rother , & polivy , 2003 ) and to those who eat bad ( i.e. , fattening ) foods ( vartanian , herman , & polivy , 2007 ) . a significant concern for most people is to be seen positively by others ; therefore , in a group setting , where social comparison is already high , offering an unhealthy food option may be too anxiety provoking and lead to increased body image insecurities , especially in those already high in eating pathology . interestingly , potential participants were less likely to attend when their friends could not attend . moreover , research has found that women feel more comfortable , have greater ease in expressing themselves , are more likely to reveal sensitive information , and report greater overall satisfaction with programming and greater group cohesion when attending prevention programs with friends ( harper , dolcini , benhorin , watson , & boyer , 2014 ) . future research should investigate successful strategies to integrate social networks not only for increased participation but also for greater satisfaction among participants . first , 56% of students reported they did not know about the body project despite receiving multiple emails ; this may have been due to emails being overlooked in the mass of institutional emails that are sent daily or the email content not attracting students . although we had a large sample size of women that did not participate in the program but were assessed through the online questionnaire , this still only represented 17% of the women living on campus and , thus , may not accurately represent the entire population of undergraduate women living on campus . third , participant perceptions of the incentives were not assessed which can assist in understanding why some incentives work better than others . this report serves to inform the effectiveness of recruitment and incentive strategies that may improve the dissemination of evidence - based prevention programs on college campuses .

abstractthe objective of this study getting individuals to participate in eating disorder prevention programs is difficult yet crucial for dissemination efforts . little research has investigated what incentive strategies can be particularly efficacious , and even less is published on their cost - effectiveness . the following study examined two types of email advertisements and six incentive strategies in an empirically supported body acceptance program disseminated at a large university . a total of 5,978 undergraduate women received email advertisements , of which 430 signed up to participate . an additional 588 who did not participate were assessed . results suggest the most effective incentives were offering gift certificates for free manicure services and free personal fashion style training gift certificates from a student organization . undergraduate women were least likely to attend due to lack of knowledge about the program , not having a friend to attend with them , or inconvenient times . implications for future research are explored .

Methods Participants Body Project Program Recruitment and procedures Incentives Online questionnaire Results Discussion

alkaptonuria ( aku ) is a rare hereditary autosomal recessive disease characterized by a defect in the gene which codes for homogentisate-1 , 2 dioxygenase resulting in the metabolic disorder of the aromatic amino acids , phenylalanine and tyrosine7 ) . the enzyme - homogentisic acid oxidase catalyzes the conversion of homogentisic acid ( hga ) to maleylacetoacetic acid , and its absence leads to increase in hga accumulation in the body . some of the excess hga excretes through the urine which turns dark on exposure to oxygen or alkalization occurs . rest of accumulated hga oxidizes initially , and deposits within the connective tissue irreversibly and subsequently turns into melanin - like pigment . it occurs especially in the cartilage of the joints and in the intervertebral discs , which is named as ochronosis1114 ) . ochronosis not only affects the cardiovascular , genitourinary and respiratory systems , but also results orthopedic disorders like spondylosis and arthropathy . the disease progresses from simple alkaptonuria to alkaptonuric ochronosis , leading to ochronotic arthropathy10 ) . cervical myelopathy is usually due to degenerative changes in the spine with the osteophytes from the posterior part of the vertebral body and uncinate process encroaching upon the neural canal . numerous reports on the successful management of ochronotic arthropathy of the peripheral joints like the knee and hip joints using total joint replacement . however , there is a paucity of reports that have shown successful surgical intervention for spinal especially cervical spine involvement . in this paper , we report a woman patient suffering from ochronotic spondylosis who was diagnosed during the late course of disease once the spondylitic myelopathy had set in resulting in increased neck and back pain , and associated with walking trouble and hands clumsiness . a 62-year - old woman was admitted in the hospital in june 2011 , with a diagnosis of cervical spondylotic myelopathy . the patient began to experience neck and back pain with stiffness 7 years ago . the symptoms developed slowly , until the gait disturbance and hand clumsiness began to appear in 2010 . however , her symptoms progressed and at the time of presentation she was wheel chair bound . she did n't have any history of trauma with no relevant past medical or similar family history . physical examination showed cyan pigmentation on the nose , sclera , and the ear auricle and also in other parts of the body ( fig . there was kyphosis at the thoracolumbar segment , resulting in a restricted movement at neck and back . the grip - and - release test was 8 movements at both hands in 10 seconds , which was obviously inferior to the normal criterion of above 20 movements . paresthesia on both hands and radial side of the left forearm and no motor weakness was observed . ct scans indicated multilevel degenerative disc disease from cervical to lumbar spine , including narrowing , calcification , osteophytosis and vacuum phenomenon ( fig . 2 , 3 ) . mri showed the cervical spinal compression in multi - segment by disc extrusion and ligamentum flavum redundancy simultaneously ( fig . 4 ) . as the color of the patient 's urine turn to dark after exposure to air after two hours so , we confirmed the presence of alkaptonuria . as the patients ' cervical spine had normal lordosis and multi - level stenosis , we performed the modified laminoplasty . this involves c3 laminectomy , c7 upper semi - laminotomy , and c46 open - door laminoplasty with implantation of man - made coral bone interposition . this was in contrast to the conventional cervical laminoplasty technique that involves a laminoplasty from c37 completely . during the operation , the ossificated nuchal ligament and the ligamentum flavum between c23 were stained black in color due to the deposition of the oxidized hga pigmentation ( fig . the cervical joa score improved from 7 preoperatively to 9 at discharge and reached 11 at one year postoperatively . she was satisfied with the treatment although she had difficulty in climbing stair case without crutches . the cervical spondylotic myelopathy ( csm ) is not an uncommon condition in the spinal area , which follows a protracted clinical course consisting of a longer period of relatively stable symptoms punctuated by exacerbation of variable duration . for patients with moderate to severe symptoms , the surgical intervention should be considered as a possible procedure to prevent further neurologic deterioration and create a chance for recovery from myelopathy4512 ) . aku , with its sequel ochronosis is a rare disease caused by an inborn deficiency of hga oxidase . although there are symptoms that may give instant clue to the diagnosis like the urine turns to dark on exposure to air and discoloration on sclera , jowl , as well as in the underwear , the patients can often neglect the symptoms and appear at late in the course of disease24 ) . in few cases it may be neglected until fourth or fifth decade when the symptoms secondary to ochronosis develop and the patients report to physicians . the disease affecting the spine follows a chronological sequence , the patient usually first suffer from ochronotic spondylosis which is accompanied with the peripheral joint arthropathy1 ) , and it progresses to cervical spondylotic myelopathy which warrants surgical intervention . the initial complaints usually include low back or neck pain with stiffness , and occasionally sciatica resulting from disc protrusion6 ) . the characteristics of ochronotic spondylosis are multiple disc space narrowing , vacuum phenomenon and calcification in a water - like pattern due to the sparing of the central nucleus pulpous39 ) . current literature has rarely reported the involvement of the cervical spine and consequent spinal cord compression requiring surgical intervention . in the present case report , findings from the mri on the cervical region revealed that the redundant ligamentum flavum appeared to be the main cause behind the cord compression . this could be secondary to the fact that the hga could bind irreversibly by a process of polymerization and oxidation to connective tissue collagen . this prevents the collagen cross - link formation , which leads to the abnormality of the ligamentum flavum3814 ) . in view of the findings from mri , we modified the standard technique to perform a modified cervical laminoplasty , which can permit extensive decompression through posterior expansion of the canal while continually maintaining cervical alignment and stability through the preservation of the posterior elements . nowadays , all the available methods are only symptomatic for the early - stage , such as dietary restriction of tyrosine and more intake of vitamin c by its antioxidant effect , etc313 ) . although the ochronotic arthropathy can severely handicap the patient because of its involvement in the spine and other joints , this incidence is quite rare . cervical laminoplasty is often the procedure of choice given the fact that the involvement is extensively causing multilevel compression . aku is a rare metabolic genetic disorder that is caused by the deficiency of homogentisic acid oxidase , affecting multiple organ systems in the body . a typical cervical spondylotic myelopathy due to the ochronotic arthropathy of the cervical spine was reported in this paper , and a modified laminoplasty proved to be the appropriate treatment that yielded satisfactory result .

ochronosis is a musculoskeletal manifestation of alkaptonuria , a rare hereditary metabolic disorder occurs due to the absence of homogentisic acid oxidase and leading to various systemic abnormalities related to deposition of homogentisic acid pigmentation ( ochronotic pigmentation ) . the present case reports the clinical features , radiographic findings , treatments and results of a cervical spondylotic myelopathy woman patient due to the ochronotic arthropathy of the cervical spine . the patient aged 62 years was presented with gait disturbance and hand clumsiness . physical examination , x - rays , computed tomography and lab results of the urine sample confirmed the presence of ochronosis with the involvement of the cervical spine . the patient underwent a modified cervical laminoplasty due to multi - segment spinal cord compression . the postoperative follow - up showed a good functional outcome with patient satisfaction . the present study concludes the conditions and important diagnostic and surgical aspects of a patient . it is necessary to identify the condition clinically and if cord compression is observed , appropriate surgical interventions needs to be instituted .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

cuttack from july 2006 to june 2007 , diagnosed as schizophrenia using international classification of diseases 10 , clinical descriptions and diagnostic guidelines , ( icd-10 criteria ) and were in the acute phase were enrolled into the study . patients fulfilling the diagnostic criteria and who gave informed consent and were in the age group of 18 - 58 formed the study subjects . patients having co - morbid substance dependence , chronic medical illnesses , mental retardation or organic brain diseases formed the exclusion criteria . modified kuppuswamy scale : this scale is based on scoring obtained in three domains : ( i ) occupation of the head , ( ii ) education of the head and ( iii ) family income per month . based on the total score obtained , socio economic status is classified into lower ( < 5 ) , upper - lower ( 5 - 10 ) , lower - middle ( 11 - 15 ) upper middle ( 16 - 25 ) and upper ( 26 - 29)positive and negative syndrome scale ( panss ) ( kay et al . 1987 ) derived originally from the brief psychiatric rating scale ( bprs ) scale , panss has 7 items each for positive and negative symptoms and 16 items for measuring general psychopathology . all the 30 items are given a score ; from 1 ( complete absence of symptoms ) to 7 ( extreme severity of symptoms ) andworld health organisation quality of life scale _ brief version ( whoqol bref ( oriya version ) formed the study tools to collect individual patient information . who - qol - bref is based on 4-domain structure with six items in physical , psychological , social and environmental domain and two items from overall qol and general health . the instrument was translated into oriya and after a series of translations and back - translations ; oriya version was used after equivalence was established . modified kuppuswamy scale : this scale is based on scoring obtained in three domains : ( i ) occupation of the head , ( ii ) education of the head and ( iii ) family income per month . based on the total score obtained , socio economic status is classified into lower ( < 5 ) , upper - lower ( 5 - 10 ) , lower - middle ( 11 - 15 ) upper middle ( 16 - 25 ) and upper ( 26 - 29 ) positive and negative syndrome scale ( panss ) ( kay et al . 1987 ) derived originally from the brief psychiatric rating scale ( bprs ) scale , panss has 7 items each for positive and negative symptoms and 16 items for measuring general psychopathology . all the 30 items are given a score ; from 1 ( complete absence of symptoms ) to 7 ( extreme severity of symptoms ) and world health organisation quality of life scale _ brief version ( whoqol bref ( oriya version ) formed the study tools to collect individual patient information . who - qol - bref is based on 4-domain structure with six items in physical , psychological , social and environmental domain and two items from overall qol and general health . the instrument was translated into oriya and after a series of translations and back - translations ; oriya version was used after equivalence was established . descriptive statistics was used and spearman 's correlation coefficient was calculated using spss version 15.0 . modified kuppuswamy scale : this scale is based on scoring obtained in three domains : ( i ) occupation of the head , ( ii ) education of the head and ( iii ) family income per month . based on the total score obtained , socio economic status is classified into lower ( < 5 ) , upper - lower ( 5 - 10 ) , lower - middle ( 11 - 15 ) upper middle ( 16 - 25 ) and upper ( 26 - 29)positive and negative syndrome scale ( panss ) ( kay et al . 1987 ) derived originally from the brief psychiatric rating scale ( bprs ) scale , panss has 7 items each for positive and negative symptoms and 16 items for measuring general psychopathology . all the 30 items are given a score ; from 1 ( complete absence of symptoms ) to 7 ( extreme severity of symptoms ) andworld health organisation quality of life scale _ brief version ( whoqol bref ( oriya version ) formed the study tools to collect individual patient information . who - qol - bref is based on 4-domain structure with six items in physical , psychological , social and environmental domain and two items from overall qol and general health . the instrument was translated into oriya and after a series of translations and back - translations ; oriya version was used after equivalence was established . modified kuppuswamy scale : this scale is based on scoring obtained in three domains : ( i ) occupation of the head , ( ii ) education of the head and ( iii ) family income per month . based on the total score obtained , socio economic status is classified into lower ( < 5 ) , upper - lower ( 5 - 10 ) , lower - middle ( 11 - 15 ) upper middle ( 16 - 25 ) and upper ( 26 - 29 ) positive and negative syndrome scale ( panss ) ( kay et al . 1987 ) derived originally from the brief psychiatric rating scale ( bprs ) scale , panss has 7 items each for positive and negative symptoms and 16 items for measuring general psychopathology . all the 30 items are given a score ; from 1 ( complete absence of symptoms ) to 7 ( extreme severity of symptoms ) and world health organisation quality of life scale _ brief version ( whoqol bref ( oriya version ) formed the study tools to collect individual patient information . who - qol - bref is based on 4-domain structure with six items in physical , psychological , social and environmental domain and two items from overall qol and general health . the instrument was translated into oriya and after a series of translations and back - translations ; oriya version was used after equivalence was established . descriptive statistics was used and spearman 's correlation coefficient was calculated using spss version 15.0 . the participants were in the age group of 18 years to 58 years with a mean age of 34 years . majority were of upper - lower socio - economic status as per modified kuppuswamy scale . 22 were of paranoid type , 11 were of unspecified variety and one was of catatonic subtype . qol was assessed with who - qol bref and then extrapolated to who - qol 100 . association between positive , negative , general psychopathology and total scores and domains of qol was assessed using spearman 's correlation coefficient . all domains of psychopathology were found to be negatively correlated with all the domains of qol . the correlation of panss general and total scores with physical , psychological and environment component of qol was statistically significant ; that with psychological and physical component was highly statistically significant . a negative linear relationship was found between total panss score with physical health and psychological health , which was found to be significant ( p<0.01 ) [ table 1 ] . association between positive and negative syndrome scale and world health organization - quality of life in case of males positive , general and total panss symptoms were found to be negatively correlated with psychological health , which was statistically significant while general and total symptoms were found to be significantly correlated with both physical and psychological health . whereas , in case of females there was a significant correlation between positive , general , and total panss scores with environmental domains of qol [ table 2 ] . association of psychopathology with quality of life disaggregated by gender and marital status in case of unmarried people positive , negative , general , and total symptoms were inversely correlated with physical health domain of qol while positive , general , and total scores on panss were inversely related with psychological health . in case of ever married people there exists a statistically significant negative relationship between general and total panss components of panss and environmental domains of qol [ table 2 ] . while examining the relationship in different education status it was found out that people who had more than 10 years of schooling had an inversely significant relationship between panss score and its components with physical and psychological domains of qol . previous researchers have found that qol in patients of schizophrenia is influenced by socio - demographic characteristics , symptoms , insight , medication side - effects . while in the acute phase , qol is associated with positive symptoms ; in the stage of clinical stability it is associated with anxiety and depression . the findings of the study are in tune with work done by previous researchers , more severe positive as well as negative symptoms predict a poorer qol . however , many researchers could not find statistically significant correlation between positive schizophrenia symptoms and qol . our study was done on patients in acute phase and shows that positive symptoms show statistically significant negative correlation with qol in psychological domains ( p<0.05 ) . negative symptoms were associated with a poor qol in physical domain , which was not statistically significant . it was found that panns general and total scores had a highly significant ( p<0.01 ) negative correlation with physical and psychological health , which is contrary to a significant correlation with social domain in maintenance phase . the emotional experience of acute phase is usually so severe that one can understand the negative impact on physical and psychological domains of qol . maintenance phase is the time when acute psychotic experiences have settled down and the person is usually struggling with negative symptoms . male gender , unmarried status , more than 10 years of schooling showed negative correlation of general and total scores of panss with physical and psychological domains of whoqol . female gender , married status and lower educational attainment were associated with better qol with only environmental domain of qol being adversely affected . shtasel et al . in their work had noted that females have better qol than males whereas meltzer et al . skantze et al . also found that qol is independent of gender and marital status . poorer qol in male subjects , higher education can be explained on the basis of level of expectation , aspiration and perceived control , which is often high in this subgroup . indian socio - cultural milieu often expects spouse to stand by the patient as marriages are considered to be sacred union , this might explain the fact that qol is better in the married than in the unmarried . previous research has demonstrated general panss scores to have more strong correlation with all domains of qol , which was evident in this study also . depression and anxiety are the strongest predictors of qol . while symptomatology of schizophrenia changes across the phases of the illness , their relative influence on various domains of qol also differs . research need to be done in identifying determinants of qol in the different phases of the illness with an aim to target these during treatment interventions . some of these respond to pharmacotherapy , some to intensive psychosocial interventions and still some other which are not amenable to treatment . have shown that following psychosocial interventions , most dimensions of self - rated qol show improvement and this improvement is largely independent of improvement in symptoms . male gender , unmarried status and schooling more than 10 years show inverse correlation of psychopathology with qol in acute phase of schizophrenia . the domains of physical and psychological well - being of who - qol were correlated with panss general and total scores whereas environmental and social health showed no correlation with panss scores . while certain factors influencing qol are not malleable , malleable factors need to be identified . more intensive longitudinal studies are needed to ascertain whether qol is a different dimension altogether , largely independent of psychopathology . longitudinal studies should be undertaken with a larger sample size to validate the results of the present study results . longitudinal studies should be undertaken with a larger sample size to validate the results of the present study results .

objective : to find the association of patient characteristic and psychopathology with quality of life in acute phase of schizophrenia.materials and methods : socio - demographic variables of patient , psychopathology and quality of life were assessed . spearman 's correlation coefficients were measured using spss version 15.0.results:quality of life of the patients varied in different domains . male gender , unmarried status and higher educational status predicted a poorer quality of life . the domains of physical and psychological well - being of who - qol were correlated with panss general and total scores whereas environmental and social health showed no correlation with panss scores.conclusion:domains of subjective quality of life in acute phase of schizophrenia are associated variedly with socio - demographic variables and symptomatology .

MATERIALS AND METHODS Study instruments Statistical analysis RESULTS DISCUSSIONS CONCLUSIONS Limitations

kawasaki disease ( kd ) is an acute systemic vasculitis of unknown cause that occurs primarily in children and rarely in adults . in the absence of specific laboratory tests , we report an unusual case of an elderly man with several unique features of incomplete kd . a 75-year - old white man was admitted to our hospital for a 3-week history of fever and poor general condition . his past medical history included hypertension , hyperlipidemia , myocardial infarction , and a coronary artery bypass graft surgery 2 years ago . the patient was febrile ( 39c ) , and physical examination revealed bilateral nonpurulent conjunctivitis , red and cracked lips ( fig . 1 ) , and slightly edematous erythema with desquamation of the hands and feet ( fig . laboratory tests revealed anemia ( hemoglobin 8.5 g / dl ) , hypoalbuminemia ( 2.6 g / dl ) , elevated erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) ( 83 mm / h and 100.3 mg / l , respectively ) , elevated alanine aminotransferase ( 56 iu / l ; normal < 40 ) , and sterile leukocyturia ( 30 cells/l ) . serological assays for human immunodeficiency virus , hepatotropic viruses , epstein - barr virus , cytomegalovirus , and antinuclear antibodies were negative . the patient was subsequently treated with an intravenous immunoglobulin infusion ( 1 g / kg for 2 days ) and high - dose aspirin ( 3 g / day ) . the fever remitted within 3 days , and within 1 week , the laboratory abnormalities returned to normal and mucocutaneous lesions improved dramatically . low - dose aspirin , which the patient had taken since his bypass surgery , was then restarted . kd is defined as the presence of a fever for 5 days and 4 of the 5 major clinical features : conjunctivitis , oral mucosal changes , polymorphous rash , palmoplantar erythema with desquamation , and cervical lymphadenopathy . incomplete kd refers to patients who do not fulfill the classic criteria of 4 of the 5 findings and in whom alternative diagnoses have been ruled out . in adults , the main differential diagnoses the higher prevalence of coronary artery lesions in patients with incomplete kd reflects difficulties in diagnosis and delays in treatment . although adults are less frequently affected by coronary aneurysms compared with children ( 5 vs. 20% , respectively ) , establishing the diagnosis of incomplete kd and initiating treatment is essential . in 2004 , a multidisciplinary committee of experts proposed an algorithm to diagnose incomplete kd in children . for pediatric patients with a fever for 5 days and < 4 classical criteria of kd , laboratory tests , including crp and esr , should be obtained . in patients with elevated crp ( 30 mg / l ) and/or esr ( 40 mm / h ) levels who fulfill 3 supplemental laboratory criteria [ including albumin 3.0 g / dl , anemia , elevation of alanine aminotransferase , 450,000/mm platelets after 7 days , white blood count 15,000/mm , and sterile pyuria ( 10 cells / high - power field ) ] , intravenous immunoglobulin and aspirin should be instituted and echocardiography should be performed . our case emphasizes the importance of considering kd at any age , even among elderly individuals . this case also highlights the usefulness of the algorithm proposed for children to diagnose incomplete kd in adults .

kawasaki disease ( kd ) is a systemic vasculitis of unknown etiology , affecting predominantly young children . here , we describe an unusual case of a 75-year - old man with several unique features of incomplete kd . healthcare professionals should therefore be aware of the importance of considering kd at any age , even among elderly individuals . this case also highlights the usefulness of the algorithm proposed for children to diagnose incomplete kd in adults .

Introduction Case Presentation Discussion Disclosure Statement

being a primary care professional ( pcp ) means being at the forefront of managing chronic , complex , and difficult areas of health . working with patients who have cancer , those who are dying and those who experience or have experienced family and/or sexual violence can be difficult and emotionally confronting . in a day 's work , pcps see many complicated and complex patients , but those who experience family violence can be particularly demanding and challenging because of chronic health issues , psychological distress , relationship and authority issues , and coping strategies such as denial or somatization . if we then add to the potential emotional impact of survivor stories on pcps , the mental and emotional work of caring for these patients , the patient - professional interaction and boundary issues , and the long - term nature of the care required , we will have the perfect recipe for increased pcp stress . work - related stress is already experienced by family physicians and nurses , with burnout being the most investigated area [ 47 ] . prevalence rates of burnout for medical residents ( including family medicine residents ) have been reported between 18 and 82% and between 10 and 55% for family physicians [ 4 , 6 , 7 ] . a number of factors have been identified as important contributors to burnout ; these include the work environment , changing policy , changes in the practice of family medicine , workload , loss of autonomy , and feeling a lack of personal accomplishment [ 4 , 7 ] . personal characteristics of the pcps themselves , such as being high achievers and perfectionists , the relationship between work and self - esteem , gender , age , and family and support networks , may also contribute to their susceptibility to stress [ 4 , 7 , 8 ] . pcp 's recognition and support of patients who have experienced violence may improve the mental and physical health through early intervention and better service access [ 912 ] . working with family violence can , in itself , be difficult and confronting for pcps because of the patient 's experience within their family . pcps may also be both victims and perpetrators of family violence and this can influence their ability to work effectively with patients . an earlier study by sugg and inui reported that 15% of male doctors and 31% of female doctors had a personal history of child abuse or intimate partner violence . pcps may be concerned that family violence is a private no - go area that can not be discussed without offending the patient . they describe feeling overwhelmed and concerned about broaching the topic with patients , that they lack the time and the confidence to deal with the problem , that they might make the situation worse , and that they can not fix it personal safety issues often confront those who work with victimized children and adults . the traumatic nature of child abuse and adult violence with its negative effects on the emotional , mental , and physical health of victim / survivors is well documented . less discussed is the impact of working in this area on the well - being of the medical professionals and their staff . this paper outlines a theoretical framework for understanding the possible reactions of pcps who work in this area . we then discuss the emotional and physical safety issues while being at work and the social , emotional , and physical impacts of child abuse and violence at work . researching and working with victims of family violence can be emotionally challenging [ 1923 ] . working with children can make it even more so because of bearing witness to the child 's abuse experiences , conflicts with parents and carers , the inability to form an effective therapeutic relationship with the child or their family , a hostile working environment , lack of or failure of services to assist the child and the family , direct threats and concerns about personal safety , and the possibility of litigation [ 22 , 2426 ] . in addition , working with victimised children and adults can evoke strong emotional reactions [ 22 , 26 ] . the general practitioners ( gps ) below describe their encounters : a mother was breastfeeding when her son 's smile triggered her memories of incest . she told me the story of her pain caused long ago by the csa perpetrated by her father . her story was powerful , making me pay attention to her story amongst so many illness stories . as i reflect on that consultation now , i think it was the intensity of her fear and distress which alerted me to the importance of her experiences . she put her baby on one side of the consulting room , as far away as possible , and just cried . ( female gp , urban ) .there were two boys and a little girl and they all lived in the same street . i went and saw the parents of the two disabled boys [ who were abusing the little girl ] and they were n't prepared to do anything , they did not want to know about it , did not feel like supervising , perhaps they did not believe the story . i reported the case to child protection and the families all said we have to move out of town if you can not do anything and we do not want to leave . the kids do not want to leave the school and why should we have to go away to keep our children safe . [ ] that process was extremely painful , you need to go through with the families , families of the abused and the family of the abuser and see it brought out in the open , to the police and then watch them go through the pain , through the system . i started doing work [ with men who perpetrate violence ] , i can remember saying how lucky i was that i worked in an area where there was no abuse what a clever decision i 'd made , to pick such a good postcode , but obviously i just did not see it [ ] i think it 's more that you are aware that it [ abuse ] goes on and i 'm looking out for it more . in the past , if someone would say something , start hinting it , you 'd run a million miles , you 'd change the topic , you 'd start talking about something else and it 's easy to do that [ ] ( male gp , urban ) . she told me the story of her pain caused long ago by the csa perpetrated by her father . her story was powerful , making me pay attention to her story amongst so many illness stories . as i reflect on that consultation now , i think it was the intensity of her fear and distress which alerted me to the importance of her experiences . she put her baby on one side of the consulting room , as far away as possible , and just cried . there were two boys and a little girl and they all lived in the same street . i went and saw the parents of the two disabled boys [ who were abusing the little girl ] and they were n't prepared to do anything , they did not want to know about it , did not feel like supervising , perhaps they did not believe the story . i reported the case to child protection and the families all said we have to move out of town if you can not do anything and we do not want to leave . the kids do not want to leave the school and why should we have to go away to keep our children safe . [ ] that process was extremely painful , you need to go through with the families , families of the abused and the family of the abuser and see it brought out in the open , to the police and then watch them go through the pain , through the system . ( female gp small rural town ) . before i started doing work [ with men who perpetrate violence ] , i can remember saying how lucky i was that i worked in an area where there was no abuse what a clever decision i 'd made , to pick such a good postcode , but obviously i just did not see it [ ] i think it 's more that you are aware that it [ abuse ] goes on and i 'm looking out for it more . in the past , if someone would say something , start hinting it , you 'd run a million miles , you 'd change the topic , you 'd start talking about something else and it 's easy to do that [ ] ( male gp , urban ) . there is an increasing literature on the emotional work of caring and the toll this work takes on those who undertake it [ 18 , 24 , 3032 ] . the effects of being a direct or indirect witness to child abuse and adult violence can result in compassion fatigue , burnout , vicarious trauma and secondary traumatic stress . these terms are often used interchangeably but are , in fact , conceptually different . compassion fatigue is a general term applied to anyone who suffers as a result of acting in a helping capacity , a natural outcome of helping and knowing about trauma [ 33 , page 92 ] and [ 34 , page 9 ] . burnout occurs when a person 's health or outlook on life is negatively affected because of the impact of their work . figley described burnout as physical , emotional , and mental exhaustion caused by long - term involvement in emotionally demanding situations , as a gradual wearing down over time . workers with burnout report low job satisfaction , feelings of powerlessness , and being overwhelmed at work . vicarious trauma is defined as the transformation of the therapist 's or helper 's inner experience as a result of empathetic engagement with survivor clients and their trauma material [ 30 , page 31 ] and results in a profound shift in the worldview of the worker . the result of vicarious trauma can potentially include the disruption of the worker 's view of themselves , the self - changing nature of care giving [ 31 , page xxxviii ] , others and the world in general . child protection workers , physicians , nurses , therapists , social workers and child abuse researchers report such experiences . the traumatic experience can extend beyond those who work directly with the victim / survivor . traumatization has been reported in those who have indirect contact : in receptionists , supervisors , and managers , in those processing accounts or records , in family members of the treating professionals , and in those working with patient histories or reports . the impact is related to the trauma they are exposed to , their own characteristics and history , the research methods they use , their support systems , and context in which they do their research . it is described as a pervasive feature of working with the traumatised and a cumulative response to traumatic material . it can be triggered by either a one - off exposure to a significant issue or repeated exposure to a range of issues and incidents . it can have a profound impact on individuals and be as debilitating as the primary trauma . other contributing factors originally proposed by pearlman and saakvitne that apply to primary care across a range of settings internationally include the chronicity of the trauma work , combining service provision and research ( particularly in resource poor nations ) , increased violence to women and children in war and conflict areas , the individual professional 's capacity for empathy , and the individual 's personal history of trauma . the vicarious trauma has been criticised because it fails to explain why similar experiences cause vicarious trauma in some while others do not experience vicarious trauma . secondary traumatic stress is a similar concept to vicarious trauma , but there are differences as it is based on a set of clinical symptoms . secondary traumatic stress closely parallels posttraumatic stress disorder in its symptomatology and is a normal response to exposure to trauma through working with survivors of traumatic events . symptoms of secondary traumatic stress impact on psychological functioning , result from cognitive shifts and disturbances in relationships . they include intrusive symptoms such as pcps re - experiencing the survivors ' trauma through thoughts , feelings , and images ; avoidance and numbing symptoms such as avoiding working in areas that recall the trauma or forgetting parts of an interview ; and symptoms of hyperarousal such as palpitations and sweating and nightmares and sleep disturbances [ 38 , 39 ] . vicarious trauma and secondary stress have provided the conceptual framework for much of the literature that underpins the current understanding of the impact on those who work with survivors of trauma . more recent work has highlighted methodological issues and the need to measure all of these areas to test their independence . in medicine and nursing , burnout is defined as feelings of emotional exhaustion , depersonalization , and a perceived lack of personal accomplishment because of a long - term engagement with emotionally demanding situations [ 2 , 4 ] . burnout as a model has the advantage of combining the personal impacts and the impact on work , but the interrelationships between the three factors are unclear . preventing , understanding , and recognising vicarious trauma , secondary traumatic stress , and burnout in their work with family violence will assist pcps to respond more effectively to it and to reduce its potential to adversely affect the way in which they work and their view of the world in general . for those already working in the area , it will assist them to better understand their experience and for those in management positions , to put in place strategies to prevent the possibility of secondary traumatization for themselves and their staff . pcps must be trained to do this work at undergraduate and postgraduate levels and as part of their ongoing professional development because pcps still report being inadequately trained to do so . both training and professional experience have been associated with increased feelings of confidence in dealing with violence as well as increased comfort in discussions with patients making pcps feel more in control and reducing pcp stress [ 5 , 13 , 40 ] . the institutions and professional organisations responsible for the education pcps must ensure that students and professionals are trained well and develop the necessary knowledge skills and attitudes as well as the facilitating support for those working with family violence . organisations which were expected to champion the health needs of women and children but failed to do so , by marginalising the need to respond to violence , blaming the victim , not addressing the training needs of pcps , and discrediting the work of those responding to and supporting women were identified as particularly difficult for workers and researchers . hierarchical organisations have been identified as a more important predictor of trauma than the individual characteristics of the workers . the education of pcps should address the needs of patients first and foremost , but the pcps sense of vulnerability , fear , and inadequacy also need to be addressed for them to work effectively in this area . like other social problems and health risk behaviours , there is no easy cure to family violence and pcps need to reframe what they consider as a successful consultation rather than being frustrated by a lack of change . pcps report of positive changes after family violence is disclosed , including patient engagement with counselling and legal services .i asked a 40 year old new patient about child abuse which i was certain she had experienced . twelve months later she came in and said you know that question you asked me twelve months ago , i could n't talk then but i can today . in retrospect i asked a 40 year old new patient about child abuse which i was certain she had experienced . twelve months later she came in and said you know that question you asked me twelve months ago , i could n't talk then but i can today . in retrospect my first consultation was very successful , i just did not know it . traumatic reactions are related to the duration of exposure to traumatic material and the type of trauma experienced . while pcps can not control the content on acute consultations , they can manage and structure their work in terms of review appointments and should consider structuring their work so that they have regular breaks and opportunities to manage less demanding problems . support and supervision from more experienced or staff trained in responding to trauma may help in the management of distressing cases . pcps need to reflect on their ability to provide care for those who have experienced family violence . while all should have a basic competency in recognising and responding to violence in positive and helpful ways , not all may be willing or able to deliver care . in such cases , knowledge of appropriate providers of ongoing care and support services is essential . support systems and networks for pcps need to be considered because these are often not routinely available to pcps . setting up a recognised family violence team at work for all who work with family violence is useful , providing peer - peer support and the ability to discuss difficult or distressing cases . psychiatric and counselling services have a much more developed culture of providing support and supervision for those working with mental illness and traumatized clients . pcps may need to consider engaging with services outside of work for support and supervision if they are going to regularly work with family violence . networks can include coworkers within primary care and may extend to other agencies working with those who experience violence ; working as part of a team is often a much more positive experience than working alone . support systems that extend beyond the clinic should not be forgotten , with friends and family being reported as an important support [ 5 , 41 ] . a key factor in the ability to care for others is the ability to care for oneself a key factor in the ability to care for others is the ability to care for oneself although not all of us working with trauma will experience secondary trauma , we are all at risk . acknowledging how emotionally difficult working in this field can be , recognising that it will affect and may distress you , learning how to recognise symptoms of secondary trauma early and the importance of self - care are essential elements in managing and preventing secondary trauma . the literature highlights a number of responses that can be used at educational , organizational , and personal levels to prevent and respond to secondary trauma , and in turn , improve the quality of care provided by pcps to people experiencing family and sexual violence . at an educational level an understanding of burnout , secondary , or vicarious trauma and their implications should be integrated into training curricula of all helping professionals working with family and sexual violence [ 44 , 4648 ] . an understanding of burnout , secondary , or vicarious trauma and their implications should be integrated into training curricula of all helping professionals working with family and sexual violence [ 44 , 4648 ] . at an organisational level develop a workplace policy that recognises secondary trauma as a potential workplace health risk ; addresses related stigma ; normalises work place stress ; and outlines appropriate responses , including professional development ; workload and case management ; peer support and ongoing routine management meetings in which secondary trauma is discussed [ 4850].develop a culture of learning , sharing and support within the workplace [ 44 , 48].provide adequate support and supervision which includes discussion of secondary trauma and its implications .provide workplace development programmes that include secondary trauma and how to identify early warning signs between oneself and others [ 46 , 49].ensure managers who are appropriately trained to recognise and respond to secondary trauma in a supportive , nonpunitive manner .review workplace policies regularly . develop a workplace policy that recognises secondary trauma as a potential workplace health risk ; addresses related stigma ; normalises work place stress ; and outlines appropriate responses , including professional development ; workload and case management ; peer support and ongoing routine management meetings in which secondary trauma is discussed [ 4850 ] . develop a culture of learning , sharing and support within the workplace [ 44 , 48 ] . provide adequate support and supervision which includes discussion of secondary trauma and its implications . provide workplace development programmes that include secondary trauma and how to identify early warning signs between oneself and others [ 46 , 49 ] . ensure managers who are appropriately trained to recognise and respond to secondary trauma in a supportive , nonpunitive manner . develop an awareness of your own personal risk of burnout and secondary trauma and accept reactions as a normal response to specialised work [ 44 , 51 ] . promote personal well - being by being physically active , engaging socially , and volunteering in your community and adopt appropriate self - care strategies [ 3 , 48 ] ( box 1 ) . skill up in the field of trauma and family violence [ 47 , 52 ] . take responsibility for your own mental health and well - being .create opportunities to debrief with colleagues and decrease social isolation [ 48 , 52].develop and maintain clear client limits and boundaries [ 47 , 48 , 51].recognise potential personality traits that may increase risk of experiencing secondary trauma .find a sense of meaning in your work and develop a connection or spirituality to something that provides guidance for and develops a positive outlook on life . .tremendously satisfying because i believe that my work is a little drop of water which when added to others over time will become a roaring mighty ocean that will spur the wind of change that will improve the lives of women everywhere ( respondent number : 37 ) . develop an awareness of your own personal risk of burnout and secondary trauma and accept reactions as a normal response to specialised work [ 44 , 51 ] . promote personal well - being by being physically active , engaging socially , and volunteering in your community and adopt appropriate self - care strategies [ 3 , 48 ] ( box 1 ) . skill up in the field of trauma and family violence [ 47 , 52 ] . take responsibility for your own mental health and well - being . create opportunities to debrief with colleagues and decrease social isolation [ 48 , 52 ] . develop and maintain clear client limits and boundaries [ 47 , 48 , 51 ] . recognise potential personality traits that may increase risk of experiencing secondary trauma . find a sense of meaning in your work and develop a connection or spirituality to something that provides guidance for and develops a positive outlook on life . .tremendously satisfying because i believe that my work is a little drop of water which when added to others over time will become a roaring mighty ocean that will spur the wind of change that will improve the lives of women everywhere ( respondent number : 37 ) . recognising that we are under stress can be difficult , particularly when professional cultures discourage it . many pcps have trained in systems that encourage them to be in control , this can be problematic when pcps are stressed and hide it from their peers and colleagues and , conversely , can be an area that colleagues fail to see occurring . it has been suggested that partners , families , and friends are better at recognising when we are stressed than we are or our work colleagues are . while prevention is the best management , even with the best preparation some pcps who work with family violence will become stressed . related stress to the healthcare system , the occupational system , and the personal system . the authors ' previous work with researchers and staff who worked with sexual violence identified work - related management strategies and personal strategies as their main support framework . in terms of the health care system , policies and procedures that recognise the time and expertise required for pcps to support and provide long - term care for survivors are necessary . ensuring that adequate counselling and effective therapy services are available to adults and children within the community is essential to support the work of the pcps . in addition , ongoing pcp education must develop and reinforce basic knowledge skills and attitudes to ensure that they have the appropriate skills to recognise and support those who experience family violence and to enable these patients to access the appropriate trauma services . work - related management strategies in managing traumatic material include ongoing development of skills because studies suggest that becoming experienced and an expert reduced trauma . spreading the workload where possible is reported as helping . those with better relationships with their coworkers and higher numbers of support systems were less likely to develop secondary traumatic stress . self - care strategies assisted pcps ; these included physical fitness , psychological strategies , spirituality / advocacy and staying connected . various forms of physical activity and eating well have been described as well as using humour and laughter , avoiding retraumatizing material like films and newspapers , being pleased with small changes , being creative , travelling and exploring new places , gardening , talking with partners and family , eating and spending time with family and friends , going to church , praying , making a difference , and writing books , papers , and pamphlets to help [ 4 , 52 , 53 ] . elwood and colleagues while recognising the importance of responding to workplace health risks and secondary trauma suggest that we should proceed with some caution . the development and implementation of workplace policies on secondary and vicarious trauma must be informed by evidence and with cognisance of available resources . much of the research done to date is limited to high - income settings and based on unclear definitions and weak methodologies . recommend more methodologically sound ; analytical research on secondary trauma , its symptoms , and responses to ensure that we do not waste limited resources on implementing ineffective interventions that are based on a poorly defined construct . these need to be addressed at the undergraduate , prevocational , and practitioner levels on an ongoing basis to develop a skilled and competent workforce where the potential for stress is minimised . as pcps engage more with recognising and responding to family violence , it will be necessary to put in place the appropriate education and healthcare system , workplace , and personal support to enable the pcps to continue to work with patients who have experienced family violence and their ongoing chronic health needs . pcps need to be aware of their own support needs and the strategies they can put in place to engage well with those who have experienced family violence , providing safe long - term chronic care and access to the required services .

primary care professionals ( pcps ) are increasingly being expected to identify and respond to family and sexual violence as the chronic nature and severity of the long - term health impacts are increasingly recognized . this discussion paper reports the authors ' expert opinion from their experiences running international workshops to prevent trauma among those who work and research sexual violence . it describes the burnout and secondary traumatic stress literature which provides the evidence supporting their work . implications for practicing basic training in response to trauma and ongoing education are a key area for responding to family violence and preventing professional stress . a professional culture that supports and values caring well for those who have experienced family violence as well as caring for the carer is needed . working in teams and having more support systems in place are likely to protect pcps from secondary traumatic stress and burnout . undergraduate and postgraduate training of pcps to develop trauma knowledge and the skills to ask about and respond to family violence safely are essential . in addition , the healthcare system , workplace , and the individual practitioner support structures need to be in place to enable pcps to provide safe and effective long - term care and access to other appropriate services for those who have experienced family violence .

1. Introduction 2. Understanding the Potential for Trauma 3. How Can PCPs Prepare to Work with Patients Who Have Experienced Family Violence? 4. Preventing Burnout and Vicarious Trauma among PCPs 5. Recognising Stress and Responding to It 6. Conclusion

behet 's disease ( bd ) is an inflammatory disorder of undetermined aetiology , which is recently and unanimously recognized as both autoimmune and autoinflammatory disease . indeed , many of its classical manifestations and the characteristics of the recurrent course overlap with those of monogenic autoinflammatory disorders [ 25 ] . it is hypothesized that the main pathogenetic elements are represented by genetic predisposition , mainly hla dependent , and environmental factors . furthermore , it is believed that a misdirected immune response , triggered by some microbial agents ( as herpes simplex virus-1 and streptococcus sanguinis ) , could play a pivotal role in the pathogenesis of bd . in this regard , the abnormal activation of either innate and adaptive immunity with consequent interaction of both t lymphocytes ( mainly th1 and th17 phenotype ) and activated neutrophils would seem to be involved in the disease onset [ 79 ] . many cytokines may contribute to the pathological mechanism of bd [ 6 , 1012 ] ; high sera title of tumor necrosis factor- ( tnf- ) alpha is found in patients with active bd and the role of tnf - alpha inhibition in the pathogenesis of ocular inflammation was described in mice models . interleukin- ( il- ) 6 has been demonstrated to be related to bd activity and central nervous system involvement , as confirmed by its high levels in csf . recent studies have suggested a role of il-1 in bd ; actually il-1 high title is found in sera and synovial fluid of bd patients . indeed il-1 may play a key role in the pathogenesis of ocular and mucocutaneous involvement , although in the latter case evidence from literature is not entirely encouraging [ 2023 ] . triple symptom complex , consisting of recurrent oral aphthosis , genital ulcers , and recurrent bilateral uveitis . besides this classical clinical trial , bd recognizes also other organ involvements , as summarised in table 1 [ 2428 ] . mucocutaneous lesions are the earliest and the most frequent manifestations of bd which may anticipate by many years other typical clinical symptoms . the most common mucocutaneous lesions are oral aphthae ( oa ) , which are included in the bd classification criteria . they manifest themselves , more frequently as minor aphthous ulcers ( < 10 mm in diameter ) or , less frequently , as major ulcers ( > 10 mm in diameter , deeper and more painful than minor ulcers ) or also as herpetiform ulcers ( numerous , shallow , and small - pinpoint ulcers occurring in coalescing clusters ) . the genital ulcers ( gu ) are the second main symptom reported in the literature . the most frequently involved body areas are the scrotum in males and the major and minor labia in females . cutaneous lesions , important characteristics of the disease , have been described as a major criterion for the classification . they mainly include erythema nodosum - like lesions , papulopustular lesions ( sterile folliculitis - like lesions on an erythematous base ) , superficial thrombophlebitis , extragenital ulceration , and other cutaneous vasculitic lesions . skin pathergy reaction represents the unifying feature of the typical bd inflammation and is characterised by the presence of an abnormal skin reaction to traumatic insults or different types of inflammatory stimuli . moreover , pathergy phenomenon has no association with specific organ involvement or disease activity and is not only restricted to the skin . the mucocutaneous manifestations are characterized by recurrent relapses ; they usually have moderate to long - term course and their spontaneous resolution is rarely described . a wide number of conventional immunosuppressive drugs could be used to treat these lesions , but several failures , with lesion relapses , are commonly reported . herewith , we provide a review of the literature published on treatment strategies for mucocutaneous bd involvement , focusing on how treatment has changed in the last decades and on possible future perspectives . the eular treatment recommendations were published ; they suggested treating skin and mucosa involvement both depending on the perceived severity and shared doctor - patient decision and according to dominant or concomitant manifestations . topical measures ( i.e. , local corticosteroids ) should be the first - line treatment for isolated oral and genital ulcers and acne - like lesions . azathioprine , inf - alpha , and tnf - alpha antagonist may be considered in resistant cases . some immunomodulator drugs , as tocilizumab and mycophenolate mofetil , have failed to reach a clinical improvement in the mucocutaneous lesions . new drugs as apremilast seem to be effective in the treatment of oral and genital aphthosis . the improvement of knowledge about the pathogenetic mechanism could add important changes in the treatment management of the bd clinical manifestation . case reports , open and double - blind trials , and cohort studies published up until 2015 were evaluated ( table 2 ) . with regard to the drugs under clinical experimentation , a wide range of drugs is available for treating mucocutaneous lesions in bd . in the past decades , traditional immunosuppressive agents were largely used to reduce the ulcers disability , but some contrasting results were observed , particularly in terms of maintaining remission effectiveness . azathioprine seems to be effective in controlling the progression of bd , especially in most critical manifestations , such as eye diseases . iarct described a favourable effect on mucocutaneous lesions , as proven by statistically significant reductions in the frequency of oral and genital ulcerations ; in particular , preventive effects were observed for gu and a healing improvement for oa . it is well known that colchicine is recommended as a first - line therapy in erythema nodosum - like lesions in bd . the effects on erythema nodosum were described in three double - blind , placebo - controlled trials [ 3537 ] . in a double - blind trial the colchicine effects on reducing of gu were proven . no effects on oa and folliculitis were observed ( the dose of colchicine was adjusted according to body weight ) . on the contrary , in another double - blind controlled crossover trial , oa , gu , and pseudofolliculitis improvements were described . two prospective studies , evaluating the association of colchicine and benzathine penicillin , have described the decrease in the frequency and duration of oa and erythema nodosum and an improvement of the frequency of gu [ 38 , 39 ] . the beneficial effects of antibacterial therapy may be supported by the hypothesized role of streptococci in bd . the use of azithromycin in bd is based on previous hypothesis that streptococcus sanguinis play a main role in pathogenesis of bd . two case series [ 42 , 43 ] described the effectiveness of azithromycin in decreasing folliculitis and in fastening the healing time of oral ulcers . minocycline is described to decrease the frequency of the oa , erythema nodosum lesions , and papulopustular lesions in an open study . despite failing in the treatment of eye involvement , one rct , a pilot study , and three open studies [ 4749 ] have demonstrated thalidomide effectiveness in the treatment of oa , gu , and papulopustular lesions , while an increase in the frequency of nodular lesion was reported . however , it is well known that thalidomide is associated with severe adverse events and birth defects , whereby its use is limited . cyclosporine , a synthesis and release inhibitor of the il-1 and il-2 , is frequently used in the treatment of eye involvements in bd , but just few evidences are described about mucocutaneous lesions . a double - blind trial , a controlled study , and an open study showed that cyclosporine is effective in the gu and dermal lesion . the administrated dose is ranged from 5 to 10 mg / kg / day . a dose of 100 mg per day has been demonstrated to be very effective in healing the mucocutaneous lesions ; as evidenced in an open study and in a double - blind placebo - controlled clinical trial , dapsone showed relevant effects by decreasing the frequency and the duration of oa and the number and the frequency of gu . rebamipide , a well - known gastric mucoprotective agent , used to treat gastritis and gastric ulcer in japan , is observed to improve the aphthae count and to relieve the pain secondary to oral ulcers in a double placebo - controlled study . the authors conclude that it could be recommended as a long - term treatment for recurrent oa , also in association with other indicated drugs . interferon - alpha , a large family of glycoproteins , seems to provide a cellular response to the foreign constituents of microbes , tumor , and antigens and is used in the treatment of several diseases . although its mechanism of action is not well defined , inf - alpha seems to restore the low natural killer cell activity in patients with bd to a near normal level . interferon alpha 2a , in bd has proven to reduce the duration and the pain of oa and the frequency of gu and papulopustular lesion , as well as erythema nodosum - like lesion , compared to placebo in a randomized placebo - controlled and double - blind study . similar results were found in 7 open studies [ 5864 ] . in two previous reviews , the author highlighted that a treatment period of 2 to 4 months is at least necessary to obtain the highest effectiveness and declared a trend to disease relapse immediately or up to 7 months after treatment discontinuation and rapidly responded after reinstitution of inf - alpha treatment . an initially high dose ( 9 million units 3 times per week ) for 3 months and then a low maintenance dose ( 3 million units 3 times per week ) are recommended . alemtuzumab is a humanized immunoglobulin g1 monoclonal antibody that targets cd 52 , a glicosilate antigen that is present on lymphocytes and macrophages . its main effect is t - cell depletion . in a case report , an 18-year - old woman with polyarthralgias , orogenital aphthosis , and erythema nodosum resisted conventional immunosuppressive therapy , describing a long lasting remission after the sixth subcutaneous administration . in an open trial , 18 patients with orogenital ulcerations , ocular involvement , and neurological involvement , treated with alemtuzumab ( 134 mg ) , in 6 months in 72% reached a complete remission and the daily dose of prednisolone was reduced ; therapy was discontinued in 33% for stable remission . , the good efficacy of biotechnological drugs in the treatment of resistant mucocutaneous lesions has been demonstrated in several published works . in particular , the use of tnf - alpha inhibitors and il-1 inhibitors has been described to improve these lesions . cytokines produced by t helper cells , including tnf - alpha are described to play an important role in the molecular genesis of bd . higher soluble tnf - alpha receptors and tnf - alpha sera levels are found in active bd , with their spontaneous secretion by monocytes . this finding supports the possible effective use of anti - tnf - alpha therapy in bd . in two clinical cases , patients with genital ulcers resistant to azathioprine and prednisone therapy , treated with infliximab ( 5 mg / kg for a total of 4 infusion ) reached a complete remission of the lesion [ 71 , 72 ] . hence , infliximab seems to produce good and long - lasting remission of mucocutaneous lesion , after its discontinuation at the 13th infusion as described in two clinical cases in which the patients were treated with infliximab , respectively , in association with azathioprine ( 0.7 mg / kg / day ) and cyclosporine ( 3 mg / kg / day ) [ 73 , 74 ] . in an open study , the researchers described a good therapeutic response in patients with refractory mucocutaneous lesions , after 12 months of etanercept ( 50 mg / wk ) therapy in addition to conventional immunosuppressive drug ( azathioprine and colchicine ) with a reduction of oral prednisolone . in a randomized controlled trial ( rct ) the effectiveness of etanercept in suppressing most of the mucocutaneous manifestations , such as the oral ulcers , the papulopustular lesions and nodular lesions has been demonstrated , and a lower probability of recurrence of oral ulcers has been described ; the genital ulcers do not seem to improve after the treatment . the improvement of genital ulcers after etanercept treatment ( 25 mg twice week ) is described in a case report . recent studies have explained the role of different chemokines in cellular and molecular pathophysiology of bd , and , in particular , interleukin-1 ( il-1 ) cytokine family is described to play a complex network of minor proinflammatory mediators and subsequent expression of integrins on leukocytes and endothelial cells , with many influences on the inflammatory response . this innovative concept introduces the identification of new potential targets for biological therapy . lately , the recombinant human il-1 receptor antagonist ( anakinra ) , the human immunoglobulin g1 ( igg1 ) anti - il-1 beta monoclonal antibody ( canakinumab ) , and the recombinant humanized anti - il-1 beta antibody ( gevokizumab ) are proven to be partially useful in the treatment of bd , while appearing to be more effective in ocular involvement . however , with regard to the mucocutaneous lesions , good responses to anakinra have been reported in a patient with oral and genital ulcers resistant to conventional therapy and in a teenager with aphthous lesions and cutaneous lesions unresponsive to traditional treatments . in a case report and in a case series , canakinumab has proved to be successfully used in oral and genital aphthosis , skin lesions , and granuloma annulare [ 21 , 80 ] . finally , apremilast , an inhibitor of phosphodiesterase 4 ( pde ) , a drug approved for psoriasis and psoriatic arthritis , seems to be a good alternative treatment in bd . in a phase ii randomized , placebo - controlled , double - blind study , the apremilast is observed to be effective in the treatment of oral ulcers and in treating genital ulcers . bd is a complex syndrome , characterized by several clinical manifestations with usually frequent relapses . the mucocutaneous lesions represent nagging and typical manifestations in bd , and their treatment management is usually driven by codominant clinical involvements , such as eyes and gastrointestinal and neurological involvement , or by the subject comorbidity . although the traditional immunosuppressive drugs , as colchicine , corticosteroids , and azathioprine , are generally effective , several treatment failures , with severe and frequent relapses , troubled the course of the disease . in the last years , new pathogenetic hypothesis supported the use of new biotechnological drugs , as il-1 inhibitors and tnf inhibitors , in the treatment of bd . many clinical trials , open studies , and clinical case reports described their efficacy in the treatment of severe mucocutaneous manifestations . further scientific demonstrations on large scale are necessary to prove their effects on reducing the frequency of mucocutaneous manifestations and on maintaining long - term effects . . a wider drug range , with less adverse events risks , may provide alternative treatment tools for the clinicians , useful in nonresponding patients or in case of adverse events . the main unmet need in the overall management of bd is the lack of a specific treatment to target strategy ; therefore , despite several available drugs , the treatment strategy of bd patients is still tailored according to the severity and type of organ involvement . the main goal of therapy in patients with bd is to induce and maintain disease remission and improve quality of life ; in this regard , biologics are rapidly becoming effective alternatives to conventional treatments , also in mucocutaneous lesions . since oa and gu represent relevant threats for quality of life impairment , biological drugs are recommended in mucocutaneous involvement , mainly when traditional drugs miss the mark .

behet 's disease ( bd ) is a multisystemic disorder of unknown etiology characterized by the triple symptom complex consisting of recurrent oral aphthosis , genital ulcers , and chronic relapsing bilateral uveitis . recurrent mucocutaneous lesions are generally considered the hallmark of the disease , being the most common symptoms presenting at the onset of disease . although the improvement of knowledge about the pathogenetic mechanism added important changes in the treatment management of bd clinical manifestations , thus avoiding the appearance of serious life - threatening complications which are disease related , the mucocutaneous lesions are still the most nagging clinical manifestations to be treated . in this work we reviewed the current state of knowledge regarding the therapeutic approaches for mucocutaneous lesions of bd mainly based on controlled studies to provide a rational framework for selecting the appropriate therapy for treating these troublesome features of the disease .

1. Introduction 2. Literature Review 3. Conclusions

meconium aspiration syndrome ( mas ) is defined as respiratory distress in an infant born through meconium - stained amniotic fluid ( msaf ) with characteristic radiological changes and whose symptoms can not be otherwise explained . because meconium is rarely found in the amniotic fluid prior to 34 weeks ' gestation , mas is often a disease of the term and near - term infant and is associated with significant respiratory morbidity and mortality . cleary and wiswell have proposed a severity criteria to define mas : ( a ) mild mas is a disease that requires less than 40% oxygen for less than 48 hours , ( b ) moderate mas is a disease that requires more than 40% oxygen for more than 48 hours with no air leak , and ( c ) severe mas is a disease that requires assisted ventilation for more than 48 hours and is often associated with pphn . in this paper , we look at the current understanding of the pathogenesis and management of mas . meconium is a viscous sticky dark green substance containing gastrointestinal secretions , bile , bile acids , mucus , pancreatic juice , blood , swallowed vernix caseosa , lanugo , and cellular debris . msaf is present in 820% of all deliveries [ 14 ] , increasing to 2352% after 42 weeks of gestation [ 5 , 6 ] . factors that promote the passage of meconium in utero include placental insufficiency , maternal hypertension , preeclampsia , oligohydramnios , and maternal drug abuse , especially of tobacco and cocaine . the risk of mas is increased in black americans , africans , and pacific islanders [ 7 , 10 ] . factors associated with the development of mas among infants with msaf include thicker consistency of meconium , nonreassuring fetal heart tracing , fetal acidosis , cesarean delivery , meconium below the cords , infants who needed intubation at birth , and a low apgar score [ 9 , 11 ] . in the united states , the incidence of mas decreased nearly fourfold from 5.8% to 1.5% between 19901992 and 1997 - 1998 and this was attributed to a 33% reduction in births at more than 41 weeks ' gestation , more frequent diagnosis of nonreassuring fetal heart rate patterns , and greater use of amnioinfusion . mas remains a serious problem in developing and newly industrialized countries , and mas accounts for about 10% of all cases of respiratory failure with 39% mortality rate . mas results from aspiration of meconium during intrauterine gasping or during the first few breaths . fetal hypoxic stress can stimulate colonic activity , resulting in the passage of meconium and also stimulates fetal gasping movements that result in meconium aspiration in - utero . mounting evidence suggests that a chronic in utero insult may be responsible for most cases of severe mas as opposed to an acute peripartum event [ 14 , 15 ] . the pathophysiologic mechanisms of hypoxemia in mas include ( a ) acute airway obstruction , ( b ) surfactant dysfunction or inactivation , ( c ) chemical pneumonitis with release of vasoconstrictive and inflammatory mediators , and ( d ) pphn with right - to - left extrapulmonary shunting . poor oxygenation is attributed to a combination of ventilation perfusion mismatching , intrapulmonary shunting related to regional atelectasis and extrapulmonary shunting related to pphn . depending on the consistency and amount of meconium aspirated , meconium may lead to either partial or complete airway obstruction leading to hyperinflation or atelectasis of the alveoli . the gas trapped may rupture resulting in air leak syndromes such as pulmonary interstitial emphysema , pneumothorax , and pneumomediatinum . presence of meconium in the alveoli can inactivate the endogenous surfactant and decrease the production of surfactant proteins a and b [ 16 , 17 ] . the exact mechanisms for meconium - induced inactivation of pulmonary surfactant are not clearly understood . however , several components of meconium , especially fat - soluble ( free fatty acids , cholesterol , and triglycerides ) , and water - soluble ( containing bilirubin , bile acids , enzymes , etc . ) meconium can impair pulmonary surfactant by a combined action of cholesterol and bile acid present in meconium . meconium may also change the viscosity and ultrastructure of the surfactant , decrease the levels of surfactant proteins , and also accelerate the conversion from large , surface active aggregates into small , less active forms . the surfactant dysfunction is enhanced by leakage of plasma protein through an injured alveolar - capillary membrane , as well as the proteolytic enzymes , and oxygen - free radical release from activated cells during the inflammation . , neutrophils and macrophages are found in the alveoli , larger airways , and lung parenchyma . meconium is also a source of proinflammatory mediators such as interleukins ( il-1 , il 6 , and il 8) , tumor necrosis factors . thus it may induce inflammation either directly or indirectly through the stimulation of oxidative bursts in neutrophils and alveolar macrophages and may injure the lung parenchyma or lead to vascular leakage causing toxic pneumonitis and hemorrhagic pulmonary edema . acute intrapulmonary meconium contamination induces a concentration - dependent pulmonary hypertensive response , with 1520% of infants with the mas showing pphn . pphn in infants with mas may be caused by ( a ) pulmonary vasoconstriction secondary to hypoxia , hypercarbia , and acidosis , ( b ) hypertrophy of the postacinar capillaries as a result of chronic intrauterine hypoxia , and ( c ) pulmonary vasoconstriction as a result of pulmonary inflammation . despite the fact that meconium itself has detrimental effects on placental and umbilical tissues in utero , very little is known regarding the meconium - stimulated cellular and biochemical alterations in fluid - filled fetal lungs . however , heavy meconium staining is supposed to inhibit , through unknown mechanisms , fetal lung fluid reabsorption at birth that may disturb the ability of the lungs to adapt properly to extrauterine life . the extent of lung destruction is not closely correlated to the quantity of meconium in lung tissue but rather to the degree of hypoxia and acidosis present at delivery . ghidini and spong postulated that severe mas may not be in fact causally related to the aspiration of meconium but rather caused by other pathologic processes occurring in utero , such as chronic asphyxia , infection , or persistent pulmonary hypertension . it is important to monitor infants born through msaf for any signs of respiratory distress for at least 24 hours . diagnosis of mas is based on the presence of respiratory distress in an infant born through msaf , with no alternate cause for respiratory distress . chest radiograph and blood gas analysis should be performed if necessary . because of diverse mechanisms causing this disease , radiographic findings are different . the classic radiographic findings in mas are overexpansion of the lungs with widespread coarse , patchy infiltrates . however , the severity of the x - ray pattern does not always correlate with the clinical picture . the lack of correlation between clinical severity and radiographic pattern suggests that mas is less dependent on the amount of meconium obstruction and parenchymal damage than on other aspects of mas , such as the presence and severity of pphn . the decrease in the incidence of mas in the last decade has been attributed to the reduction in postterm delivery , aggressive management of abnormal heart rate monitoring , and decreased number of infants with a low apgar score . meta - analysis of 14 randomized controlled trials ( rcts ) suggests that elective induction of labor for pregnancies at or beyond 41 weeks is associated with significant reduction in the incidence of mas ( rr = 0.43 , 95% ci 0.230.79 ) and fewer perinatal deaths ( rr = 0.31 ; 95% ci : 0.110.88 ) compared to expectant management . intrapartum monitoring has been recommended to screen for early signs of fetal hypoxia , a risk factor for mas . there is no evidence that electronic fetal heart rate monitoring ( efm ) with or without fetal blood gas and acid - based assessment reduces the risk of fetal or neonatal mortality or morbidity . fetal scalp ph determination and newer modes like fetal pulse oximetry will improve decision making in timing of delivery and may reduce the incidence of msaf and mas . amnioinfusion has been proposed to reduce the risk of mas by diluting the meconium , thus reducing its mechanical and inflammatory effects . amnioinfusion also helps by cushioning the umbilical cord , thus correcting the recurrent umbilical compressions that lead to fetal academia . in a meta - analysis of rcts , reported that intrapartum amnioinfusion was significantly associated with reduced risk of mas ( or 0.30 ; 95% ci 0.19 , 0.46 ) , meconium below the vocal cords , and neonatal acidemia . in a recent cochrane meta - analysis of 13 studies , the author stratified the studies based on the clinical settings . amnioinfusion reduces the risk of mas only in clinical settings with limited peripartum surveillance ( rr 0.25 , 95% ci 0.13 to 0.47 ) , but not in clinical settings with standard peripartum surveillance . however , american college of obstetricians and gynecologists conclude that routine prophylactic amnioinfusion for the dilution of msaf is not recommended for the prevention of mas . in a large multicenter rct involving 2514 term infants with msaf comparing intrapartum suction versus no suction , the incidence of mas ( 4% versus 4% ) , mortality , the need of mechanical ventilation , and duration of oxygen therapy were similar in both groups . therefore , routine intrapartum oropharyngeal and nasopharyngeal suctioning for infants born with clear or meconium - stained amniotic fluid is no longer recommended . neonatal resuscitation program ( nrp ) recommends intubation and direct endotracheal suction soon after delivery for nonvigorous infants born through msaf , who have depressed respiratory efforts , poor muscle tone , and/or heart rate less than 100/minute . cochrane meta - analysis of four randomized studies did not show a difference in the incidence of mas between intubated and nonintubated vigorous infants . hence , if the baby born through msaf has a normal respiratory effort , normal muscle tone , and a heart rate greater than 100 beats per minute , direct endotracheal suction is not recommended . only suctioning of mouth and nose using a bulb syringe or large bore suction catheter is indicated . according to the international consensus on cardiopulmonary resuscitation and emergency cardiovascular care science , the available evidence does not support or refute the routine endotracheal suctioning of depressed infants born through msaf . all infants at risk for mas who show signs of respiratory distress should be admitted in the neonatal intensive care units . maintenance of optimal thermal environment and minimal handling is essential because these infants are agitated easily , which causes right - to - left shunting , leading to hypoxia and acidosis . maintenance of adequate oxygenation , optimal blood pressure , correction of acidosis , hypoglycemia and other metabolic disorders is the mainstay of treatment . ventilator management of the neonate with mas is challenging because of the complicated pulmonary pathophysiology resulting from areas of atelectasis and areas of hyperinflation , in association with ventilation - perfusion mismatch and airway compromise . approximately 40% of babies with mas require mechanical ventilation and additional 10% require continuous positive airway pressure . there is little evidence from the clinical trials regarding the ventilator treatment of infants with mas . ventilation should be aimed at increasing oxygenation while minimizing the barotrauma that lead to air leak syndromes . some infants only require oxygen by hood . in infants with mas who have hypoxemia ( pao2 < 50 mm hg ) , hypercarbia ( paco2 > 60 mm hg ) , or acidosis ( ph less than 7.25 ) in an oxygen - enriched environment with an inspired oxygen fraction ( fio2 ) > 0.6 are often considered candidates for mechanical ventilation . in infants with mas without associated pphn , it is sufficient to maintain a ph of 7.37.4 , with a pao2 targeted between 60 and 80 mmhg and a paco2 of 4050 mmhg . infants may be started with a moderate peak inspiratory pressure ( pip ) preferably not exceeding 25 cm h2o , a relatively rapid ventilator rate ( 4060/min ) , a moderate positive end expiratory pressure ( 46 cm h2o ) , and an adequate expiratory time ( 0.50.7 sec ) to prevent gas trapping and air leaks . if gas trapping is noticed , expiratory time may be increased and peep should be decreased ( 3 - 4 cm h2o ) . in infants with mas and concomitant but the strategy of achieving hypocapnia and alkalosis by hyperventilation has adverse effects including cerebral vasoconstriction leading to long - term neurologic morbidity as well as air leaks [ 35 , 36 ] . in such situations theoretically high frequency ventilation ( hfv ) minimizes the barotrauma and may reduce air leak syndrome in mas . no prospective randomized trials have compared conventional ventilation versus hfv in mas . in pilot studies using inhaled nitric oxide ( ino ) , kinsella and abman found that the combination of hfv and ino caused the greatest improvement in oxygenation in some patients with severe pphn . they speculated that improved lung inflation during hfv may augment the response to ino by decreasing intrapulmonary shunting and improving ino delivery to the pulmonary circulation [ 37 , 38 ] . partial liquid ventilation was found to be a better method of delivering surfactant in an adult rat model of mas when compared with conventional mechanical ventilation . there is no randomized clinical trial about the use of partial liquid ventilation in human neonates with mas . in vitro studies have shown that meconium interferes with surfactant in several ways : inactivation of its function depending on the concentration , direct toxicity on type ii pneumocytes , displacement of surfactant from the alveolar surface , and decrease of surfactant protein a and b levels . canadian pediatric society position statement recommends that intubated infants with mas requiring more than 50% oxygen should receive exogenous surfactant therapy . bolus surfactant therapy for mas has been associated with reduction in the severity of respiratory distress and decrease in the number of infants with progressive respiratory failure requiring ecmo . meta - analysis of 4 rcts showed reduction in the severity of respiratory illness and decrease in the number of infants with progressive respiratory failure requiring ecmo ( rr 0.64 , 95% ci 0.460.91 ) . however , there was no significant difference in mortality , hospital stay , length of ventilation , duration of oxygen use , pneumothorax , pulmonary interstitial emphysema , or chronic lung disease . clinical trial of surfactant lavage using lucinactant in conventionally ventilated infants with mas found no difference between lavage infants and controls in terms of ecmo requirements , air leak , or duration of ventilation . similarly , dargaville and colleagues reported that lung lavage with dilute surfactant ( survanta ) in ventilated infants with severe mas does not decrease the duration of respiratory support , but may produce a reduction in mortality , especially in units not offering ecmo . in 2003 , cochrane meta - analysis of two trials [ 44 , 45 ] including 85 infants with mas showed that there was no difference in mortality but a small increase in the duration of oxygen treatment in steroid - treated group . since then , two more trials reported that steroid therapy in mas was associated with a decrease in the duration of oxygen therapy and hospital stay [ 47 , 48 ] . steroids may be beneficial in severe mas with apparent lung edema , pulmonary vasoconstriction , and inflammation . at present , there is no conclusive evidence to propose routine steroid therapy in the management of mas . further research is needed regarding the dosing , timing , and ways of administration of steroids considering their individual properties and possible acute and long - term side effects . the presence of meconium increases the chances of positive cultures from amniotic fluid in preterm and term infants . however , studies evaluating the development of sepsis in infants with msaf failed to demonstrate that relationship . three randomized control studies reported that routine antibiotic prophylaxis is not recommended in the management of mas for those without perinatal risk factors [ 5153 ] . antibiotic therapy did not affect the clinical course and outcome related to infection in mas without perinatal risk factors for infection and without ventilator use . the role of antibiotics in the management of mas may need to be reevaluated in well - designed trials . unless there is definite risk for infection , prophylactic use of antibiotics in mas did not reduce infection . if antibiotics are started for suspected infection due to perinatal risk factors , consider discontinuing antibiotics once the blood culture results are negative . randomized clinical trials have demonstrated that ino therapy decreases the need for ecmo in addition to mortality in full - term and near - term neonates with hypoxic respiratory failure and pphn . for hypoxic respiratory failure due to mas , infants responded well to combined ino and hfv as compared to either treatment alone . the response to combined treatment with hfv and ino reflects both decreased intrapulmonary shunt and augmented nitric oxide delivery to its site of action . ecmo has been used as a final rescue therapy in infants with severe and refractory hypoxemia associated with mas . use of ecmo has been decreased significantly in developed countries with the availability of ino and hfv . the survival rate has approached 95% of infants with mas who underwent ecmo . in the ecmo registry , the highest survival rates ( > 90% ) were seen in the patients with mas who qualified for ecmo . all infants with mas should be monitored using noninvasive monitors ( pulse oximeter , transcutaneous o2/co2 methods ) and blood gas sampling should preferably be done with an indwelling arterial line . sedation and analgesia are used frequently in infants with mas and pphn to alleviate pain and discomfort that may lead to hypoxia and right - to - left shunting . opioids , particularly morphine or fentanyl , are frequently used to optimize gas exchange and also to avoid asynchrony , reflex catecholamine release , and aggravation of pulmonary vascular resistance . depolarizing muscle relaxants ( pancuronium , vecuronium ) were widely used in the past along with opioids to decrease agitation and subsequent hypoxic episodes in ventilated infants . the benefits of neuromuscular blockade include improved oxygenation , decreased oxygen consumption , and decreased accidental extubations . however , the use of neuromuscular blockade remains controversial and is reserved for the infant who can not be treated with sedatives alone . neuromuscular blockage can promote atelectasis of dependent lung regions and ventilation perfusion mismatch and may also be associated with increased risk of death . infants who do not show initial response to ino and those that deteriorate subsequently while on ino therapy continue to have significant pphn and need other alternative therapy . alternatives available include ( a ) phosphodiesterase-5 inhibitors like sildenafil , zaprinast , milrinone , dipyridamole , ( b ) prostaglandins like prostacyclin or pge1 , ( c ) tolazoline , magnesium sulfate , ( d ) no precursor l - arginine , ( e ) free radical scavengers like superoxide dismutase , ( f ) experimental agents like bosentan ( endothelin antagonist ) . currently mas treatments are all supportive in nature and do not directly affect the injurious actions of meconium on the lung . there is still no effective and safe treatment or prophylactic measure for mas once the meconium has passed below the vocal cords into the lungs . it has been suggested that fetal pancreatic digestive enzymes play an important role in the lung damage after meconium aspiration by causing disruption of intercellular connections and cell detachment from the basement membrane . a protease inhibitor cocktail prevented the cell detachment induced by meconium suggesting that they may be useful in the treatment and/or prophylaxis . recent data show that some of the cell death induced by meconium occurs by apoptosis , and therefore has the potential for pharmacologic inhibition through the use of apoptosis blockers or other strategies . despite improvement in obstetrical and neonatal care , mas continues to be a neonatal disorder with high morbidity and mortality . the lung injury caused by meconium is complex and can be attributed to mechanical obstruction of airways , surfactant inactivation , chemical pneumonitis , and pphn . among preventive strategies , elective induction of labor for pregnancies at or beyond 41 weeks is associated with significant reduction in the incidence of mas and amnioinfusion reduces the risk of mas only in clinical settings with limited peripartum surveillance . intrapartum management includes endotracheal suctioning to clear meconium only in nonvigorous infants born through msaf . invasive ventilation if required should use lower pip , moderate peep , higher rates ( 4060/min ) , and adequate expiratory time and permissive hypercapnea should be tolerated to facilitate gentle ventilation . mas complicated with pphn and not responsive to conventional ventilation may require hfv and ino . ino therapy has decreased the need for ecmo in mas complicated by hypoxic respiratory failure and pphn . surfactant replacement should be considered in ventilated infants requiring more than 50% fio2 . unless there is definite risk for infection , prophylactic use of antibiotics in mas does not reduce infection or alter the clinical course of illness . ecmo has been used as a final rescue therapy in infants with severe and refractory hypoxemia associated with mas . the role of steroids and other adjuvant pharmacotherapies like magnesium sulfate , free radical scavengers , and protease inhibitors is still experimental and they are not routinely recommended . as mas is a major cause of mortality in developing countries , studies focusing on prevention and early treatment

meconium aspiration syndrome ( mas ) is a common cause of severe respiratory distress in term infants , with an associated highly variable morbidity and mortality . mas results from aspiration of meconium during intrauterine gasping or during the first few breaths . the pathophysiology of mas is multifactorial and includes acute airway obstruction , surfactant dysfunction or inactivation , chemical pneumonitis with release of vasoconstrictive and inflammatory mediators , and persistent pulmonary hypertension of newborn ( pphn ) . this disorder can be life threatening , often complicated by respiratory failure , pulmonary air leaks , and pphn . approaches to the prevention of mas have changed over time with collaboration between obstetricians and pediatricians forming the foundations for care . the use of surfactant and inhaled nitric oxide ( ino ) has led to the decreased mortality and the need for extracorporeal membrane oxygenation ( ecmo ) use . in this paper , we review the current understanding of the pathophysiology and management of mas .

1. Introduction 2. Epidemiology of MAS 3. Pathophysiology of MAS 4. Diagnosis of MAS 5. Management of MAS 6. Conclusions

estimates were made of the proportion of chemicals that were carcinogenic , anticarcinogenic , or either in 397 long - term bioassays conducted by the national toxicology program ( ntp ) . the estimates were obtained from the global pattern of p - values obtained from statistical tests applied to individual experiments . these tests accounted for multiple comparisons using a randomization procedure and were found to operate at the correct level of significance . representative estimates of the proportion of carcinogens [ with 90% confidence intervals ( ci ) ] compared to the ntp estimates were as follows : male mice , 0.32 ( ci , 0.19 - 0.44 ) , ntp = 0.29 ; female mice , 0 . 28 ( ci , 0.15 - 0.41 ) , ntp = 0.34 ; male rats , 0.35 ( ci , 0.23 - 0.47 ) , ntp = 0.36 ; female rats , 0.34 ( ci , 0.21 - 0.46 ) , ntp = 0.28 ; all sexes and species , 0.59 ( ci , 0.49 - 0.69 ) , ntp = 0.51 . representative estimates of the proportion of anticarcinogens were as follows : male mice , 0 . 34 ; female mice , 0.27 ; male rats , 0.40 ; female rats , 0.44 ; all sexes and species , 0.66 . thus , there was as much or more evidence in this study for anticarcinogenesis as carcinogenesis . even though the estimators used were negatively biased , it was estimated that 85% of the chemicals were either carcinogenic or anticarcinogenic at some site in some sex - species group . this suggests that most chemicals given at high enough doses will cause some sort of perturbation in tumor rates.imagesfigure 1figure 2figure 3figure 4figure 5

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food additives are widely used in foodstuffs to prevent from spoilage and improve color , flavor , and texture of foods . however , these additives in foods may affect individuals who are sensitive with some type of allergy , asthma , and hay fever . consequently , authorities have set threshold values for acceptable daily intake , varying from country to country . for instance , the list of authorised food additives and maximum permitted levels in european union are laid down in the annexes of council directive [ 1 , 2 ] . to ensure food safety from farm to fork , it is also essential to develop effective and reliable analytical methods for the monitoring of the additive levels in food . therefore , various analytical methods have been reported for the simultaneous determination synthetic food additives , such as thin layer chromatography , uv - visible spectrophotometry [ 5 , 6 ] , voltammetry [ 7 , 8 ] , differential pulse polarography , capillary electrophoresis , hplc - dad [ 1114 ] , hplc - ms , and hplc - ms - ms [ 16 , 17 ] . until now , although many analytical techniques have been developed for the determination of various food additives in foods , there is no report about simultaneous determination of this combination in food samples . among these analytical methods , hplc coupled with uv / vis or diode array detectors ( dads ) are the most commonly used methods due to their sensitivity , selectivity , and high resolution . so , development of effective chromatographic separation method involves judicious selection of experimental conditions that is suitable for the separation of interested components at an adequate resolution with reasonable run time . in this it not only is a timesaving method but also it has an ability to reveal possible interactions between variables [ 19 , 20 ] . hence , experimental designs have been increasingly used to determine the optimum conditions of chromatographic separation of some analytes in food , drug , and biological fluid samples with a minimum number of experiments for over the past decade [ 2128 ] . in this paper , a new rp - hplc method was developed , using experimental design , for simultaneous determination of five synthetic food additives in soft drinks , including three synthetic colorants ( carmoisine , allura red , and ponceau 4r ) , two preservatives ( potassium sorbate and sodium benzoate ) , and caffeine . for the optimization procedure , box - behnken design ( bbd ) was used to construct mathematical models that predict how changes input or controlled by variables ( ph , flow rate , and mobile phase ratio ) affected the resolution in defined experimental region . further , the method validation has been carried out according to the international conference on harmonization guidelines . the optimized and validated method was successfully applied to some commercial soft drinks containing potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine . chromatographic analyses were performed using a shimadzu hplc system ( kyoto , japan ) consisting of a model lc20 at pump unit , spd-20a uv - vis detector , 7725 20 l sample injection , a computer , and an inertsil octadecylsilane- ( ods- ) 3v column ( 5 m , 250 mm 4.6 mm ; gl sciences , tokyo , japan ) . the statistical analysis for the analytical responses and validation data , va , usa ) was used for the graph plotting and for estimating the responses of experimental variables . milli - q water was used to prepare the solutions and mobile phases ( millipore , milford , ma , usa ) . sodium acetate trihydrate , glacial acetic acid , and hplc - grade acetonitrile were acquired from merck ( darmstadt , germany ) . potassium sorbate ( 99.0% purity ) , sodium benzoate ( 99.0% , purity ) , carmoisine ( 98.0% purity ) , allura red ( 98.0% purity ) , ponceau 4r ( 99.0% purity ) , and caffeine ( 100.0% purity ) were purchased from sigma - aldrich ( st . louis , missouri , usa ) . standard stock solutions of potassium sorbate , sodium benzoate , and caffeine were prepared at a concentration of 250 g / ml . standard stock solutions of carmoisine , allura red , and ponceau 4r were prepared at a concentration of 100 g / ml . fresh working solutions in the concentration range of 210 g / ml for carmoisine , allura red , and ponceau 4r and 525 g / ml for caffeine , potassium sorbate , and sodium benzoate were prepared by the dilution of the standard stock solutions in milli - q water . soft drink samples were purchased from local supermarkets in istanbul , turkey , and were degassed in an ultrasonic bath for 5 min . then , 1 ml of the sample was transferred to a 10 ml volumetric flask and diluted to the volume with milli - q water . prior to the analysis , both soft drink samples and standard solutions were filtered through 0.45 m millipore filters and then injected into hplc system . the optimum separation of all analytes was achieved with 0.025 m sodium acetate / acetic acid buffer , ph 6.0 , acetonitrile gradient that follows 05 min , 95 : 75 ( v / v ) ; 510 min , 70 : 30 ( v / v ) . the mobile phase flow rate was 1.0 ml / min and the injection volume was 20 l in all the chromatographic runs . the detection was made with a variable ultraviolet - visible detector fixed at 230 nm . a box - behnken design ( bbd ) using three variables at three levels ( coded levels : 1 , 0 , and + 1 ) was used for the optimization of simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine by hplc . the variables and levels selected for optimization procedure were ph ( a ; 6.0 , 6.5 , and 7.0 ) , flow rate ( b ; 1.0 , 1.2 , and 1.4 ) , and mobile phase ratio ( in terms of acetate buffer ) ( c ; 85 , 90 , and 95 ) ( table 1 ) . while the first step has an effect on the chromatographic separation , the second step has an effect on the run time of the method . therefore , experimental variables of the first step of hplc method were taken into account . 15 experimental runs were performed at random and overall resolution ( r ) was chosen as the response for the separation of the compounds . experimental design matrix used and the results obtained by bbd were listed in table 2 . in - house validation of the method evaluated parameters are linearity of calibration curve , limit of detection ( lod ) , limit of quantification ( loq ) , and precision , accuracy , and stability . the linearity of the hplc method for the determination of five food additives and caffeine was evaluated in a concentration range of 210 g / ml for carmoisine , allura red , and ponceau 4r and 525 g / ml for potassium sorbate , sodium benzoate , and caffeine covering the normal range of concentrations obtained when analyzing soft drinks . calibration equations were calculated by the least squares treatment of the peak area of the food additives and caffeine . the limit of detection ( lod ) and limit of quantitation ( loq ) were calculated as lod 3x/s and loq 10x/s , where is the standard deviation of intercept and s is the slope . in order to test the prediction performance of the proposed methods , intraday ( three times in a day operation under the same conditions ) and interday ( four different days ) studies were performed at three different concentrations ( level 1 : 10 g / ml ; level 2 : 15 g / ml ; level 3 : 20 g / ml for potassium sorbate , sodium benzoate , and caffeine ; level 1 : 4 g / ml ; level 2 : 6 g / ml ; level 3 : 8 g / ml for carmoisine , allura red , and ponceau 4r ) . accuracy of the method was ascertained by a recovery study by adding a known amount of reference standards to the soft drink samples . firstly , 0.5 ml of the soft drink sample was transferred to a 10 ml volumetric flask and the reference standards were added on it at three different concentration levels . then , added samples were diluted to the volume with milli - q water , filtered , and analyzed . chromatographic optimization requires selecting suitable criteria for the evaluation of the resultant chromatograms in order to choose the optimum conditions . bbd is an independent , rotatable , or nearly rotatable second - order design based on three - level incomplete factorial designs . it is more efficient compared to other response surface designs , such as central composite designs . it can also provide sufficient information to test the lack of fit , and therefore it is one of the best quadratic models for response surface method and has been widely used in analytical fields . because of the nonlinearity of the model , a polynomial function to contain second - order model is postulated to describe the evolution phenomenon:(1)yi = b0+i=1nbixi+i=1nbiixi2+1ijnbijxixj+i , where n is the number of variables , b 0 is the constant term , b i , b ii , and b ij represent the coefficient of the first - order terms , quadratic terms , and interaction terms , respectively , and i is a term that represents other sources of variability not accounted for the estimation , such as background noise . the experimental results are shown in table 2 . the regression model for the response was tested through analysis of variance ( anova ) . from the results of anova ( table 3 ) , it can be deduced that linear contribution of mobile phase ratio ( c ) and quadratic contribution of mobile phase ratio ( cc ) influence the resolution significantly . interactions of the individual variables in this study are not significant to resolution in the selected range . fitted quadratic model equation is also presented in ( 2 ) . figure 1 shows the analysis of individual variables of experimental design . from figure 1 , it can be seen how the value of the resolution may increase if we take higher mobile phase ratio ( c ) . also , we can infer that although ph ( a ) and flow rate ( b ) do not greatly influence the resolution better resolutions are obtained for low values of ph and flow rate(2)r^=0.67172.99a153.92b+1089.10c+152.74a2345.80ac152.83b2307.58bc+1088.49c2 . the regression models obtained were used to calculate the response surface for each variable separately . figure 2 illustrates the response surface plots for the resolutions . in particular , the effect of ph ( a ) and mobile phase ratio ( c ) on resolution is shown in figure 2(b ) . this plot shows that the highest resolution is obtained at greater values of the mobile phase ratio . the relation between the effects of the other variables on the resolution is also plotted in figures 2(a)2(c ) . according to the results of the optimization procedure , the optimum variables corresponded to ph , 6.0 ; flow rate , 1.0 ml / min ; mobile phase ratio , 95% . a typical chromatogram obtained under optimum conditions the results of the linearity , lods , and loqs are summarized in table 4 . a good linear relationship is displayed between the corresponding peak areas and the concentrations of the compounds based on the correlation coefficients ( r > 0.9962 ) . the lods of the six compounds were in the range of 0.100.19 g / ml , and the loqs of the six compounds were in the range of 0.330.63 g / ml . a summary of intraday ( the rsd of the recoveries of the nine samples ) and interday precision ( the rsd of the recoveries of the twelve samples ) are listed in table 5 . these results show that the proposed method is precise for the simultaneous determination of these compounds . the recoveries of the six compounds to determine the accuracy of the method are summarized in table 6 . the proposed method resulted in satisfactory recoveries for all additives and caffeine , ranging from 97.67% to 105.56% . the recoveries demonstrated that the matrixes have negligible effect on the quantification of these compounds and the method is accurate within the desired range . under refrigerated and room temperature conditions , all food additives and caffeine in mobile phase and water were stable for at least 1 month . these results show that the proposed method is precise , accurate , and sensitive for the simultaneous determination of the six compounds and can be used for routine analysis of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in soft drinks . the proposed hplc method was applied to the simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in different soft drinks . five replicates determination was made and the results are summarized in table 7 . the concentration of food additives in soft drinks ranged from 24.26 0.47 g / ml to 254.13 1.24 g / ml . the amounts of food additives and caffeine in all soft drink samples were below the limit value defined in the legislation on the food additives [ 1 , 2 ] . an efficient , accurate , and reliable method for the simultaneous determination of five food additives and caffeine in soft drinks was developed using hplc . box - behnken design was applied to the optimization of the chromatographic separation conditions and this design reduced to the number of experiments required . it can be concluded that a slight change in mobile phase ratio has a direct effect on the resolution . high percentage recovery data also shows that the proposed method is free from the interference . consequently , this study will provide a sensitive and rapid method for the detection of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in soft drinks .

box - behnken design was applied to optimize high performance liquid chromatography ( hplc ) conditions for the simultaneous determination of potassium sorbate , sodium benzoate , carmoisine , allura red , ponceau 4r , and caffeine in commercial soft drinks . the experimental variables chosen were ph ( 6.07.0 ) , flow rate ( 1.01.4 ml / min ) , and mobile phase ratio ( 8595% acetate buffer ) . resolution values of all peak pairs were used as a response . stationary phase was inertsil octadecylsilane- ( ods- ) 3v reverse phase column ( 250 4.6 mm , 5 m ) dimensions . the detection was performed at 230 nm . optimal values were found 6.0 ph , 1.0 ml / min flow rate , and 95% mobile phase ratio for the method which was validated by calculating the linearity ( r 2 > 0.9962 ) , accuracy ( recoveries 95.75% ) , precision ( intraday variation 1.923% , interday variation 1.950% ) , limits of detection ( lods ) , and limits of quantification ( loqs ) parameters . lods and loqs for analytes were in the range of 0.100.19 g / ml and 0.330.63 g / ml , respectively . the proposed method was applied successfully for the simultaneous determination of the mixtures of five food additives and caffeine in soft drinks .

1. Introduction 2. Experimental 3. Results and Discussion 4. Conclusion

sacral fractures are complex injuries that rarely occur in isolation,1 2 3 and therefore they often require a multidisciplinary treatment approach including orthopedic trauma surgeons and spine surgeons ( either orthopedic surgeons or neurosurgeons ) . many classifications have been proposed for sacral fractures , but none are comprehensive or universally accepted.4 5 6 7 8 9 the development of a comprehensive , reliable , and universally accepted classification of these fractures is critical to allow for effective communication between treating physicians and trainees as well as researchers ; furthermore , a well - designed classification may lead to the development of an optimal treatment algorithm for these challenging injuries . the existing sacral injury classifications are based on either fracture morphology or an inferred mechanism of injury . the existing systems are either very broad and simplistic , such as the commonly used denis classification that separates fractures broadly based on the location of the fracture with respect to the foramen , or they are exceedingly specific so that they can only be applied to a very select type of sacral fracture , such as the isler classification , which is only relevant for a vertical fracture through the foramen . alternatively , sacral fractures may be incorporated into pelvic injury classifications , but despite being one of the critical structures involved in posterior pelvic ring stability,10 only a rudimentary evaluation of sacral injuries is considered in pelvic fracture classifications.8 finally , despite the fact that the treatment decisions are often affected by the neurologic status of the patient and associated soft tissue injuries , none of the existing classifications formally consider these variables.4 5 6 7 8 9 recognizing the limitations of the existing classifications , the aospine trauma knowledge forum is partnering with pelvic trauma experts from aotrauma to develop a comprehensive sacral fracture classification similar to the aospine thoracolumbar injury classification system and the aospine subaxial cervical spine injury classification system.11 12 13 14 importantly , one of the main goals of this project is to develop a classification system that will achieve global acceptance from both spine surgeons and orthopedic traumatologists . however , given the complexity of sacral fractures , multiple controversial areas will have to be addressed . in an effort to develop a classification that will be able to obtain global acceptance , a survey was sent to all aospine and aotrauma members and their input was sought on controversial aspects of sacral fracture classification . the goal of this study is to determine the global perspective on controversial aspects of sacral fractures . in july 2015 , a survey ( see appendix a in online supplementary material ) was sent to all members of aospine and aotrauma . the survey was only sent out a single time , and each e - mail address was only permitted to answer the survey once . along with demographic information including region , experience , and specialty , the following four questions were asked : do you think that the appropriate order for sacral fracture severity from least to most severe is transverse fractures , unilateral vertical fractures , and fractures that lead to spinopelvic instability ? in an isolated vertical fracture of the sacrum , do you agree that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen ? do you think the integrity of the l5s1 facet is adequately considered if a unilateral vertical fracture where the ipsilateral superior s1 facet is discontinuous with the medial portion of the sacrum is considered differently from a fracture where the ipsilateral superior s1 facet is in continuity with the medial portion of the sacrum ? do you think a nondisplaced sacral u fracture that may be seen in low - energy insufficiency fractures is a clinically relevant entity that deserves its own spot in a classification ? ( a ) axial computed tomography ( ct ) image displaying a vertical fracture through the foramen . ( b ) axial ct image of the same fracture at the cephalad aspect of the sacrum , demonstrating that the fracture exits medial to the l5s1 facet . ( c ) axial ct image of the sacrum demonstrating a vertical fracture through the sacral foramen . chi - square tests were initially used to compare the surgeons ' answers to the four key questions of the survey . further on , the data was modeled through multivariable logistic regression analysis . specifically , four logistic regression models were performed , with ao region , specialization , years of clinical practice , and number of traumatic sacral fractures treated by the surgeon in the previous year considered as independent variables , whereas each one of the four key questions was used as the dependent variable . all statistical analyses were performed using sas version 9.2 ( sas institute inc . , cary , north carolina , united states ) . the survey was sent to 15,229 aospine and aotrauma members ; 671 surgeons ( 4.4% ) from all six ao regions of the world responded to the survey , and 474 surgeons ( 3.1% ) answered every question . overall , 86.9% of respondents felt that there was an increase in severity as fractures moved from a transverse fracture to a unilateral vertical fracture , and finally to a fracture that results in spinopelvic instability ; 77.8% of respondents thought that in an isolated vertical fracture of the sacrum , the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen . almost 4/5 of the respondents felt that it was appropriate to consider a unilateral vertical fracture where the ipsilateral superior s1 facet is discontinuous with the medial portion of the sacrum differently from a fracture where the ipsilateral superior s1 facet is in continuity with the medial portion of the sacrum , and 83.8% of surgeons agreed that a nondisplaced sacral u fracture is a clinically relevant entity that deserves its own spot in the classification ( fig . overall results of the survey suggest that there is widespread agreement with the proposed morphologic classification of sacral fractures . when the results were stratified by region ( table 2 ) , the regional differences were seen in the responses to the question concerning the risk to neurologic injury with vertical sacral fractures ( p = 0.014 ) and the question concerning the integrity of the l5s1 facet ( p = 0.039 ) . specifically , only 62.1% of surgeons from north america agreed that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen , and although this number represents almost two thirds of all north american surgeons , it is substantially lower than the rest of the world . additionally , although 69.4% of the surgeons from the middle east felt that the integrity of the l5s1 facet was adequately considered if a unilateral vertical fracture where the ipsilateral superior s1 facet is discontinuous with the medial portion of the sacrum is considered differently from a fracture where the ipsilateral superior s1 facet is in continuity with the medial portion of the sacrum , this number was substantially less than surgeons from every other region in the world . note : due to extremely low frequencies , africa was not included in the comparison . experience was accounted for in two ways : first respondents were stratified by years in practice , and additionally they were stratified by the number of traumatic sacral fractures they treated in the previous year ( table 3 ) . no significant variability was found in the responses based on years in practice , but as surgeons treated more sacral fractures , they were less likely to agree that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen ( p = 0.003 ) . however , despite this variability , more than two out of three surgeons who treat more than 10 sacral fractures a year agree that that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen . finally , the results were compared between spine surgeons ( orthopedic or neurosurgeon ) and orthopedic trauma surgeons . significantly more spine surgeons than trauma surgeons agreed that the risk of neurologic injury is highest in a vertical fracture through the foramen ( 84.7 versus 75.4% , respectively , p = 0.033 ) ; however , still more than three in four trauma surgeons agreed that fractures through the foramen have the highest rate of neurologic injuries . more spine surgeons than trauma surgeons agreed with the organization of b - type injuries ( 84.7% versus 75.4% , respectively , p = 0.03 ) ; however , still more than three in four trauma surgeons agreed with the organization of b - type injuries . subsequently , four multivariable logistic regression models were performed to identify demographic variables that were independent predictors of the survey responses . surgeons from the middle east were more likely than european participants to disagree with the statement that the appropriate order for sacral fracture severity from least to most severe is transverse fractures , unilateral vertical fractures , and fractures that lead to spinopelvic instability ( odds ratio = 0.36 ; 95% confidence interval [ ci ] 0.16 to 0.86 ; p = 0.020 ) . surgeons from either the middle east ( odds ratio = 0.42 ; 95% ci 0.20 to 0.91 ; p = 0.028 ) or north america ( odds ratio = 0.43 ; 95% ci 0.21 to 0.87 ; p = 0.019 ) were less likely than those from europe to agree that in an isolated vertical fracture of the sacrum , the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen , as were surgeons who treated > 10 sacral fractures the last year ( odds ratio = 0.51 ; 95% ci 0.29 to 0.90 ; p = 0.020 ) . being from the middle east ( p = 0.023 ) was the only variable found to affect the response to the question regarding the integrity of the l5s1 , and no variables independently affected the surgeons ' response to the question pertaining to nondisplaced u - type fractures . ao region , specialty , years of clinical practice , and number of traumatic sacral fractures treated in the previous year were regarded as independent variables , and each response to the survey questions was the dependent variable . statistical significance . the results of this study demonstrate that even in the controversial aspects of sacral fractures , there is a broad global consensus on how a classification should approach these topics . although there was some variability in response based on region , experience , and specialty , the vast majority of all groups of surgeons agreed that there is an increase in severity as fractures move from transverse to vertical to fractures that result in spinopelvic instability . interestingly , surgeons from north america were most likely to disagree with the statement that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen despite the fact that the seminal study identifying the low risk of neurologic injuries with isolated vertical fractures medial to the foramen was performed in north america.15 additionally , although the majority of surgeons from the middle east agreed with all of the questions asked , they had a significantly different response to all of the questions except the question pertaining to u - type sacral fractures . finally , although surgeon specialty did appear to significantly affect the results in the univariable analysis , this variability was not significant in the multivariable regression analysis . the results of this survey suggest that the development of a comprehensive sacral injury classification system that may be acceptable to surgeons worldwide is possible . the controversies investigated in this study are in large part a result of the inadequacy of the current classifications . currently , the most commonly used sacral classification is the denis classification , which divides factures of the sacrum based on their location , irrespective of their orientation . zone 1 injuries are lateral to the foramen , and they account for 50% of all sacral fractures . because an isolated zone 1 injury is completely lateral to the foramen , the rate of neurologic injury is low ( 6% ) . zone 2 injuries are fractures that involve the sacral foramen but do not extend medial to the foramen ; they account for approximately one third of all sacral fractures and are associated with a neurologic injury ( most commonly an l5 , s1 , or s2 nerve root injury ) in 28% of cases ; finally , zone 3 injuries are all sacral fractures that have extension medial to the foramen ( regardless of orientation ) , and therefore these fractures involve the spinal canal . these injuries are the most rare , but may result in a neurologic injury in more than 50% of cases.4 although the denis classification is commonly used , it drastically oversimplifies sacral fractures , which has made meaningful interpretation of the existing literature on sacral fractures difficult . this difficulty is most clearly seen in denis zone 3 injuries , as these injuries may be relatively benign isolated vertical fractures or highly unstable u - type fractures . bellabarba et al reported on 10 vertical fractures medial to the foramen without a transverse component , and none of the patients in this case series had an associated neurologic injury.15 comparatively , roy - camille et al,6 with further modifications proposed by strange - vognsen and lebech,7 proposed a separate classification for zone 3 u - type fractures . these injuries are highly unstable injuries that are associated with bilateral spinopelvic instability , and they have a high rate of neurologic injury . despite the differences in presentation and appropriate treatment between the injuries described by bellabarba et al and roy - camille et al , all of these injuries are still classified as zone 3 injuries in the denis classification.4 6 7 15 the failure of the existing classifications to clearly differentiate sacral fractures has made an evidence - based algorithm for the treatment of these injuries challenging . the results of the current study suggest that there is a broad global consensus among trauma and spine surgeons on how controversial aspects of sacral fractures should be classified . only with a comprehensive and reproducible sacral fracture classification one of the most important findings of this study is that 77.8% of respondents agree that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen . because the denis classification is widely used , the authors of the current study did not anticipate such an overwhelming percent of the global community would agree that the risk of neurologic injury is highest in a vertical fracture through the foramen and lowest in a vertical fracture medial to the foramen . these findings suggest that changing the order of vertical sacral fractures in a new classification will likely be met with broad acceptance . first it is survey of surgeons , and the responses given to a survey are not always consistent with a surgeon 's actual behavior . so despite the fact that there is a global consensus on how controversial areas of sacral injuries should be handled in a new classification system , this consensus does not ensure acceptance of a new classification . additionally , although this survey had over 600 responses , the overall response rate was still very low ( 4.4% ) . furthermore , this study only sought input on areas of sacral fractures that the aospine trauma knowledge forum thought were controversial , and undoubtedly there may be other controversial aspects of sacral injuries . finally , as this survey was only sent to members of aospine and aotrauma , the results are representative of the worldwide ao community . because sacral injuries are complex injuries that require a multidisciplinary treatment approach , a comprehensive and universally accepted classification system is critical to ensure accurate communication between the treating physicians . this study establishes the most accepted manner in which controversial areas in the injury patterns should be addressed in a new sacral fracture classification .

study design survey study . objective to determine the global perspective on controversial aspects of sacral fracture classifications . methods while developing the aospine sacral injury classification system , a survey was sent to all members of aospine and aotrauma . the survey asked four yes - or - no questions to help determine the best way to handle controversial aspects of sacral fractures in future classifications . chi - square tests were initially used to compare surgeons ' answers to the four key questions of the survey , and then the data was modeled through multivariable logistic regression analysis . results a total of 474 surgeons answered all questions in the survey . overall 86.9% of respondents felt that the proposed hierarchical nature of injuries was appropriate , and 77.8% of respondents agreed that that the risk of neurologic injury is highest in a vertical fracture through the foramen . almost 80% of respondents felt that the separation of injuries based on the integrity of l5s1 facet was appropriate , and 83.8% of surgeons agreed that a nondisplaced sacral u fracture is a clinically relevant entity . conclusion this study determines the global perspective on controversial areas in the injury patterns of sacral fractures and demonstrates that the development of a comprehensive and universally accepted sacral classification is possible .

Introduction Methods Results Discussion Conclusion Supplementary Material

a 70-year - old man complained of a calloused right hemiscrotum for three weeks and pain for 24 h. in the previous year , the patient had tongue cancer that required surgical removal . examination of the patient s social history revealed that he had retired 10 years previously and had since lived with his wife . a detailed medical history revealed that he did not have an apparent dietary or travel history that indicated exposure to l monocytogenes . however , the patient did report that he consumed sashimi ( raw fish ) daily . the patient presented with a blood pressure of 123/77 mmhg , a heart rate of 81 beats / min , a temperature of 36.4c , and a conscious and alert state . on examination , erythema and blackish discolouration of the skin around the right buttock toward the back of the scrotum with a foul odour he immediately underwent surgical debridement of the necrotic tissue , suprapubic cystostomy and resection of an incidentally identified rectal tumour ( figure 1 ) . blood cultures were performed , and intravenous antimicrobial treatment was administered empirically using meropenem ( 1 g every 12 h ) , clindamycin ( 600 mg every 8 h ) and vancomycin ( 15 mg / kg of body weight every 12 h ) . gram stain of necrotic tissue specimens obtained during surgery demonstrated dominance of gram - positive rods , and the blood culture and the necrotic tissue specimens yielded l monocytogenes . species identification of l monocytogenes was performed using the microscan walkaway 96si pc3.1j panel ( siemens healthcare diagnostics , usa ) . the serotype of the isolate was assessed using commercial listeria antisera ( denka seiken , japan ) according to the manufacturer s instructions and was determined to be 4b . the treatment was subsequently switched to ampicillin / sulbactam at a dose of 3 g intravenously every 4 h. the antimicrobial treatment regimen was continued for a period of 21 days . in addition , the rectal tumour was identified as an adenocarcinoma , and the patient consequently underwent stoma and abdominoperineal resection along with d2 lymph node dissection . the incidence of l monocytogenes infection is much lower in japan than in western europe and north america . a questionnaire - based nationwide surveillance of hospitals in japan estimated the incidence of l monocytogenes infection to be 0.65 cases per 1,000,000 individuals ( 1 ) . however , the incidence was 2.9 cases per 1,000,000 individuals in the united states ( 2 ) , and 6.3 cases per 1,000,000 individuals in 2006 in europe ( 3 ) . it is speculated that l monocytogenes is primarily transmitted vertically or feco - orally through the consumption of contaminated foods including processed meats , unpasteurized milk and soft cheeses . however , the patient in the current case lacked a diet or travel history indicative of l monocytogenes exposure . furthermore , older age and immunosuppression due to an associated rectal adenocarcinoma were considered to be possible risk factors . there are no published reports of l monocytogenes causing necrotizing skin and soft tissue infection . in our case , fournier s gangrene was possibly caused by undiagnosed rectal adenocarcinoma and l monocytogenes invasion from the tumour , similar to a previously reported case ( 4 ) . further investigation of colorectal lesions as a portal for entry of l monocytogenes is , therefore , warranted . individuals of japanese descent often consume a unique diet comprising large quantities of raw ready - to - eat ( rrte ) seafood , including sashimi and sushi . examination of rrte seafood in japan revealed that raw minced tuna , which is a common appetizer in japan , and fish roe products are frequently contaminated with l monocytogenes ( 5.7% to 12.1% of the time ) ( 5 ) . the rrte seafood has been reported to be relatively safe from contamination as long as it is consumed immediately after purchase . however , bacterial cell number will rapidly increase , especially when rrte seafood is not properly maintained under refrigeration ( 5 ) . in japan , elderly individuals generally do not eat large quantities of food and the japanese culture is careful not to let food waste ( a practice referred to as mottainai ) . therefore , elderly individuals living alone or with their partner will often store foods in a refrigerator for extended periods of time and eat them slowly . furthermore , frequent opening and closing of the refrigerator door may increase the temperature inside the refrigerator above the recommended temperature for extended periods of time . therefore , it is quite conceivable that consumption of rrte seafood , such as sashimi , may result in ingestion of l monocytogenes , resulting in infection . the united states food and drug administration has established preventive regulatory guidelines for raw seafood , such as rte foods , that can support l monocytogenes growth ( 6 ) , and these guidelines have been effective in reducing the incidence of l monocytogenes infection ( 7 ) . however , in japan , regulation of foods linked with l monocytogenes infection is limited to dairy products and rte meat products . it should also be noted that there are no official statistics regarding the prevalence of l monocytogenes infection from rrte seafood in japan owing to the lack of a mandatory reporting system . therefore , we propose that such a system be established for the control of l monocytogenes infection in japan . in the present case , the l monocytogenes isolate was determined to be of serotype 4b , which is the most frequently occurring form , accounting for 64% of cases in the united kingdom ( 8) and 59.9% of cases in japan ( 9 ) . interestingly , the 4b serotype is not the leading serotype isolated from samples in western countries . in addition , serotype 4b has been found to account for only 10.2% of reported cases associated with commercial foods in japan . in a 2002 review , kathariou ( 10 ) suggested that such a discrepancy between food incidence and prevalence of illness may exist because serotype 4b is more virulent to humans than any of the other known serotypes . the strain isolated from blood culture was susceptible to penicillin g ( minimum inhibitory concentration [ mic ] 0.25 mg / ml using e - test [ biomrieux , france ] ) and ampicillin ( mic 0.25 mg / ml ) according to the breakpoints of clinical laboratory standards institute m45-a2 . l monocytogenes isolated from humans has been rarely reported to be resistant to penicillins ( 11 ) . however , most ( 92% ) l monocytogenes isolated from dairy farms is resistant to ampicillin ( mic 2 mg / ml ) ( 12 ) . furthermore , statistical analysis showed an increase in mics for aminopenicillins in l monocytogenes isolated from humans , which was not associated with clinical failure ( 11 ) . this could be explained by increased use of beta - lactams in human and animals , particularly in recent years . this indicates that continuous surveillance of the susceptibility of l monocytogenes to antibiotics may be useful . to our knowledge , this is the first report of fournier s gangrene caused by l monocytogenes infection . old age and immunodeficiency due to undiagnosed rectal adenocarcinoma may have supported the direct invasion of l monocytogenes from the rectal adenocarcinoma , thereby causing infection . the consumption of rrte seafood should be considered to be a possible risk factor associated with l monocytogenes infection . moreover , the findings suggest that l monocytogenes should be considered to be a possible pathogen when patients with known risk factors for listeriosis present with skin and soft tissue infection .

fournier s gangrene is a necrotizing infection of the scrotal region , and is typically caused by exterobacteriaceae and anaerobic bacteria . this report describes a case involving a 70-year - old man of japanese descent who developed fournier s gangrene that was found to be caused by listeria monocytogenes . the patient s unique risk factors are discussed .

CASE PRESENTATION DISCUSSION

direct restoration is the most commonly used technique for both anterior and posterior teeth . however , with direct restoration , excessive polymerization shrinkage increases the stress at the composite - tooth interface and can compromise the integrity of the bond . in large posterior cavities , especially in those with cervical margins located in dentin , excessive shrinkage can produce marginal defects and gaps that promote microleakage and result in marginal discoloration , post - operative sensitivity , secondary caries and pulpal irritation . when some mistakes in placement and finishing are added to these material problems , resulting marginal leakage and poor anatomical form and proximal contacts can reduce restoration longevity . recent developments in resin - based composite resins have made it possible to fabricate esthetic indirect adhesive restorations that aim to overcome the shortcomings of direct composite resin restorations , such as polymerization shrinkage and inadequate degree of conversion . the high percentage by volume of inorganic fillers in indirect restorative material when compared with composite resins used in direct restorations results in improved mechanical and physical properties . however , indirect restoration requires more dentin exposure than direct restoration and thus increases dentin sensitivity . previous studies have shown the clinical performance of indirect composite restorations to be significantly affected by both the bonding of the luting agent to the tooth and the restorative material and the surface treatment prior to bonding . in their systematic review of materials , van meerbeek et al . whereas , etch and rinse systems require dentin to be acid - etched , rinsed and dried before the bonding agent is applied , self - etching bonding systems require no dentin pre - conditioning . the simultaneous etching and resin infiltration that occurs with self - etching systems provides the important advantage of reduced technical sensitivity . whereas , shortall et al . reported indirect adhesive restoration to be a better alternative to direct composite restoration for larger cavities , wakiaga et al . reported no reliable evidence of either a direct or indirect type of veneer restoration being superior in terms of restoration longevity . however , recent advances in indirect restoration technology may improve the clinical life of indirect restorations over direct restorations . , schaumburg , il , usa ) claim that these products show great success for occlusal restorations . the aim of this study was to test the null hypothesis that restoration preparation technique ( direct / indirect ) , surface conditioning ( etch and rinse / self - etch ) and curing of bonding agents ( light cure / dual cure ) had no effect on the retention strength of resin composite used with seven different adhesive systems . this study was conducted using 112 newly extracted non - carious , human molar teeth obtained according to protocols approved by the relevant institutional review board ( atatrk university faculty of dentistry ) . tissue remnants and debris were removed and teeth were disinfected in 1% thymol and stored in distilled water until required for use . specimens were decoronated and embedded in self - curing acrylic resin in 3 cm diameter teflon molds . following polymerization , teeth were sectioned using a water - cooled saw ( struers minitom , struers , copenhagen , denmark ) parallel to the occlusal surface to expose mild - coronal dentin . standardized dentin surfaces were created by polishing specimens with waterproof polishing papers ( # 600 , # 800 # 1200 sic ) . polished teeth were randomly divided into two groups according to restoration technique ( direct restoration vs. indirect restoration ) and subdivided into seven subgroups ( n = 8) according to surface conditioning techniques used . direct restorations were constructed using a hybrid resin composite valux plus ( 3 m dental products , st paul , mn , usa ) and the indirect restorations were made using tescera atl system ( bisco inc . dentin bonding agents included four etch and rinse adhesives ( scotchbond multi - purpose plus [ sbmp ] , all - bond 3 [ ab ] , adper single bond [ sb ] and prime bond nt [ pbnt ] ) and three self - etch adhesives ( clearfil liner bond [ lb ] , futurabond dc [ dc ] , g bond [ gb ] ) . of these , sbmp , sb and gb are polymerized by light - curing , whereas ab , pbnt , lb and dc are polymerized by dual - curing . bonding systems , surface conditioning , curing type and manufacturer composite resin cylinders were built up on the dentin surfaces using a bonding jig ( ultradent products inc . , south jordon , ut and usa ) and an incremental technique . excess restorative material was carefully removed using a sharp explorer and the cylinders were cured for 20 s using a light - emitting diode unit ( led , elipar free light ii 3 m - espe , st . composite resin cylinders were constructed in the same size with bonding jig 's space used in direct technique . the specimens were light - cured and heat - cured using the tescera atl light box and heat box , respectively , in line with the manufacturer 's recommendations . following polymerization , cylinder surfaces were sandblasted with 50 m aluminum oxide powders ( microetcher , danville engineering , san ramon , ca ) and rinsed with water . indirect cylinders were cemented to the dentin surfaces using dual - curing luting cement duo - link ( bisco inc . , schaumburg , illinois , usa ) under a constant pressure of 5 kgf and then light - cured for 60 s using led . all specimens were stored for 24 h at 37c and 100% relative humidity and then subjected to thermocycling ( 5/55c , 1,000 cycles , 30 s dwell time ) . shear bond - strength testing was performed using a universal testing machine ( instron corporation , canton , ma , usa ) at a crosshead speed of 0.5 mm / min . fractured surfaces were examined under a stereomicroscope ( sz - tp olympus , japan ) at 20 magnification and failure modes were classified as either adhesive ( failure at the dentin / composite interface ) , cohesive ( failure within the resin composite or dentin ) or mixed ( partial adhesive / partial cohesive fracture ) . furthermore , two samples from each subgroup were evaluated under scanning electron microscopy to see the failing surfaces [ figure 1 ] . scanning electron microscopy evaluation of dentin surfaces after shear testing : ( a1 , a2 ) 90 and 1500 magnification of a mix failing mode respectively in group indirect + all bond 3 ; ( b1 , b2 ) 130 and 1500 magnification of an adhesive failing mode respectively in group direct + prime and bond nt ; ( c1 , c2 ) 90 and 2500 magnification of a mix failing mode respectively in group ndirect + scotch bond multipurpose plus ; a : adhesive resin ; d : dentin ; c : composite resin mean bond strengths for direct and indirect restorations were calculated for each subgroup ( n = 8) and the data were pooled according to restoration technique ( direct / indirect ) , surface conditioning ( etch and rinse / self - etch ) and curing of bonding agents ( light cure / dual cure ) . independent sample t - tests were used to compare mean bond strengths of pooled data according to restoration technique , surface conditioning and curing of bonding agents . anova and tukey 's test were used to compare mean bond strengths of each dentin bonding agents ; and the chi - square test was used to analyze the distribution of fracture modes . all statistical analysis was conducted with the level of significance set at p = 0.05 . composite resin cylinders were built up on the dentin surfaces using a bonding jig ( ultradent products inc . , excess restorative material was carefully removed using a sharp explorer and the cylinders were cured for 20 s using a light - emitting diode unit ( led , elipar free light ii 3 m - espe , st . composite resin cylinders were constructed in the same size with bonding jig 's space used in direct technique . the specimens were light - cured and heat - cured using the tescera atl light box and heat box , respectively , in line with the manufacturer 's recommendations . following polymerization , cylinder surfaces were sandblasted with 50 m aluminum oxide powders ( microetcher , danville engineering , san ramon , ca ) and rinsed with water . adhesives were applied on the dentine surfaces according to the manufacturers instructions . indirect cylinders were cemented to the dentin surfaces using dual - curing luting cement duo - link ( bisco inc . , schaumburg , illinois , usa ) under a constant pressure of 5 kgf and then light - cured for 60 s using led . all specimens were stored for 24 h at 37c and 100% relative humidity and then subjected to thermocycling ( 5/55c , 1,000 cycles , 30 s dwell time ) . shear bond - strength testing was performed using a universal testing machine ( instron corporation , canton , ma , usa ) at a crosshead speed of 0.5 mm / min . fractured surfaces were examined under a stereomicroscope ( sz - tp olympus , japan ) at 20 magnification and failure modes were classified as either adhesive ( failure at the dentin / composite interface ) , cohesive ( failure within the resin composite or dentin ) or mixed ( partial adhesive / partial cohesive fracture ) . furthermore , two samples from each subgroup were evaluated under scanning electron microscopy to see the failing surfaces [ figure 1 ] . scanning electron microscopy evaluation of dentin surfaces after shear testing : ( a1 , a2 ) 90 and 1500 magnification of a mix failing mode respectively in group indirect + all bond 3 ; ( b1 , b2 ) 130 and 1500 magnification of an adhesive failing mode respectively in group direct + prime and bond nt ; ( c1 , c2 ) 90 and 2500 magnification of a mix failing mode respectively in group ndirect + scotch bond multipurpose plus ; a : adhesive resin ; d : dentin ; c : composite resin mean bond strengths for direct and indirect restorations were calculated for each subgroup ( n = 8) and the data were pooled according to restoration technique ( direct / indirect ) , surface conditioning ( etch and rinse / self - etch ) and curing of bonding agents ( light cure / dual cure ) . independent sample t - tests were used to compare mean bond strengths of pooled data according to restoration technique , surface conditioning and curing of bonding agents . anova and tukey 's test were used to compare mean bond strengths of each dentin bonding agents ; and the chi - square test was used to analyze the distribution of fracture modes . all statistical analysis was conducted with the level of significance set at p = 0.05 . mean shear bond strengths by restoration technique , surface conditioning and curing of bonding agents are given in table 2 . according to t - test , direct restorations were found to have significantly higher shear bond strengths than indirect restorations ( p < 0.05 ) ; etch and rinse surface conditioning resulted in significantly higher shear bond strengths than self - etching ( p < 0.05 ) ; and dual - cured bonding agents resulted in significantly higher shear bond strengths than light - cured bonding agents ( p < 0.05 ) . shear bond strength values ( mean and standard deviation ) of composite resin restorations by restoration technique , surface conditioning and curing of bonding agent mean values of shear bond strength and standard deviations by restoration technique and bonding agent are given in table 3 . mean values ranged from a high of 26.50 6.05 mpa ( direct restoration / pbnt ) to a low of 16.96 4.09 mpa ( direct restoration / gb ) . for direct restorations , pbnt had the highest bond strength ( 26.50 6.05 mpa ) and gb had the lowest bond strength ( 16.96 4.09 mpa ) . bond strengths of pbnt , ab and sbmp were significantly higher than bond strengths of gb . no other significant differences in bond strengths were observed among the other subgroups for direct restorations . shear bond strength values ( mean and standard deviation ) for restoration technique ( direct / indirect ) and bonding agent for indirect restorations , dc had the highest bond strength ( 26.15 5.59 mpa ) and gb had the lowest bond strength ( 18.07 3.41 ) and the difference between them was statistically significant ( p < 0.05 ) . no other significant differences in bond strengths were observed among the other sub - groups for indirect restorations . overall , direct restorations with sbmp and pbnt showed significantly higher mean shear bond strengths than indirect restorations with ab , sb , lb and gb and direct restorations with ab showed significantly higher mean shear bond strengths than both direct and indirect restorations with gb . indirect restorations with pbnt also showed significantly higher mean shear bond strengths than direct restorations with gb . for all groups , adhesive failure was the most common mode of fracture and no significant differences in fracture modes were observed among the groups ( p > 0.05 ) . according to the findings of this study , the null hypothesis stating that the shear bond strength of composite resins is not affected by restoration preparation technique , surface conditioning or curing of the bonding agent must be rejected . adhesive dentistry involves the physical bonding of restorative materials to dental substrates in order to return esthetics and functioning to previously damaged teeth . since the introduction of acid - etching into the field of dentistry , various adhesive methods have been developed to bond composites to tooth structure . given the importance of bond strength between the adhesive system and the dentin surface , this subject continues to remain a topic of extensive research . studies have shown that indirect composite restorations , introduced in the 1990 's , exhibit better clinical performance than direct restorations in terms of proximal contact , occlusal anatomy and marginal adaptation . with indirect restorations , except for a thin layer of high - flow composite resin ( so - called resin cement ) used to lute the restoration to the tooth surface , however , it is still possible for polymerization stress to occur during the curing of the resin cement , causing a disruption between the restoration and the cavity walls that will subsequently lead to marginal leakage , particularly if the margins are located in dentin . douglas et al . concluded that the indirect method of placement of composite restorations offers considerable improvement in microleakage performance , particularly on the dentin - restorative interface . however , our finding that direct restoration produces higher shear bond strengths than indirect restoration is in conflict with douglas et al . , given that microleakage is directly related to the strength of the bond between the dentin surface and the adhesive system / resin cement . in clinical practice , thus , the clinical life of indirect restoration can be affected by the physical properties of the restorative material . in the present study , indirect restorations were produced using the tescera atl system , according to its manufacturers , which offers improvements in terms of durability and appearance . the present study found that the surface conditioning had a significant effect on the shear bond strength of resin composite to dentin , etch and rinse adhesives exhibited higher bond strength than self - etch adhesives . this finding is in line with a previous study that showed etch and rinse systems resulted in higher bond strengths than self - etch systems , but conflicts with other studies that showed no differences between self - etch and total - etch adhesives in terms of bonding to dentin . in our study , the bond strength of direct restorations was significantly lower when gb was used as an adhesive in comparison to all the other adhesives tested . these findings may be due to the adhesives content and the surface - etching technique of the bonding systems . it is possible that dual - cure bonding systems can significantly increase the retention of indirect restorations . the present study found dual - cure bonding agents to have higher shear bond strength than light - cure bonding systems . this is in line with previous studies that showed both dual - cure bonding agents and light - cured bonding agents have sufficient bond strength to tooth structures . further investigation is needed into the various adhesive methods used to lute indirect restorations as there seems to be no current consensus in the literature regarding which technique can best improve adhesive strength . in terms of failure mode , the present study found the majority of failures to be adhesive failures at the resin cement - restoration interface . in contrast to a recent study that found a higher rate of adhesive failures at the resin cement / veneer interface for indirect restorations , our study showed no differences in failure modes between direct and indirect restorations . despite improvements in adhesive technology used for luting indirect restorations , the results of the present study indicated direct restoration to be a more reliable method than indirect restoration . although etch and rinse bonding systems showed higher shear bond strength to dentin than self - etch systems , both systems can be safely used for the adhesion of direct as well as indirect restorations .

objective : the aim of this study was to test the null hypothesis that different surface conditioning ( etch and rinse and self - etch ) and curing techniques ( light cure / dual cure ) had no effect on the shear bond strength of direct and indirect composite inlays.materials and methods : a total of 112 extracted human molar teeth were horizontally sectioned and randomly divided into two groups according to restoration technique ( direct and indirect restorations ) . each group was further subdivided into seven subgroups ( n = 8) according to bonding agent ( etch and rinse adhesives scotchbond multi - purpose plus , all - bond 3 , adper single bond and prime bond nt ; and self - etch adhesives clearfil liner bond , futurabond dc and g bond ) . indirect composites were cemented to dentin surfaces using dual - curing luting cement . shear bond strength of specimens was tested using a universal testing machine . two samples from each subgroup were evaluated under scanning electron microscopy to see the failing modes . data was analyzed using independent sample t - tests and tukey 's tests.results:surface conditioning and curing of bonding agents were all found to have significant effects on shear bond strength ( p < 0.05 ) of both direct and indirect composite inlays . with direct restoration , etch and rinse systems and dual - cured bonding agents yielded higher bond strengths than indirect restoration , self - etch systems and light - cured bonding agents.conclusions:the results of the present study indicated that direct restoration to be a more reliable method than indirect restoration . although etch and rinse bonding systems showed higher shear bond strength to dentin than self - etch systems , both systems can be safely used for the adhesion of direct as well as indirect restorations .

INTRODUCTION MATERIALS AND METHODS Direct technique Indirect technique RESULTS DISCUSSION CONCLUSIONS

especially c jejuni and c coli , are considered to be amongst the most prevalent foodborne pathogens associated with sporadic diarrhoea in humans ( engberg et al . , 2001 ; colonise the intestines of food animals and they can contaminate meat during slaughter or post - slaughter processing ( hermans et al . , 2011 ; mackiw et al . , although campylobacter infections are usually self - limiting and do not require antibiotic treatment , in some cases such as prolonged enteritis and septicaemia , antimicrobial treatment is needed . macrolides and fluoroquinolones are the drugs of choice in treatment of human campylobacteriosis ( van looveren et al . , 2001 ; guevremont et al . , 2006 ; moore et al . , 2006 ) , however emergence of resistance to these agents has prompted worries related to their use ( moore et al . , 2005 ) . in 2009 , zanoni and colleagues described a new campylobacter species isolated from rabbit caecal contents named c. cuniculorum . so far there are no data on antimicrobial susceptibility in this novel campylobacter species , so the aim of this study was to define for the first time the antimicrobial susceptibility in c. cuniculorum isolated from rabbits for meat . a selection of 29 c. cuniculorum isolates from a total of 29 epidemiologically non - correlated rabbits farms during a previous study by revez et al . ( 2013 ) was used for this study : one strain for each farm was randomly selected for the evaluation of antibiotics susceptibility by using agar dilution method . the tested isolates were collected from april 2007 to november 2008 from 29 farms , 27 ( 18 intensive and 9 rural ) were located in 7 different italian regions while 2 farms ( rural ) were located in portugal ( revez et al . , 2013 ) . the number of mares in the intensive farms ranged from 300 to 700 subjects ; while in rural ones they ranged from 5 to 15 . the minimum inhibitory concentration ( mic ) value of ampicillin , ciprofloxacin , chloramphenicol , erythromycin , gentamicin , nalidixic acid , enrofloxacin and tetracycline was determined using a modified agar dilution method for c. jejuni and related species described by the clinical and laboratory standards institute ( wayne , pa , usa ; clsi , 2008 ) in order to be applied to the study of antimicrobial resistance of c. cuniculorum . the method was modified as follows : i ) the base medium was nutrient broth n.2 ( oxoid , basingstoke , uk ) supplemented with 1.5% bacto agar ( difco - bd , milan , italy ) and 5% defibrinated sheep blood ; ii ) the plates were incubated at 37c1 under microaerobic atmosphere with hydrogen for 72 h. these changes have been introduced because several isolates of c. cuniculorum did not grow on mueller hinton agar with 5% defibrinated sheep blood ; moreover , the reading times were increased since visible growth does not appear before 72 h of incubation for this species . all antimicrobial agents were purchased from sigma - aldrich ( saint louis , mo , usa ) and the antibiotic concentrations ranged from 0.015 to 128 g ml . c jejuni atcc 33560 , escherichia coli atcc 25922 , and staphylococcus aureus atcc 29213 were used as a quality control strains . in this were those used by the national antimicrobial resistance monitoring system ( atlanta , ga , usa ; narms ) as reported in the us centers for disease control narms annual report ( 2010 ) for campylobacter spp . for ampicillin , erythromycin and gentamicin we adopted the breakpoints described by clsi ( 2008 ) for campylobacter spp . since a standardised mic breakpoint for enrofloxacin is not available for campylobacter spp . the following resistance breakpoints were used : ampicillin32 , chloramphenicol 32 , ciprofloxacin4 , enrofloxacin4 , erythromycin32 , gentamicin8 , nalidixic acid64 , and tetracycline 16 . the results of mic testing for each single isolate are reported in table 1 , while the distribution of mic values of the 29 isolates tested is shown in table 2 . a monomodal distribution ( with one distinct peak ) for the mics was found for all the antibiotics tested except for ciprofloxacin , enrofloxacin and erythromycin , which showed a bimodal ( with two distinct peaks ) appearance with a second peak at 4 - 128 , 1 - 16 and 128-128 g ml , respectively . the bimodal distribution means that there are two distinct microbial populations , showing a different behaviour to antimicrobials , suggesting an acquired resistance . noteworthy 15 and 13 isolates , all obtained from rabbits reared in intensive farms , showed a resistance to erythromycin and enrofloxacin characterised by a bimodal frequency with a high level of mic in the second peak . ( 2013 ) , investigating the occurrence of e - pro - teobacteria in caecal contents of rabbits , isolated c. cuniculorum in 83 out of 87 animals tested , in a large number of colonies , suggesting that this microorganism , when present , colonises the caecum at a high concentration ( revez et al . , 2013 ) . the results of this study , even if not statistically analysed , show high resistant level in c. cuniculorum isolated from rabbits reared in intensive farm ; indeed , all the 18 isolates from intensive farms resulted resistant to two antibiotics at least . on the contrary , out of the 11 isolates from rural farms , only one resulted resistant to only one antibiotic . these data suggest that modern food animal production managements contribute to produce favourable conditions for the emergence and spread of antibiotic resistant bacteria due to the larger use of antimicrobial agents to control infections . moreover , trends in antimicrobial resistance have shown a clear association between use of antibiotics in the veterinary industry and resistant isolates of campylobacter spp . in humans ( alfredson and korolick , 2007 ; angulo et al . , 2004 ) . nowadays , there is no information on the pathogenic role of this new campylobacter species , but the importance of antibiotic resistances that could be transmitted to other pathogen campylobacter species may represent a risk of human concerns . resistance to fluoroquinolones and macrolides however , the resistance to tetracycline show the potential for resistance transmission to other campylobacter species ( aarestrup and engberg , 2001 ) . in the present study , fluoroquinolone and macrolide showed a bimodal distribution suggesting an acquired resistance due to a gene mutation . fluoroquinolones and macrolides are the antimicrobials chosen for the treatment of campylobacteriosis ; in campylobacter spp . , fluoroquinolone resistance seems to be due to mutations in the gyra gene encoding part of the gyra subunit of dna gyrase ( aarestrup and engberg , 2001 ; alfredson and korolik , 2007 ) . relatively to macrolide resistance in campylobacter species , modification of the target , represented by point mutation or methylation of 23s rrna gene , seems to be the main mechanism involved . as far as c. cuniculorum is concerned , in - depth studies should be performed to clarify molecular mechanisms by sequencing the involved genes in c. cuniculorum isolates and performing comparisons of these sequences in sensible and resistant isolates . for all the other antibiotics tested , a monomodal distribution of mic values was observed and , on the basis of the clinical breakpoints , we may assume that all c. cuniculorum isolates are sensitive to chloramphenicol and ampicillin . sixteen out of the 29 tested isolates resulted resistant to nalidixic acid and tetracycline . regarding the nalidixic acid , 12 out of 16 isolates resistant to nalidixic acid were resistant to ciprofloxacin too , so , the quinolone resistance - determining region of gyra gene could be involved in the acquisition of this resistance . with regard to tetracycline resistance , it is found to be located in c jejuni and c coli on a self - transmissible plasmid encoding a ribosomal protection protein , designated as tet ( o ) , thus suggesting a potential role of c. cuniculorum in passing this resistance to other campylobacter species . in conclusion , for the first time this study shows data on c. cuniculorum antimicrobial susceptibility , suggesting a probable higher risk of antibiotic resistance in rabbits reared in intensive farms than those reared in rural farms . the evidence of tetracycline resistance in c. cuniculorum that could be transmitted to other human pathogen campylobacter species may represent a risk for human health .

the present study aimed to investigate the antimicrobial susceptibility in campylobacter cuniculorum . to do so , 29 isolates from rabbits reared in 18 intensive and 11 rural farms not epidemiologically correlated were tested . minimum inhibitory concentration of 8 antimicrobial agents was determined using the agar dilution method recommended by the clinical and laboratory standards institute ( wayne , pa , usa ) , modified for what supplements in the base medium and incubation conditions concern for c. cuniculorum isolates . the isolates obtained from rural farming resulted susceptible to all the antimicrobial agents tested , with the exception of one isolate resistant to nalidixic acid . all the isolates obtained from intensively farmed rabbits were sensitive to chloramphenicol and ampicillin ; 16 isolates were resistant to tetracycline ; 15 to nalidixic acid and erythromycin ; 13 and 10 isolates to ciprofloxacin and enrofloxacin , respectively ; and only 1 to gentamicin . the resistance of several isolates to macrolides and fluoroquinolones , which are the drugs of choice in treatment of human campylobacteriosis , could pose a risk to human health if a pathogenic role of c. cuniculorum was demonstrated .

Introduction Materials and Methods Results Discussion Conclusions

alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) are the most common age - related neurodegenerative diseases , while other less common , albeit devastating , neurodegenerative diseases include huntington 's disease ( hd ) , amyotrophic lateral sclerosis ( als ) , prion diseases , and frontotemporal dementia ( ftd ) . although the specific protein aggregates and selective cellular vulnerabilities differ , shared disease mechanisms are increasingly apparent among neurodegenerative diseases and next to aberrant protein aggregation also include anatomically selective cell - to - cell propagation . major themes of research on these diseases have included therapeutic neurotransmitter replacement , most successful with dopamine for pd , elucidating the biology of aberrant protein misfolding , and trying to understand how ageing promotes the development of these diseases . more recently , synapses have moved more to the center of research on these diseases [ 1 , 2 ] . neurites ( axons and dendrites ) and synapses are a unique feature of neurons and play fundamental roles in brain function . furthermore , the aggregation - prone proteins linked pathologically and genetically to neurodegenerative diseases are normally present particularly at synapses . for example , the pd - linked protein -synuclein is known to normally reside primarily in presynaptic compartments [ 3 , 4 ] , although , as the name indicates , a nuclear role also characterizes this protein that aggregates in the distinctive cytoplasmic lewy bodies and lewy neurites that characterize pd and the related lewy body dementia ( lbd ) . an important role at synapses for the ad - linked -amyloid ( a ) , and the amyloid precursor protein ( app ) from which it is derived , is also increasingly becoming apparent ( figure 1 ) . app is transported down axons and dendrites to synapses [ 2 , 5 ] , where the proteases that generate a are also localized . the precise processing and trafficking of app and a in pre- versus postsynaptic compartments and how these relate to the mechanism of synaptic damage in ad remain to be elucidated . evidence supports that a accumulation in synapses alters synaptic function by altering important synaptic proteins and receptors . a major hurdle for research on neurodegenerative diseases has been that the normal physiological roles and functions of the aggregation - prone proteins have been difficult to ascertain . a potential reason for this could very well be that synapses are so complex and are only gradually being elucidated . although mouse knockout studies do not support that loss of function of the disease - linked proteins is the salient issue in these various diseases [ 7 , 8 ] , it is nevertheless possible that their propensity to aggregate is a feature that makes these proteins normally important at synapses . it is further possible that a better understanding of the normal function of neurodegenerative - linked proteins at synapses will be important in order to uncover better therapeutic targets and devise more effective therapies for these diseases . synaptic activity and plasticity are of central importance in the brain and at synapses , and it has become clear that neurodegenerative disease - linked proteins are modulated by synaptic activation [ 9 , 10 ] . the major nonneuronal cells of the brain , the astrocytes and microglia , are also increasingly linked to synaptic function and thereby might impact the pathophysiology of these diseases that appear to initiate at synapses . modulation of synapses has also been shown to directly impact synapse damage in the brain of transgenic mouse models of neurodegenerative diseases . the endosome - lysosome system and the ubiquitin proteasome system ( ups ) play many essential roles in cells and are increasingly implicated in neurodegenerative diseases of ageing . in neurons , these systems , best known for their role in protein degradation , are also important for the normal function of synapses . the diversity of rare genetic neurodegenerative storage diseases of childhood linked to aberrant protein or lipid accumulation in the endosome - lysosome system supports the potential disease relevance of this system also in the common age - related degenerative diseases of the brain . the endosome - lysosome system is involved in many central functions , including cellular internalization , degradation , and release . in down syndrome ( ds ) , characterized by trisomy of chromosome 21 , which invariably leads to age - related ad - like pathology and dementia , abnormal endosome enlargement has long been known to precede the characteristic neuropathological amyloid plaques and tau tangles . the related autophagy system is intimately linked with the endosome - lysosome system and is important for the engulfment and degradation of larger subcellular structures , including whole organelles . autophagy is a prominent neuropathological feature of neurodegenerative diseases , where autophagic vesicles , limited by double or multilamellar membranes , accumulate in neurons and their processes . autophagic vesicles are particularly abundant in dystrophic neurites in ad brain and transgenic mouse models of ad . autophagic vesicles are thought to form from the formation of double membranes in the cytoplasm . autophagic vesicles are thought to subsequently fuse with endosomes or lysosomes followed by degradation of their contents by lysosomal proteases . although autophagy is considered a mechanism of normal cellular degradation , autophagy is rarely seen on electron micrographs of normal brain ( personal observations ) . the autophagy system is further implicated in neurodegenerative diseases of ageing , since inhibition of the mammalian target of rapamycin ( mtor ) , which is well known to induce autophagy , was found to prolong the lifespan in lower organisms and mammals . more recent studies show that pharmacological mtor inhibition by rapamycin is protective in transgenic mouse models of alzheimer 's disease [ 18 , 19 ] . a challenge for the development of treatments of neurodegenerative diseases based on mtor inhibition is that mtor is part of a central signaling system regulating many cellular functions . neurites can extend considerable distances and synapses are thus at a greater distance from their cell bodies than are components of other cells . therefore , protein degradation at synapses depends on retrograde transport via endosomes back to the cell body . late endosomes can take on some limited lysosomal functions , but compared to lysosomes they are much less efficient at degradation , particularly of aggregation prone insoluble proteins . endosomes also play important roles in the regulated delivery , recycling , and degradation of receptors at synapses that are important for synaptic plasticity . interestingly , the inner vesicles that characterize the late endosomal multivesicular body ( mvb , alternatively called multivesicular endosome ) are released as exosomes , and increasing evidence suggests that exosomes may be important in the release and propagation of neurodegenerative disease - linked proteins . for example , ad - linked a peptides were shown to normally localize and then accumulate and aggregate particularly in mvbs of dystrophic neurites and synapses of ad transgenic mouse models and human ad brains , where they associated with localized subcellular pathology , even prior to extracellular amyloid plaques [ 2325 ] . for example , mutations in parkin represent an example of a ups - linked protein ( component of a ubiquitin ligase ) that is mutated in familial forms of pd . moreover , the ups and the endosome - lysosome system are linked , since transmembrane proteins at the cell surface , including app and neurotransmitter receptors at synapses , can be ubiquitinated and routed via the endocytic pathway to the lysosomes for degradation [ 13 , 26 ] . ad - linked a normally localizes to and with ad pathogenesis preferentially accumulates at the outer limiting membranes of mvbs where the endosomal sorting complexes required for transport ( escrt ) reside , which is involved in targeting ubiquitinated transmembrane proteins for degradation . experimental evidence supports that accumulating a42 associated with mvbs impairs mvb sorting by disrupting the ups . altered regulation of synaptic protein trafficking from early aberrant protein / peptide accumulation near synapses might lead to the earliest synapse dysfunction in neurodegenerative diseases . interestingly , mutations of chmp2b , a component of escrtiii , have been linked with familial forms of ftd . although ftd , the 2nd most common cause of dementia before the age of 65 , is not genetically linked with a/app , it has been linked to familial mutations in tau , the main constituent of the other characteristic neuropathology of ad , the neurofibrillary tangles . cell - to - cell propagation of tau has recently become a hot topic in the field of neurodegenerative diseases . the endocytic pathway is also central to cholesterol and lipid uptake and trafficking in neurons , which are implicated in ad [ 30 , 31 ] and are important in injury and synapse remodeling in neurons . the lipoprotein carrier apolipoprotein e4 ( apoe4 ) polymorphism is the major genetic risk factor for typical late onset ad . cholesterol traffics via the endocytic pathway into cells , and together with the endoplasmic reticulum ( er ) and mitochondrial - er membranes , this interconnected pathway that regulates cellular cholesterol metabolism has been related to a . recent research supports that apoe , generated mainly by astrocytes , is important for regulating neuronal a [ 33 , 34 ] . interestingly , the apoe receptor , low - density lipoprotein receptor ( ldlr ) related protein ( lrp ) , is routed from the plasma membrane to the limiting membrane and then internal vesicles of mvbs and interacts with app via fe65 . as noted previously , a normally localizes to mvbs and with ad pathogenesis accumulates at mvbs , and upon release from the cell , mvb inner vesicles are then called exosomes . thus , it is conceivable that apoe biology intersects with a/app in endosomes and might even modulate propagation of secreted misfolded proteins via exosomes and/or protein degradation via the mvb - lysosome pathway . in addition , endosomes have a lower ph , which is known to promote aggregation and amyloid formation of misfolding proteins , including a and prp [ 3638 ] . finally , evidence consistent with leakage of endosome - lysosome contents by a has been reported [ 39 , 40 ] . consistent with such leakage , immunoelectron microscopy showed marked accumulation of a42 directly associated with outer membrane disruption of mvbs in ad transgenic mice . thus , multiple lines of evidence point to an important role of the endosomal - lysosomal system and in particular of endosomes at neurites and synapses in common neurodegenerative diseases of ageing . prions are the unusual proteins that are best known for their ability to propagate disease between members of a species and between different species . formerly classified as atypical slow virus diseases , this nomenclature was abandoned , since they propagate as proteins and in contrast to viruses lack nucleic acids . however , clinically and pathologically , prion diseases are most similar to common neurodegenerative diseases , such as ad . the normal role of the prion protein is also poorly understood , although it is expressed at particularly high levels in the brain and is thought to primarily localize to synapses . analogous to ad and pd , the majority of prion diseases are sporadic , without a clear infectious or genetic cause . although the term prion is widely associated with fear , based on its infectivity from highly publicized outbreaks of prion disease , prion proteins are normal proteins that can even provide protective functions [ 42 , 43 ] . thus , prions , like amyloids , should not be only viewed in a negative light , and prp likely plays important physiological roles , which potentially may be particularly relevant at synapses . the mere evolutionary presence of prp , which is conserved in mammals , as well as some deficits noted in prp deficient mice , supports a physiological role for prp . a more recent development in research on neurodegenerative diseases is the surprising realization that other disease - linked aggregation prone proteins , such as a [ 46 , 47 ] , -synuclein [ 48 , 49 ] , and tau , can also propagate in experimental systems . despite the many years of research on prion propagation , the cellular mechanism(s ) of propagation the recent surge of research on this topic particularly in ad , pd , als , and hd will undoubtedly lead to important new insights . some overlooked earlier papers have provided clues to cellular mechanisms of propagation in these diseases . for example , a literature from the 1990s showed that exogenously added extracellular ad - linked a142 peptides induced a marked upregulation of newly generated a42 within the treated cells . moreover , it was shown that extracellular a142 failed to alter synapses in the absence of app or in neurons where de novo generation of a was inhibited . similarly , depletion of endogenous prp protects against scrapie - induced prp pathogenesis [ 51 , 52 ] . while neurons generate a from app within neurons , secreted a can also be internalized by neurons . more recent evidence points to synapses as selective sites of neuron - to - neuron spread of a . one could speculate that release of exosomes might be particularly prominent near synaptic terminals , although it was estimated that only about 1% of released a was associated with exosomes . overall , the cell biology of such synapse - associated endocytic and exocytic pathways in neurons is less well understood than of other cells . a challenge in considering prion - like cell - to - cell propagation is to explain where and how the initial pathological conformation of disease - linked peptide forms and what determines the anatomical selectivity of spread by various disease - linked proteins . recent evidence supports the surprising scenario that in the setting of aberrant intracellular protein aggregation , the secretion of ad - linked a is actually impaired . in general , the release of more toxic soluble oligomers appears to be at much lower levels than those of monomeric proteins ; for example , the concentration of a oligomers is about 1% of the monomeric forms in cerebrospinal fluid . thus , it follows that if abnormal aggregation can initially arise spontaneously in one anatomical region , it might be possible that other vulnerable cells might also have de novo appearance of abnormal aggregated conformations rather than a prior requirement for propagation from other cells . it is also possible that despite the reductions in the normal secretion of monomers , cell - to - cell transmission of more aggregated oligomers may be the driving force in disease propagation , which even at low levels might still act as the nidus to drive further aggregation in the recipient cells . there is no convincing evidence that proteins linked to the common age - related neurodegenerative diseases can spread from person to person as was recently highlighted in a study of patients who had received growth hormone . thus , neuroscientists need to contribute to reducing the excessively alarmist view that is linked with the term prion in the public . actual prion diseases are remarkably rare in humans and even the highly publicized outbreaks were relatively small in scale . for example , the number of human cases of variant creutzfeldt - jakob disease cases per year associated with the outbreak of bovine spongiform encephalopathy in great britain peaked in the year 2000 with 28 deaths ( http://www.promedmail.org/direct.php?id=20120809.1236446 ) . yet , this overall low incidence of human cases should not take away from the importance of proper safety precautions associated with avoiding contamination with prions or in efforts to prevent prion outbreaks in animals and man . neurodegenerative diseases of ageing are a growing disease epidemic that is placing an increasing financial and emotional toll on societies . our slow progress in developing treatments that will eventually slow down or even halt the progression of these debilitating diseases likely hinges on a better understanding of the complexity of the ageing brain , cell biology , and synapses . rare gene mutations or more common polymorphisms have provided new clues in our understanding of the biochemical pathways that determine these important diseases . synapses are turning out to be potentially critically vulnerable sites prone to diseases of protein misfolding . the major degradative organelles , the lysosomes , localize to the cell body of neurons and are thus removed from distal neurites and synapses in which various endosomal organelles provide diverse functions , including secretion and degradation . cellular degradation systems , such as the endosome - lysosome system and the ups , may be particularly vulnerable to the development of age - related dysfunction of the nervous system and thereby might predispose to aberrant protein aggregation with ageing . major contributors to the ageing process , including mitochondrial dysfunction , cardiovascular disease , and inflammation , likely impact the declining function of these important cellular degradation pathways . it will be critical to better define the cellular and biochemical pathways implicated in neurodegenerative diseases as well as to elucidate the normal biology of synapses . it is also possible that the aggregation - prone properties of misfolding proteins linked to neurodegenerative diseases hinge on their normal role at synapses and their propensity to aggregate . furthermore , it will be important to better define the more precise cellular mechanisms leading to cell - to - cell propagation of neurodegenerative disease - linked proteins .

age - related misfolding and aggregation of disease - linked proteins in selective brain regions is a characteristic of neurodegenerative diseases . although neuropathological aggregates that characterize these various diseases are found at sites other than synapses , increasing evidence supports the idea that synapses are where the pathogenesis begins . understanding these diseases is hampered by our lack of knowledge of what the normal functions of these proteins are and how they are affected by aging . evidence has supported the idea that neurodegenerative disease - linked proteins have a common propensity for prion protein - like cell - to - cell propagation . however , it is not thought that the prion - like quality of these proteins / peptides that allows their cell - to - cell transmission implies a role for human - to - human spread in common age - related neurodegenerative diseases . it will be important to better understand the molecular and cellular mechanisms governing the role of these aggregating proteins in neural function , especially at synapses , how their propagation occurs and how pathogenesis is promoted by aging .

1. Synapses 2. Endosomes 3. Prions 4. Conclusion

atherosclerotic plaques with a large lipid core are a frequent cause of acute coronary syndromes . in 1993 near - infrared spectroscopy ( nirs ) was first used for atherosclerotic plaque imaging in an experimental animal model . in further ex vivo validation studies , nirs was found to accurately detect the lipid ( cholesterol ) content of human atherosclerotic plaques . in 2001 , a device prototype for intracoronary imaging was developed . nirs provides a chemogram of the wall of the coronary artery and aims to detect lipid - rich plaques . this nirs imaging system consists of a 3.2 french catheter ( infraredx , burlington , massachusetts , usa ) , a pullback , rotation device , and a dedicated console comprising a laser light source and a computer for algorithmic data processing . the catheter comprises the rotating core with optical fibers that deliver and collect near - infrared light . the light is emitted into a sample by the nir spectrometer , which subsequently measures the proportion of light that is returned over the range of optical wavelength . the nirs system uses wave lengths of 800 to 2500 nm , converting a diffuse reflectance signal from an object to produce a spectrum . subsequently , a computer analyzes the spectra and produces an algorithm to demonstrate a chemogram , that corresponds strongly to the lipid content ( figs . 1 and 2).fig . chemogram is shown detecting a colour map of the artery wall and indicates the location and intensity of coronary lipid content . the horizontal - axis of the chemogram represents the pullback position and the vertical - axis represents the circumferential position in degrees ( 0360 ) . adapted and modified from : choi b et al . eur heart j 2013;34:20472054fig . 2near - infrared spectroscopy ( nirs ) imaging indicates the lipid - core containing plaques are substantially more frequent in human coronary arteries with endothelial dysfunction . adapted and modified from : choi b et al . chemogram is shown detecting a colour map of the artery wall and indicates the location and intensity of coronary lipid content . the horizontal - axis of the chemogram represents the pullback position and the vertical - axis represents the circumferential position in degrees ( 0360 ) . adapted and modified from : choi b et al . eur heart j 2013;34:20472054 near - infrared spectroscopy ( nirs ) imaging indicates the lipid - core containing plaques are substantially more frequent in human coronary arteries with endothelial dysfunction . adapted and modified from : choi b et al . non - invasive imaging modalities including computed tomography or magnetic resonance have a limited resolution for precise plaque characterization . therefore , catheter - based intravascular imaging methods , such as optical coherence tomography ( oct ) and nirs are highly valuable for detailed characterization of coronary atherosclerotic plaques , and have become available within the last years . as described above , the nirs imaging system allowing for plaque assessment was carefully validated against atherosclerotic plaque autopsy specimens , and can nowadays be used in humans in vivo [ 7 , 8 ] . nirs provides excellent spectra through blood and despite heart movement by using short scanning acquisition cycles . the prospect study suggested , that a plaque burden of > 70 % , thin - cap fibroatheroma , and minimal luminal area 4.0 mm are predictors of overall long - term adverse cardiovascular events . nirs has been established as a very accurate modality to characterize plaques with different lipid content . madder et al . have shown , that target lesions responsible for acute coronary syndrome are frequently composed of lipid core plaque with a high lipid core burden index ( lcbi ) . the presence of lipid core plaque may address the high - risk setting for subsequent coronary events , which needs to be further investigated in future studies [ 11 , 12 ] . interestingly , both culprit and non - culprit lesions contained lipid core plaque more frequently in acs patients as compared to patients with stable angina .table 1intracoronary human nirs studiesstudyyearn * patientsaimconclusioncaplan j.d . 20066initial clinical experience in stable cadhigh quality nirs spectra can be obtained in patients in vivo jacc waxman , s. et .al 2009106to determine whether catheter - based nirs signals from coronaries of patients are similar to those from autopsy specimens and to assess initial safety of nirs devicespectral data were safely obtained by nirs similarly to those from autopsy specimens ; results demonstrated the feasibility of invasive detection of coronary lcp jacc cardiovasc imaging raghunathan , d. et .al 201130to examine whether an association exists between the presence and extent of lcp detected by nirs performed before pci with postprocedural mipci of lcp - positive lesions is associated with increased risk for mi after pci am j cardiol goldstein , j.a . et 201162to analyze the relationship between the presence of a large lcp detected by nirs and periprocedural minirs provides rapid , automated detection of extensive lcps that are associated with a high risk of periprocedural mi circ cardiovasc interv brugaletta , s. et .al 201131to compare the findings of nirs , ivus virtual histology and grayscale ivus obtained in matched coronary vessel segments of patients undergoing coronary angiographylarger plaque area by grayscale ivus was more often associated with either elevated percentage vh necrotic core or lcp by nirs jacc cardiovasc imaging madder , r.d . 201260to determine the frequency of lcp at target and remote sites in acs vs. stable anginatarget lesion responsible for acs were frequently composed of lcp ; lcps often were found in remote , non - target areas ; lcps were more common in patients with acs vs. stable angina patients circ cardiovasc interv pu , j. et .al 201266to evaluate nirs combined with ivus to provide novel information of human coronary plaque characterizationcombining nirs and ivus contributes to the plaque characterization eur heart j dixon , s.r . et .al 201269to compare the target lesion length using nirs combined with angiography vs. angiography alonepatients undergoing stent implantation could have lcp extending beyond the intended treatment margins as defined using qca alone am j cardiol brugaletta , s. et .al 2012202to explore a relationship between lipid plaque composition by nirs and angiographic severity of coronary artery diseasepatients with highest syntax score have a higher lcbi jacc cardiovasc imaging brilakis , e.s . et .al 20129to investigate whether use of an embolic protection device might prevent complications of lcp interventionsuse of embolic protection devices frequently resulted in embolized material retrieval after stenting of native coronary artery lesions with large lcp catheter cardiovasc interv brugaletta , s. et .al 201268to assess lcp distribution in nonculprit coronary arteries using nirslcp were mainly located in proximal portions of the lad and lcx , and more uniformly distributed in the rca ; jacc cardiovasc imaging kini , a.s . 201387to determine the impact of short - term intensive statine treatment on intracoronary plaque lipid contentshort - term intensive treatment with statine may reduce lipid content in obstructive coronary lesions jacc papayannis , a.c . et .al 20139to examine the association between presenting lcp ( by nirs ) and poststenting thrombus formation ( by oct)stenting large lcps may be associated with intrastent thrombus formation catheter cardiovasc interv townsend , j.c . et .al 2013100to investigate , whether coronary bifurcations have higher levels of intracoronary lcp than non - bifurcation regionscoronary bifurcations do not appear to have higher levels of intracoronary lcp than their comparative non - bifurcation regions am j cardiol maini , a. et .al 201377to evaluate lcp modification with coronary revascularization and its correlation with periprocedural miplaque modification may be performed successfully using interventional methods and can be evaluated with nirs ; axial plaque shifting is an acute prognostic marker for postprocedure mi j inv cardiol madder , r.d . 201320to describe nirs findings of culprit lesions in stemiplaques causing stemi have a high lcbi jacc cardiovasc interv zynda , t.k . 201378to determine if there was a relationship between angiographic lesion complexity and the extent of lcp identified by catheter - based nirsangiographic syntax score weakly correlated with lipid core burden index catheter cardiovasc interv choi , b.j . 201332to investigate whether coronary endothelial dysfunction is associated with the lcp in patients with early cadpatients with early cad and endothelial dysfunction had a higher lipid content in the vascular wall than patients with normal endothelial function ; eur heart j acs acute coronary syndrome ; cad coronary artery disease ; ivus intravascular ultrasound ; lad left artery descending ; lcbi lipid core burden index ; lcp lipid core - containing plaque ; lcx left circumflex artery ; mi myocardial infarction ; nirs near - infrared spectroscopy ; pci percutaneous coronary intervention ; qca quantitative coronary angiography ; rca right coronary artery ; stemi st - segment elevation myocardial infarction ; * number of patients intracoronary human nirs studies acs acute coronary syndrome ; cad coronary artery disease ; ivus intravascular ultrasound ; lad left artery descending ; lcbi lipid core burden index ; lcp lipid core - containing plaque ; lcx left circumflex artery ; mi myocardial infarction ; nirs near - infrared spectroscopy ; pci percutaneous coronary intervention ; qca quantitative coronary angiography ; rca right coronary artery ; stemi st - segment elevation myocardial infarction ; * number of patients moreover , the study by pu et al . have recently supported the notion , that combining nirs with ivus can lead to a better plaque characterization . in addition , the three - dimensional reconstruction of coronary anatomy by angiocare software permits the identification of the lipid core plaque location and therefore the association among vessel geometry , endothelial shear stress and plaque composition . the impact of short - term intensive statin treatment on a lipid plaque content has been investigated in a first study using nirs demonstrating changes in lipid composition . several small studies have examined the potential impact of plaque evaluation by nirs for interventional coronary therapy [ 1618 ] . it is well known , that 3 - 15 % of percutaneous coronary interventions ( pcis ) are complicated by periprocedural myocardial infarction ( mi ) due to the distal embolization by intraluminal thrombus and/or lipid - core plaque content [ 1921 ] . first studies suggest that edge dissections or plaque disruptions may be more common when stents end within a lipid core plaque . have documented , that in 16 % of lesions lipid core plaque may extend beyond the margins of the lesion as assessed by angiography . this further points to limitations of qca alone in the evaluation of lesion- and subsequently stent length . notably , the nirs - identified lipid core plaque has been described to be strongly associated with a high risk of thrombus formation or periprocedural myocardial infarction [ 17 , 18 , 24 ] . nirs could be an interesting tool to investigate novel lipid - modulating and other cardiovascular therapies aiming to prevent adverse coronary events [ 17 , 23 ] . however , a potential limitation of nirs may be its inability to assess the depth of a lipid core and the measurement of lipid volume has not been validated so far . nirs may therefore be used in combination with other imaging modalities , such as ivus or oct . in this respect , a combined imaging catheter has recently been developed ( tvc imaging system , mc 7 system , infraredx , burlington , massachusetts , usa ) , combining both , an ivus probe and nirs light source . this catheter is being used in the ibis-3 ( integrated biomarker and imaging study 3 ) to investigate the effects of rosuvastatin therapy on the lipid - rich coronary atherosclerotic plaques .

acute coronary syndromes are frequently caused by vulnerable coronary plaques with a lipid - rich core . in 1993 near - infrared spectroscopy ( nirs ) was first used to detect the lipid ( cholesterol ) content of atherosclerotic plaques in an experimental animal study . nirs was then carefully validated using human atherosclerotic plaques ( ex vivo ) , and has subsequently been developed for intracoronary imaging in humans , for which now an fda - approved catheter - based nirs system is available . nirs provides a chemogram of the coronary artery wall and is used to detect lipid - rich plaques . using this technology , recent studies have shown that lipid - rich plaques are very frequent in the culprit lesion of patients with an acute coronary syndrome , and are also common in non - culprit coronary lesions in these patients as compared to patients with stable coronary disease . first studies are evaluating the impact of statin therapy on coronary nirs - detected lipid cores . intracoronary nirs imaging represents a highly interesting method for coronary plaque characterization in humans and may become a valuable tool for the development of novel therapies aiming to impact on the biology of human coronary artery plaques , likely in combination with other intracoronary imaging techniques , such as optical coherence tomography .

Introduction NIRS Catheter Experience Using NIRS for Coronary Plaque Imaging in Humans in Vivo Implications for Guiding Therapy Future Perspectives

according to the statistics , accidents and disasters are increasing worldwide , including in our country . in 2011 , about 332 natural disasters were recorded in the world , although this amount was less than the number in the years 2001 - 2010 , but the human and economic impact of these crises were much greater than in the previous years . about 86.3% of the victims of the crisis reported had been allocated to asia . in the latest report by the center of epidemiological crisis , in 2012 , iran was not among the first ten countries in terms of deaths caused by the crisis , but between 1980 and 2010 , about 1,376,263 people in iran were affected by natural crises every year . earthquakes , droughts , and floods were the major disasters in terms of mortality , adverse economic effects , and the victim population that were considered in this country . therefore , addressing the crisis and improving the response to critical events in the country is very important . there is no single agreed definition for crisis and there are several definitions in literature in this regard . the center of international strategy for disaster reduction and the international federation of red cross have defined crisis as : disorganization in the function of community , which results in the loss of human , economic , and environmental resources , and is usually beyond the ability of the community to deal with it . moreover , due to the many influences that it leaves behind on people 's lives , wide efforts must be made to minimize the mortality . this has a direct connection to the readiness and competence of the health teams in response to the disaster . numerous studies have indicated several roles for nurses , including their role in the crisis team . florence nightingale showed the world that nurses have an important role in disaster response , and nurses with expertise should be used in various stages of the crisis , including disaster response . in studies on the experiences of nurses working in emergency departments , most of the participants emphasized on the key role of skilled and trained nurses in crisis interventions and expressed that nurses should have the knowledge and competencies for professional services in critical situations . despite the important role of nurses in response to the crisis , little information on specialized skills or competencies needed to participate effectively in these situations are available . few nurses have the experience of providing care in critical situations and in providing care in response to a crisis . studies conducted on the nurses experiences in the presence of these situations suggest that most of them , after participating in the crises response team , declared that they did not have the adequate ability to meet the crisis . this can be attributed to the lack of relevant content and appropriate education in the field of their formal education . hsu believes that the available information about the competencies needed in response to the crisis are not strongly evidence - based , and most of the nurses do not earn these capabilities and only after attending a critical situation they discover that these skills are also required . therefore , determining the competencies required by nurses to effectively participate in response to disaster team is necessary . magnaye suggested that in this area , during response to crisis , nurses require specialized skills and competencies in order to care for the victims . furthermore , technical skills are the most important among other skills . however , the details of these competencies are not clearly explained . in iran , at the current moment , nursing in a crisis is not well - defined and the competencies required of nurses to provide services in a crisis have not been well - established . therefore , integrated educational programs based on the needs in this area are not available . nurses only receive a limited amount of training during their undergraduate courses on emergency and nursing care in these situations . moreover , their training is not based on their needs for successful participation during critical times . studies on the role of nurses , the skills and competencies required in the emergency department , a nurse 's role in the crisis team , and their response to crises are very limited . the technical competencies required for nurses to play a role in the crisis have not been fully clarified . published studies on the experiences of nurses in critical situations indicated the need for their preparation for technical protocols and for their having a set of specialized skills . however , there was no study found to clearly explain the role of nurses , their competency dimensions , and the skills needed . most of the studies were on the assessment of the readiness of nurses to deal with a crisis and also to arrive at a conclusion about their preparation for the same , and also to check if they lacked any skill . given the circumstances of iran , there is a need for some change with regard to the policy and challenges of the health and education system . given that determining the qualifications can be used to assess the training needs of nursing for their role in the crisis , and also to benefit in the nursing curriculum and design , appropriate continuing education programs are required in iran for preparing nurses for critical situations , therefore , this study explains the technical competencies required for a nurse in response to the crisis through a qualitative study . this qualitative analytical research is part of a larger study that has been done for a deeper understanding of the nurses competencies in disaster response . the participants were 35 licensed nurses , who had recent experience in healthcare delivery , during an iranian earthquake ( 2003 bam or 2012 ahar ) or some other kind of natural or man - made disaster , within the past 10 years . the selection of participants was determined using a purposeful sampling method , as per the instructions of the nursing office . nurses were invited to participate in the study after a telephone conversation , during which they were told of the study 's objectives . the ethical issues in this study involved the assurance of confidentiality and autonomy for the participants . they were also informed that participation in the study is voluntary , so they could refuse to participate or withdraw from the study at any time . moreover , the participants were reassured that their responses would be kept confidential and their identities would not be revealed in research reports and publications of the study . lastly , the participants who agreed to participate in the study were asked to sign a written consent . the data have been saturated after 30 semi - structured interviews and five more have been done to confirm the categories . disaster has been defined as a natural or man - made incident that leaves at least 10 dead or more than 100 injured . these incidents are not manageable by the local management system and need more help from outside . each interview began with the question , please tell me about your experience of providing care in a disaster? the nurses responses were clarified and expanded upon by follow - up questions . after taking permission , the interviews were recorded using a digital voice recorder . the researcher listened to the recordings and transcribed them word - for - word , in a word 2007 software after developing a general idea . an analysis unit was established , where the transcripts were read line - by - line . the important sentences and phrases were underlined and the main ideas derived from them were labeled as codes . drawing conclusions from the coded data was the latest step that determined the characteristics and dimensions of the categories , the relationship between them , and gave a holistic view about the competence concept . peer checking and maximum variation of the sampling attested to the conformability and credibility of the findings and underpinned the effort to obtain as wide and varied an experience of the phenomenon as possible . there was good variation within the sample selected in , age , years of experience in nursing , and exposure to working in different disaster relief situations as a healthcare provider , healthcare manager or emergency nurse . this procedure , combined with the available transcribed data and notes from the analysis process , are believed to assure dependability . they had between seven and twenty - eight years of work experience in different nursing wards . all had at least one year of experience encountering and delivering care in a disaster situation ( natural disasters such as earthquakes , floods or large road accidents ) . after data analysis of the special nursing competencies in disaster management , two important themes emerged , including : ( 1 ) basic and special nursing knowledge and ( 2 ) skill to care for injured people . these results , as themes , are sequentially presented in the following sections with exemples of interviews . themes and subthemes of special nursing competencies in disaster the research findings indicated that every individual needs some important scientific knowledge as a nurse , to play his / her role in a disaster successfully . this theme consisted of the following subthemes : ( a ) having basic scientific knowledge and ( b ) knowledge about the disaster and specific interventions related to a disastrous situation . participants believed that nurses need to have some basic information and be familiar with physiology , epidemiology , pathophysiology , and pharmacology . for example , a participant stated : there are some important and basic information that a nurse needs to have in any situations in order to deliver an appropriate care . when nurses teach to become a nurse in their study courses , they need to learn physiology very good . they also need to pick up useful information about the pathophysiology of disease and the psychology . after that , they can play their practice correctly , these skills are like alphabets make a bases for other work practices ( participant 12 ) . during the process of comparative data analysis , it was found that the participants addressed a number of special scientific knowledge and interventions that nurses needed to have in a disastrous situation . they emphasized on issues , such as , knowing about various types of disasters , their complications , the related care involved , what could threaten individuals lives in a disaster , and drug protocols : a nurse who wants to work in a disaster need to have some information about disaster , several types of disasters , what is the difference between natural and man - made disasters , each one may lead to what kind of problems ? and how i need to deal with them almost all participants highlighted that nurses do need some essential skills to effectively work in a disaster and efficiently help injured people . this theme consisted of the following subthemes : ( a ) skills in a triage ; ( b ) skill in secondary assessment ; ( c ) skills to work with pre - hospital emergency equipments and instruments ; ( d ) treatment skills ; ( e ) physical care skills ; ( f ) psychological care skills , and ( g ) care recording skills . these subthemes are presented in the following sections . participants also explained that prioritizing the patients and the interventions that needed to be conducted at the first encounter were important factors , along with other significant factors , such as , the skill to triage adults and children , skill to conduct a quick ( primary ) triage , skill to conduct a management and technical triage ( secondary ) , skill to conduct a quick assessment of the situation of the injured , skill to identify life - threatening problems , skill to explore the mechanism of injury , and the skill to make the appropriate diagnosis . the participants frequently stated that the management and technical triage was the most essential competency required by the nurses in order to rank the injured people , as also time management and saving injured lives : the important thing is that to know the triage we have the management and technical triage it is very important for nurse to be able to have a general assessment of patient in the disaster scene , and then assess the injured person and the mechanism leading to the injury . after that , they will be able to make correct differential diagnoses and do first line interventions. ( participant 14 ) . assessment of an injured person is very vital and a nurse has to do an appropriate and quick assessment assess to understand what they need to do at this point of time , prioritize individuals needs and intervene accordingly the sub - theme skill in secondary assessment was created to explain skills like monitoring in secondary assessment , diagnosing secondary shock , and identifying the individuals health needs . in response to questions put to the interviewees , the participants expressed that nurses needed to be prepared for a secondary assessment of an injured person , in order to identify individual 's problems and to evaluate the interventions that were conducted : .the secondary assessment is very important competency as in these regular assessments you can identify problems might happen for the person in the near future such as shock this can prevent worsening the situation if you do the things on the right time ( participant 17 ) . it became clearer during the interviews that some important skills during disasters included factors such as , being able to work with tools inside an ambulance , manipulate immobilizing tools and splints , and use communication appliances . one nurse , for example , who had the experience of working in the bam earthquake , stated : we were not be able to make any communication with the local area and even in kerman nobody knew exactly about the extension of the earthquake , what injured people need ? the reason was mainly because no one could maintain a communication using such devices the other skills that the nurse participants elaborated on were , carrying out therapeutic actions such as first aid , treatment of shock , drug administration , management of internal and external bleeding , cardiopulmonary resuscitation , and airway management . for example , it would be very harmful for a person with severe bleeding to transfer to another area to manage . the person with bleeding need to manage in the scene iv therapy and opening airways sometimes are quite necessary . even in some cases , the person is pregnant and the nurse need to collaborate in a delivery it is very important skill in a disaster ( participant 23 ) . nurse participants also defined physical care as a very important skill , when working in a disaster . this skill was composed of abilities like caring for patients of different age groups , with chronic conditions and injures , care of burns and toxicities , wound care , transfer of the injured using an inappropriate method and position , dressing and suturing , conducting nasogastric tubing , bandaging , using splints and immobilizers , and appropriate care in nuclear and biological disasters . the following is what a nurse participant highlighted : nurses need to do dressing , suturing and bandaging . in our country we do not have nuclear disasters so far but nurses need to be prepared to work in such situations if they happen ( participant 4 ) . it was emphasized by several participants that appropriate transfer of the injured was of great importance , as this could lead to severe complications like paraplegia : patient transfer and transport is very important . after bam earthquake , we faced with some spinal cord injuries more than other . probably with a correct way of transfer , we could prevent most of these cases ( participant 19 ) . this skill is composed of competencies that include , diagnosing psychological shocks in a crisis , transformative disorders , posttraumatic stress disorders ( ptsd ) , and people who need to refer to groups for psychological support , skills to psychologically support the victims and their families , maintain assurance and tranquility in all individuals on the disaster scene , give a sense of safety to the injured person , and understand the whole situation of the victims and their families : as a human being it is very important to understand victims and diagnose their psychological problems . you may need to transfer them to a social worker or a consultant to help them ( participant 31 ) . the most important problem that we dealt with was ptsd . we got used to such a language and tried to just listen and support them as well as physical care , psychological support is also very important in addition to explaining the important skills mentioned earlier , during the course of the interviews , the participants suggested that documentation of the interventions was quite important and worthy of consideration . this included an accurate and delicate documenting of all nursing interventions and their outcomes , particularly key notes such as the time and the dosage of drugs used . failure to document correctly could cause legal issues later : documenting the events are very vital , it is a skill . we need to document all details , when the actions took place , when the interventions conducted . while we are in a disaster but we might need to answer to several questions afterwards a victim or their family might question you in the future the research findings indicated that every individual needs some important scientific knowledge as a nurse , to play his / her role in a disaster successfully . this theme consisted of the following subthemes : ( a ) having basic scientific knowledge and ( b ) knowledge about the disaster and specific interventions related to a disastrous situation . participants believed that nurses need to have some basic information and be familiar with physiology , epidemiology , pathophysiology , and pharmacology . for example , a participant stated : there are some important and basic information that a nurse needs to have in any situations in order to deliver an appropriate care . when nurses teach to become a nurse in their study courses , they need to learn physiology very good . they also need to pick up useful information about the pathophysiology of disease and the psychology . after that , they can play their practice correctly , these skills are like alphabets make a bases for other work practices ( participant 12 ) . during the process of comparative data analysis , it was found that the participants addressed a number of special scientific knowledge and interventions that nurses needed to have in a disastrous situation . they emphasized on issues , such as , knowing about various types of disasters , their complications , the related care involved , what could threaten individuals lives in a disaster , and drug protocols : a nurse who wants to work in a disaster need to have some information about disaster , several types of disasters , what is the difference between natural and man - made disasters , each one may lead to what kind of problems ? and how i need to deal with them participants believed that nurses need to have some basic information and be familiar with physiology , epidemiology , pathophysiology , and pharmacology . for example , a participant stated : there are some important and basic information that a nurse needs to have in any situations in order to deliver an appropriate care . when nurses teach to become a nurse in their study courses , they need to learn physiology very good . they also need to pick up useful information about the pathophysiology of disease and the psychology . after that , they can play their practice correctly , these skills are like alphabets make a bases for other work practices ( participant 12 ) . during the process of comparative data analysis , it was found that the participants addressed a number of special scientific knowledge and interventions that nurses needed to have in a disastrous situation . they emphasized on issues , such as , knowing about various types of disasters , their complications , the related care involved , what could threaten individuals lives in a disaster , and drug protocols : a nurse who wants to work in a disaster need to have some information about disaster , several types of disasters , what is the difference between natural and man - made disasters , each one may lead to what kind of problems ? and how i need to deal with them almost all participants highlighted that nurses do need some essential skills to effectively work in a disaster and efficiently help injured people . this theme consisted of the following subthemes : ( a ) skills in a triage ; ( b ) skill in secondary assessment ; ( c ) skills to work with pre - hospital emergency equipments and instruments ; ( d ) treatment skills ; ( e ) physical care skills ; ( f ) psychological care skills , and ( g ) care recording skills . participants also explained that prioritizing the patients and the interventions that needed to be conducted at the first encounter were important factors , along with other significant factors , such as , the skill to triage adults and children , skill to conduct a quick ( primary ) triage , skill to conduct a management and technical triage ( secondary ) , skill to conduct a quick assessment of the situation of the injured , skill to identify life - threatening problems , skill to explore the mechanism of injury , and the skill to make the appropriate diagnosis . the participants frequently stated that the management and technical triage was the most essential competency required by the nurses in order to rank the injured people , as also time management and saving injured lives : the important thing is that to know the triage we have the management and technical triage it is very important for nurse to be able to have a general assessment of patient in the disaster scene , and then assess the injured person and the mechanism leading to the injury . after that , they will be able to make correct differential diagnoses and do first line interventions. ( participant 14 ) . assessment of an injured person is very vital and a nurse has to do an appropriate and quick assessment assess to understand what they need to do at this point of time , prioritize individuals needs and intervene accordingly was created to explain skills like monitoring in secondary assessment , diagnosing secondary shock , and identifying the individuals health needs . in response to questions put to the interviewees , the participants expressed that nurses needed to be prepared for a secondary assessment of an injured person , in order to identify individual 's problems and to evaluate the interventions that were conducted : .the secondary assessment is very important competency as in these regular assessments you can identify problems might happen for the person in the near future such as shock this can prevent worsening the situation if you do the things on the right time ( participant 17 ) . it became clearer during the interviews that some important skills during disasters included factors such as , being able to work with tools inside an ambulance , manipulate immobilizing tools and splints , and use communication appliances . one nurse , for example , who had the experience of working in the bam earthquake , stated : we were not be able to make any communication with the local area and even in kerman nobody knew exactly about the extension of the earthquake , what injured people need ? the other skills that the nurse participants elaborated on were , carrying out therapeutic actions such as first aid , treatment of shock , drug administration , management of internal and external bleeding , cardiopulmonary resuscitation , and airway management . for example , it would be very harmful for a person with severe bleeding to transfer to another area to manage . the person with bleeding need to manage in the scene iv therapy and opening airways sometimes are quite necessary . even in some cases , the person is pregnant and the nurse need to collaborate in a delivery it is very important skill in a disaster ( participant 23 ) . nurse participants also defined physical care as a very important skill , when working in a disaster . this skill was composed of abilities like caring for patients of different age groups , with chronic conditions and injures , care of burns and toxicities , wound care , transfer of the injured using an inappropriate method and position , dressing and suturing , conducting nasogastric tubing , bandaging , using splints and immobilizers , and appropriate care in nuclear and biological disasters . the following is what a nurse participant highlighted : nurses need to do dressing , suturing and bandaging . in our country we do not have nuclear disasters so far but nurses need to be prepared to work in such situations if they happen ( participant 4 ) . it was emphasized by several participants that appropriate transfer of the injured was of great importance , as this could lead to severe complications like paraplegia : patient transfer and transport is very important . after bam earthquake , we faced with some spinal cord injuries more than other . probably with a correct way of transfer this skill is composed of competencies that include , diagnosing psychological shocks in a crisis , transformative disorders , posttraumatic stress disorders ( ptsd ) , and people who need to refer to groups for psychological support , skills to psychologically support the victims and their families , maintain assurance and tranquility in all individuals on the disaster scene , give a sense of safety to the injured person , and understand the whole situation of the victims and their families : as a human being it is very important to understand victims and diagnose their psychological problems . you may need to transfer them to a social worker or a consultant to help them ( participant 31 ) . the most important problem that we dealt with was ptsd . we got used to such a language and tried to just listen and support them as well as physical care , psychological support is also very important in addition to explaining the important skills mentioned earlier , during the course of the interviews , the participants suggested that documentation of the interventions was quite important and worthy of consideration . this included an accurate and delicate documenting of all nursing interventions and their outcomes , particularly key notes such as the time and the dosage of drugs used . failure to document correctly could cause legal issues later : documenting the events are very vital , it is a skill . we need to document all details , when the actions took place , when the interventions conducted . while we are in a disaster but we might need to answer to several questions afterwards a victim or their family might question you in the future participants also explained that prioritizing the patients and the interventions that needed to be conducted at the first encounter were important factors , along with other significant factors , such as , the skill to triage adults and children , skill to conduct a quick ( primary ) triage , skill to conduct a management and technical triage ( secondary ) , skill to conduct a quick assessment of the situation of the injured , skill to identify life - threatening problems , skill to explore the mechanism of injury , and the skill to make the appropriate diagnosis . the participants frequently stated that the management and technical triage was the most essential competency required by the nurses in order to rank the injured people , as also time management and saving injured lives : the important thing is that to know the triage we have the management and technical triage it is very important for nurse to be able to have a general assessment of patient in the disaster scene , and then assess the injured person and the mechanism leading to the injury . after that , they will be able to make correct differential diagnoses and do first line interventions. ( participant 14 ) . assessment of an injured person is very vital and a nurse has to do an appropriate and quick assessment assess to understand what they need to do at this point of time , prioritize individuals needs and intervene accordingly the sub - theme skill in secondary assessment was created to explain skills like monitoring in secondary assessment , diagnosing secondary shock , and identifying the individuals health needs . in response to questions put to the interviewees , the participants expressed that nurses needed to be prepared for a secondary assessment of an injured person , in order to identify individual 's problems and to evaluate the interventions that were conducted : .the secondary assessment is very important competency as in these regular assessments you can identify problems might happen for the person in the near future such as shock this can prevent worsening the situation if you do the things on the right time ( participant 17 ) . it became clearer during the interviews that some important skills during disasters included factors such as , being able to work with tools inside an ambulance , manipulate immobilizing tools and splints , and use communication appliances . one nurse , for example , who had the experience of working in the bam earthquake , stated : we were not be able to make any communication with the local area and even in kerman nobody knew exactly about the extension of the earthquake , what injured people need ? the reason was mainly because no one could maintain a communication using such devices the other skills that the nurse participants elaborated on were , carrying out therapeutic actions such as first aid , treatment of shock , drug administration , management of internal and external bleeding , cardiopulmonary resuscitation , and airway management . for example , it would be very harmful for a person with severe bleeding to transfer to another area to manage . the person with bleeding need to manage in the scene iv therapy and opening airways sometimes are quite necessary . even in some cases , the person is pregnant and the nurse need to collaborate in a delivery it is very important skill in a disaster ( participant 23 ) . nurse participants also defined physical care as a very important skill , when working in a disaster . this skill was composed of abilities like caring for patients of different age groups , with chronic conditions and injures , care of burns and toxicities , wound care , transfer of the injured using an inappropriate method and position , dressing and suturing , conducting nasogastric tubing , bandaging , using splints and immobilizers , and appropriate care in nuclear and biological disasters . the following is what a nurse participant highlighted : nurses need to do dressing , suturing and bandaging . in our country we do not have nuclear disasters so far but nurses need to be prepared to work in such situations if they happen ( participant 4 ) . it was emphasized by several participants that appropriate transfer of the injured was of great importance , as this could lead to severe complications like paraplegia : patient transfer and transport is very important . after bam earthquake , we faced with some spinal cord injuries more than other . probably with a correct way of transfer , we could prevent most of these cases ( participant 19 ) . this skill is composed of competencies that include , diagnosing psychological shocks in a crisis , transformative disorders , posttraumatic stress disorders ( ptsd ) , and people who need to refer to groups for psychological support , skills to psychologically support the victims and their families , maintain assurance and tranquility in all individuals on the disaster scene , give a sense of safety to the injured person , and understand the whole situation of the victims and their families : as a human being it is very important to understand victims and diagnose their psychological problems . you may need to transfer them to a social worker or a consultant to help them ( participant 31 ) . the most important problem that we dealt with was ptsd . we got used to such a language and tried to just listen and support them as well as physical care , psychological support is also very important in addition to explaining the important skills mentioned earlier , during the course of the interviews , the participants suggested that documentation of the interventions was quite important and worthy of consideration . this included an accurate and delicate documenting of all nursing interventions and their outcomes , particularly key notes such as the time and the dosage of drugs used . failure to document correctly could cause legal issues later : documenting the events are very vital , it is a skill . we need to document all details , when the actions took place , when the interventions conducted . while we are in a disaster but we might need to answer to several questions afterwards a victim or their family might question you in the future the participants descriptive analysis showed that skilled nursing competence in disasters included knowledge and skill of taking care of victims . based on the research findings , having a basic scientific knowledge , being familiar with the crisis , and specific actions in critical situations were the essential capabilities that a nurse should have at a disaster scene . he also emphasized the importance of having a basic scientific knowledge and information on the crisis situations and mentioned it as one of the underlying competencies . the skill of taking care of the crisis victims was one of the main factors extracted for classes on this study . the participants mentioned different skills that came under the nurse 's special skills for participation in critical situations , of which the recording skill was one . participants believed that record - keeping and accurate reporting skills at the indicated times was imperative for nurses during crisis management , as having a lack of ability to record could later cause legal problems for the person providing the care . in this context , yin in his study in 2011 , on chinese nurses , stated that record - keeping of the care provided was one of the specialized skills required by chinese nurses during care in critical situations and that nurses should receive adequate training in this area . the skills of working with equipment in a crisis were an essential component of the competencies for a nurse , in order to work in crisis team and provide care . magnaye reported in his study in philippines that nurses needed the skill to work with equipment in a crisis response , in order to take care of the crisis victims , and this was one of the technical skills . another matter that was referred to in the application of knowledge in critical situations was the skill to perform a triage . participants considered triage as one of the chief professional skills required of the nurses during a crisis . the world health organization and the international association of nursing , in 2009 , considered the triage skill as an immediate action necessary in crisis situations , which was consistent with the experience of the participants in the present study with regard to nursing management competencies in a crisis . the participants expressed that the nurse should have a secondary assessment skill in order to identify the patient 's problems and the impact of the actions taken . yin et al . , in a study in china in 2011 , and the daily in a review study , in 2010 , in america , listed the secondary assessment of the patient 's condition and the impact of actions taken among the most important measures to be taken by a nurse in critical situations . this was also one among the competencies cited by the participants of the current study . the therapeutic interventions by nurses showed the success of applying specialized knowledge and included skills when performing life - saving actions , such as , initial rescue , control of bleeding , and cardiopulmonary resuscitation . the recent findings were somewhat similar to the findings of al khalaileh , in 2010 , in jordan . he reported that all nurses should have specialized competencies such as , the ability for cardiopulmonary resuscitation . he also emphasized on the key role played by skilled and trained nurses , who provided service in critical situations . the america association of schools of public health , in 2011 , provided a framework for preparing graduate students to become successful and effective in critical situations . among the provided competencies , expertise in performing cardiopulmonary resuscitation and remedial measures were listed as essential points in training , which were similar to the competencies cited by the participants of the present study . one of the most important measures in critical situations was appropriate and on - time care for the victims . in a recent study on the skills , among a range of actions in the form of competencies , physical care was emphasized on . in this regard , the emergency nurses association of america , in 2008 , emphasized that the skill in physical care was an important capability for crisis nurses and that all nurses should have sufficient skills to provide physical care during an emergency and in critical situations . yin in a similar study , in 2011 , in china , mentioned several skills as essential skills for nurses in crisis times , and first aid and skills to perform procedures were among these skills . these could contribute to achieving the best results in delivery of services , in a crisis . therefore , nurses as important members of the treatment team should be able to carry out these actions well . the recent study participants believed that a nurse , in addition to taking good care of the patients , also had to be a good psychiatric nurse . in crisis situations , nurses had to deal with humans who had both physical and spiritual dimensions , and if attention was only given to the physical aspect , the desired outcome would not be achieved . only if these two aspects were considered together the participants believed that a nurse , in a crisis , should be able to play the role of a psychiatric nurse from the diagnosis onward and try to eliminate the psychological needs of the victims . he / she should be effective in reconstructing and restoring the victims mental state to normal . in studies regarding the experiences of nurses working in emergency departments , most of the participants emphasized on the key role played by nurses in supporting the mental health of the victims and their families during a crisis . they also expressed that nurses should have the knowledge and professional competencies for mental health care in crisis situations . moreover , given that the nurses are among the first people to arrive at the location of the crisis , in addition to physical care , emotional and spiritual care of those injured should also be performed . denise danna writes in a similar study , in 2010 , in america , that any crisis led to social and psychological dysfunction . in these situations , the nurses should play a caring role , besides being a psychiatric nurse and providing mental health care . comparing the results of the recent study , which was the first a comprehensive study conducted in iran , relating to competencies of nurses in crisis , and the competencies provided by the international council of nursing ( icn ) , in 2009 , showed that in the area of specialized competencies required of nurses in the disaster response phase , most of the competencies extracted were similar . competencies , such as , skill with the equipment , secondary assessment skills , and therapeutic skills were among the competencies that were not mentioned in the icn report . however , in studies conducted in other countries , great emphasis has placed on care skills during nuclear crisis or bioterrorism . on account of a lack of these crises in iran , in the recent study , most of the participants had had experience in natural crises , such as , floods and earthquakes ; therefore , most of the qualifications obtained were in connection with such crises . the subjective nature of the data collection and the small sample size limited the generalizability of the study results . however , the selection of samples from individuals with a history of participation in critical situations and with different academic ranks in nursing , caused the results to be applicable to a large extent in the same units . the main goal of nursing in a crisis situation is to achieve the best possible level of health for the individuals and communities affected by the crisis . therefore , it is important to have qualified professionals to provide care in critical situations . the present study has examined the professional and technical competencies required of nurses to provide care in critical situations . according to the findings , all nurses , in order to be ready to participate in critical situations , must receive some technical skills in addition to the basic skills they receive during the training that they use in every day care , to be able to effectively act during a crisis . it is also necessary for nurses , before being placed in real crisis situations , to obtain skills using hospital emergency equipment , triage skills , and psychological care , and they should also have scientific information regarding crisis situations , and the actions pertaining to crisis management . knowledge and care skills together are the components used to deal with crisis and play a role in crisis situations . considering that nurses do not acquire some of these specialized and technical competencies during their training , it is necessary that this training be included in the academic and in - service training for all nurses . the findings of the present study can serve as a guide for the design of training programs for nurses , in order to prepare them for critical situations , and can also be a basis for further studies .

background : today disasters are a part of many people 's lives . iran has a long history of disaster events and nurses are one of the most significant groups within the iranian disaster relief operations , providing immediate and long - term care for those affected by the disaster . however , the technical competence of iranian nurses and their training for this work has received little attention . this article presents the results of a study that aims to explore this context.materials and methods : a qualitative study was conducted using in - depth interviews to collect data from 30 nurses , who were deliberately selected from the health centers affiliated to the isfahan university of medical sciences . themes were identified using the conventional qualitative content analysis . the trustworthiness of the study was supported by considering the auditability , neutrality , consistency , and transferability . the study lasted from 2011 to 2012.results:data analysis undertaken for the qualitative study resulted in the identification of five main themes , which included : ( 1 ) management competences , ( 2 ) ethical and legal competences , ( 3 ) team working , and ( 4 ) personal abilities and the specific technical competences presented in this report.conclusions:this report presents an overview of the nursing technical capabilities required for iranian nurses during disaster relief . it is argued that additional competencies are required for nurses who care in high - risk situations , including disasters . nurses need to prepare themselves more effectively to be responsible and effective in nursing care .

I M R Basic and special nursing knowledge Having basic scientific knowledge Knowledge about disaster and specific interventions related to a disastrous situation Skill to care of injured people Skills in triage Skill in secondary assessment Skills to work with pre-hospital emergency equipments and instruments Treatment skills Physical care skills Psychological care skills Skill of documentation D C

this 41-year - old housewife , educated up to sixth standard , hailing from middle socioeconomic status , with nil contributory family history , and suffering from hypertension and myopia in left eye ( + 0.75 ) and hypermetropia in right eye ( 0.75 ) , reported to outpatient clinic with history of 10-years duration characterized by unpleasant , frequent , and distressing doubts related to dirt and contamination . these resulted in performance of long , nonfunctional repetitive activities like cleaning and saying same words again and again . it was also reported both by husband and the patient herself that these symptoms have a seasonal pattern appearing in october and complete resolution in april - may . this seasonal pattern was so well - recognized that no treatment was taken for initial 3 - 4 years as they considered it an effect of change of season and that symptoms would disappear once the winter is over . no associated stress or precipitating factor was identified that might lead to reoccurrence of these symptoms every year in winter . gradually over the years she noted that the severity of her illness increased and was causing socio - occupational dysfunctions . therefore , this year she reported for psychiatric treatment at the beginning of october . on mental state examinations she had obsessions of contamination , compulsions for washing , and assurance seeking . the severity of symptoms was assessed by administrating yale brown obsessive compulsive scale ( y - bocs ) . the total y bocs score was 30 ( obsessions score 17 and compulsions score 13 ) , other routine investigations were within normal limit . other than ocd in subsequent follow - ups , patient was also diagnosed as having cervical spondylitis . she was on fluoxetine 20 mg once daily for 1month , but did not show much improvement . therefore , keeping in view the seasonal variation in the pattern of oc symptoms , she was also considered for phototherapy . she was also taken up for exposure and response prevention ( erp ) therapy twice a week for 1hour , where therapist assisted the patient in exposure . her y - bocs score came down to 8 ( 5 for obsession and 3 for compulsion ) and she was discharged on fluoxetine 20 mg once daily maintenance treatment . epidemiological studies have found the prevalence rates of sad to be generally higher in northern locations like north america and europe , and generally lower in warmer , tropical countries . however , the influence of latitude in prevalence of ocd is yet to be established by cross - sectional surveys from different countries . studies also suggest that seasonal ocd is rare in prevalence and difficult to diagnose . one reason might be the requirement of long history with respect to seasonality is required for establishing diagnosis . in a large - scale study of children in united states aged 9 - 17 years , significant seasonal variations were found in overanxious disorder , ocd , separation anxiety disorder , social phobia , and major depressive disorder , with worsening of symptoms in august - october . similarly , a retrospective study of 34 patients with ocd found that 53% patients with ocd reported a marked seasonal variation in their symptoms . while the reasons for the seasonal variation in ocd symptoms is not known , alterations in monoaminergic neurotransmission in the brain are believed to be the reason for seasonal variations in mood disorders . in a study of 101 healthy men , it was found that the turnover of serotonin by the brain was lowest in winter . moreover , the rate of production of serotonin by the brain was directly related to the duration of bright sunlight , and increase rapidly with greater duration of sunlight . these findings are supportive of the notion that changes in release of serotonin by the brain may underlie seasonal variations in ocd . the alteration of brain serotoninergic systems in ocd supports the use of light therapy for the treatment of ocd . in fact , just like in the present case , a reduction in oc symptoms by bright light therapy has been reported earlier in a patient having seasonal variation in ocd . can these variations in symptoms be solely attributed to climate ( light ) changes or there are factors that still remain unexplored . it is also questionable whether the response is because of anti - obsessional drugs or it was just a placebo effect of the drug therapy . a careful prospective study of seasonal pattern of ocd will help in understanding symptoms , course , and suitable treatment options in such cases . different clinical course and treatment response indicates different and/or overlapping neurobiological basis for the seasonal ocd . is seasonal variation of ocd different from ocd unrelated to seasons ? our case had contamination obsession and washing compulsion , but whether seasonal ocd manifest in other types like aggression , sexual , hoarding or symmetry , and whether it may be classified as reactive or further it can be speculated that , like sad , seasonal ocd may be of some diagnostic value or can be taken as part of spectrum or a course specifier in nosology .

a case of obsessive - compulsive disorder ( ocd ) with seasonal variation in symptoms of 10-years duration is reported because of its rarity . the phenomenology of the observed disorder was obsessions related to dirt and contamination resulting in washing compulsions with onset in october and complete resolution in april - may every year . the patient responded to phototherapy along with exposure and response prevention therapy and pharmacotherapy .

CASE REPORT DISCUSSION

helicobacter cinaedi has come to be recognized as an important organism , especially among immunocompromised patients . h. cinaedi sometimes causes cellulitis but the main symptom is only fever without any focal findings in most cases . furthermore , unlike most other infectious diseases , the clinical course is not acute . for these reasons , when an sle patient becomes febrile , we should determine whether there has been an exacerbation of sle itself or whether the condition is a complication of an infectious disease . this is vital because opposite strategies are used in the treatment of sle and infectious diseases . in the present case , h. cinaedi bacteremia occurred in a patient with sle , who initially only showed fever . these symptoms mimicked a flare of the disease , which led us initiate the bolus administration of methylprednisolone . her symptoms included a typical rash , severe multiple organ failure , consciousness disturbance and retinal vasculitis . a laboratory analysis revealed reduced c3 , c4 and ch50 levels ( 20 mg / dl , 7 mg / dl and 10 u / ml , respectively ) and anti - nuclear antibody positivity ( 640 , homogenous , speckled ) , and anti - dna antibody negativity . she was in a severe condition and was refractory to the initial treatment of prednisolone ( 1 mg / kg / day ) . plasmapheresis , high - dose methylprednisolone ( 1 g bolus for 3 days ) and an increased dose of prednisolone ( 1.3 mg / kg / day ) were necessary to achieve remission from sle . after the initial therapy , her condition remained stable with the administration of prednisolone ( 10 mg / day ) for more than 10 years . a regular blood examination showed a gradual increase in the patient 's c - reactive protein ( crp ) level from 3 months before her admission , which had previously been normal . at first , an exacerbation of sle was suspected and the amount of prednisolone was increased to 15 mg / day . one month before the admission , she suffered from a high grade fever ( 39 ) at night with shivering , chills and mild nausea . a complete blood cell count revealed anemia ( hb , 7.3 g / dl ) with an increase in the number of fragmented red blood cells , leukocytosis ( wbc , 18,880 /l ) with neutrophilia ( 93.5% ) , and severe thrombocytopenia ( platelet count , 37,000 /l ) . tests for anti - nuclear antibodies and anti - dna antibodies were negative and the patient 's complement values were normal ( c3 , 87 mg / dl , c4 , 24 mg / dl , and ch50 , 51 u / ml ) . an interferon- release assay tb was negative and no vegetation was observed by transthoracic echocardiography . 1 ) and the values of alkaline phosphatase and -glutamyltranspeptidase were also elevated ; however , she had no abdominal pain . the patient 's blood cultures being negative , and the focus of infection had not been detected on the fourth day of admission . we were therefore forced to start methylprednisolone ( 1,000 mg / day ) due to suspected acalculous cholecystitis which was thought to have occurred due to a flare of sle or a complication of vasculitis . the next day , the patient 's blood cultures were found to be positive ( after 5 days of incubation ) and microscopy showed gram - negative spiral bacteria ( fig . the isolate was finally identified as h. cinaedi by a pcr and 16s rrna gene sequencing . we considered that the patient 's thrombocytopenia had been caused by h. cinaedi bacteremia , and stopped the bolus administration of methylprednisolone ; prednisolone was reduced to the maintenance dose . with the administration of meropenem , the patient 's temperature decreased and there was an improvement in her laboratory data with regard to her platelet count , crp level , and the number of fragmented red blood cells . the antibiotic was changed and de - escalated to ampicillin , which caused a systemic drug eruption . doripenem was administered for 3 weeks ; thus , the total duration of treatment reached 5 weeks , at which point , the antibiotics were stopped due to pancytopenia . one month after the treatment , the h. cinaedi bacteremia relapsed with a high - grade fever and cellulitis ( fig . meropenem was administered intravenously for another 2 weeks , and 6 weeks of oral minocycline were given , no relapse occurred after this treatment . abdominal ct on admission showing the thickness of the gallbladder wall ( white arrowheads ) . psl : prednisolone , mpsl : methylprednisolone , mepm : meropenem , abpc : ampicillin , ipm / cs : imipenem / cilastatin , azm : azithromycin , drpm : doripenem , wbc : white blood cell ( /l ) , hb : hemoglobin ( g / dl ) , plt : platelet ( 10/l ) , ast : aspartate aminotransferase ( u / l ) , alt : alanine aminotransferase ( u / l ) , alp : alkaline phosphatase ( u / l ) , crp : c - reactive protein ( mg / dl ) , closed triangle : positive blood culture , open triangle : negative blood culture cellulitis complicated with secondary bacteremia was seen on both extremities and the patient s back . h. cinaedi is a gram - negative spiral bacterium that belongs to the helicobacteriaceae family . it is hypothesized that h. cinaedi infection occurs via the oral route . in some reports , the possibility of zoonotic transmission from animal feces to humans has been discussed . however , the patient had no pets and no contact with animals . the median time for h. cinaedi to become positive in blood cultures is 5 days ( 2 - 12 days ) , and the positive ratios from 5 to 7 days were only 55% and 87% , respectively ( 1 ) . in order to successfully detect h. cinaedi bacteremia , at least 7 days of incubation should be necessary , although only 1 - 3 days are sufficient for most other organisms . the other characteristics of h. cinaedi bacteremia are nonspecific symptoms and a tendency of relapse . it has been hypothesized that h. cinaedi infection is associated with cellulitis , arthritis and gastroenteritis ( 2 - 4 ) ; however , fever is the only symptom in many cases . actually , in ten cases of h. cinaedi bacteremia that were observed in our hospital in the eight years from 2007 to 2014 , the only symptom was fever in five cases . the four cases of ten showed skin lesions ; however , these were very mild in all of the cases the appropriate antimicrobial agents and the duration of the treatment for h. cinaedi infection have not been established . although we treated the patient for a sufficient length of time ( 5 weeks ) , she nevertheless relapsed . cinaedi bacteremia cases are usually reported in immunocompromised patients such as those with hiv infection , hematological disorders , malignancies , alcoholism and receiving immunosuppressive therapy ( 5,6 ) . some other infectious diseases have been reported to mimic sle or a flare of sle , human parvovirus b19 , cytomegalovirus , visceral leishmaniasis , which is only in endemic area , and so on ( 7,8 ) . some bacterial infections may mimic a flare of sle , and h. cinaedi might be one of them . however , as far as we are aware , reported cases on h. cinaedi bacteremia with sle are very rare ( 9 ) . we finally diagnosed gallbladder thickening and thrombocytopenia , which were associated with h. cinaedi infection itself , not with a flare of sle . although the association with hepatobiliary diseases and enterohepatic helicobacter species have been mentioned , most cases were caused by h. bilis or h. hepaticus ( 10 ) , and not h. cinaedi or cinaedi , there have been only a few reports on hepatobiliary diseases in animals ( 11 ) and only one report on hepatobiliary diseases in humans . our case might represent a possible presentation of the hepatobiliary infections caused by h. cinaedi in humans . in conclusion , we should be mindful of the presence of h. cinaedi bacteremia , especially when we see febrile immunocompromised patients such as patients with sle . cinaedi takes longer to diagnose , the most popular symptom is only fever and it is sometimes associated with various symptoms . once an h. cinaedi infection is diagnosed , it is necessary to treat the patient with antibiotics for more than one month due to the tendency to relapse .

a 40-year - old woman with systemic lupus erythematosus ( sle ) presented with high - grade fever and severe thrombocytopenia . acalculous cholecystitis and thrombocytopenia were initially suspected to be complicated with sle and vasculitis . contrary to our expectation , however , the patient was finally diagnosed with helicobacter cinaedi bacteremia . sle patients show various symptoms , especially when their condition is complicated with vasculitis , which mimics h. cinaedi bacteremia . it is therefore difficult to provide a definite diagnosis . physicians should be mindful of the presence of h. cinaedi infection .

Introduction Case Report Discussion The authors state that they have no Conflict of Interest (COI).

interactions of rna with other biomolecules are fundamental in cellular processes . in particular , noncoding rna protein interactions play important roles in protein synthesis ( 1,2 ) , gene expression ( 3,4 ) , rna processing ( 57 ) , developmental regulation ( 8,9 ) , etc . some lncrnas function as transcriptional regulators through direct association with transcription factors ( 1012 ) , while others indirectly interact with proteins in cooperation with genomic dnas ( 13 ) . the functional importance of many ncrna protein interactions for correct transcriptional regulation has been demonstrated ( 1419 ) , suggesting wide - ranging effects of ncrna protein interaction . in addition to targeting or being targeted by proteins or protein - coding transcripts , ncrnas could also potentially target other ncrnas , resulting in a layer of regulatory interactions between noncoding rna classes ( 11,20 ) . consequently , cataloguing interactions of ncrnas and other biomolecules is significant for gaining insight into biological processes and understanding the mechanism by which ncrnas carry out their regulatory function . given the importance of ncrna interactions in various pathways , we embarked on a project to build a comprehensive catalogue of such data and established the npinter database ( 21 ) . a large amount of new research has led to deeper insight into ncrna interactions at a variety of levels ; thus , npinter has been updated to v2.0 to accommodate for such expanding data resources . following data collection as described below , noncoding molecules in npinter were automatically filtered and assigned identifiers from noncode ( 22 ) or mirbase ( 23 ) , while protein - coding molecules were assigned identifiers from uniprot ( 24 ) , refseq ( 25 ) or unigene ( 26 ) . with the exception of rrnas and trnas , all reported interactions of ncrnas were included . the aim of the database is to provide a platform that will facilitate both bioinformatic as well as experimental research . in addition to a user - friendly interface and a convenient search option that allow efficient recovery of related interactions and other information , npinter v2.0 also provides a visualization platform for related interactions . based on the first version of npinter database , new datasets from literature and other related databases were collected . in this update , data from literature accounts for the major source . we first retrieved literature published in the last 5 years from pubmed , employing key words including clip interaction , non - coding rna bind protein post - transcriptional , rna rna interaction , lncrna protein interaction , lncrna bind , rna protein cross linking , rip non - coding rna , etc , and found 1270 relevant articles . information on reported interactions , either verified by experiments or derived from binding sites generated by sequencing , was manually extracted . for the latter , we only extracted processed data by authors , rather than raw sequencing data . binding sites were first compared with refseq coding genes to eliminate those located within coding regions and then screened against noncode , which serves as an ncrna reference database . binding sites that lie within noncode ncrnas were retained and assigned noncode ids , while others were discarded . as interaction partners , proteins , protein - coding rnas and dna were assigned uniprot ids , refseq ids and unigene ids , respectively , while other noncoding rnas and dna were still assigned noncode ids or mirbase ids . we also integrated data from external resources , mainly lncrnadisease ( 27 ) , which curated 478 experimentally supported lncrna interactions at various levels , including binding , regulation and co - expression . curation on data from literature and integration of data from lncrnadisease , npinter v2.0 provides a multilevel snapshot of the interactome . a process of redundancy elimination was then performed on the whole dataset , including previously existing data and newly collected data as mentioned above . the workflow used for the generation of npinter is schematically shown in figure 1 . rna binding sites of individual proteins identified using genome - wide techniques were screened against noncode and only those correspond to noncode rnas were extracted . rna binding sites of individual proteins identified using genome - wide techniques were screened against noncode and only those correspond to noncode rnas were extracted . the purpose of the database is to serve as a knowledge base for experimentally - oriented studies and as a resource for bioinformatics applications . the first release of npinter in 2006 contained 700 published functional interactions from six model organisms . npinter v2.0 presently contains 201 107 ncrna interactions distributed on 18 organisms , collected from 529 published articles . the significant growth in the amount of data is primarily because of systematic identification of protein binding sites on the transcriptome through a combination of clip and rna sequencing , whereas other interactions , including ncrna rna interactions and transcript factor ( tf)ncrna interactions , were obtained mainly from interaction studies on individual ncrna . the basic information on each interaction contains an interaction i d , name of ncrna and its interaction partner , organism in which the interaction was identified , level and class of interaction manually defined in npinter , as well as tags manually added . for example , at the binding level , which accounts for most of the npinter datasets , ncrnas may interact with dna ( 13,28 ) , ncrna ( 29 ) , mirna ( 30 ) , mrna ( 31 ) and protein ( 32 ) . at the level of indirect interactions , ncrnas may either regulate or be regulated by an interaction partner ( 33,34 ) . the level of interaction defined in npinter represents the types of interacting molecules and characteristics of the interaction , including rna protein , rna rna and dna tf. in general , npinter classifies all interactions into three classes , which are regulatory and co - expression. tags are added to give brief introductions to each interaction , suggesting in which biological process the interaction participates . mirna , mirna target interaction , ncrna affects synthesis or function of protein , ncrna is regulated , ncrna protein binding , expression , processing , or function of ncrna is affected , ncrna targets mrna , other linkages , promoter as action site , regulatory and rna rna interaction. each interaction can be labelled simultaneously with several tags . the npinter database is composed of three linked tables : the interaction table gives a description of details of the interaction between two molecules . for example , for the interaction between kcnq1ot1 and dnmt1 in mouse , information in this table states that this interaction occurs at the level of rna protein and is classified into protein binding.the molecule table describes information on interacting molecules , containing identifiers from unigene , noncode , mirbase , refseq or uniprot as well as the name , aliases and description of each molecule in npinter.the reference table gives the details of literature references in the interaction table . each record in the table includes the medline standard article code ( pmid ) as well as general publication information . for example , for the interaction between kcnq1ot1 and dnmt1 in mouse , information in this table states that this interaction occurs at the level of rna protein and is classified into binding class with a tag ncrna protein binding. the molecule table describes information on interacting molecules , containing identifiers from unigene , noncode , mirbase , refseq or uniprot as well as the name , aliases and description of each molecule in npinter . each record in the table includes the medline standard article code ( pmid ) as well as general publication information . users can also query the database through the search interface , using the name or aliases of molecules , molecule ids or any other descriptive words . the whole datasets of npinter can be directly downloaded from the webpage , and the results of each search can be exported . in the updated version of the database , we have also integrated the online blast service ( ncbi wwwblast version 2.2.17 ) , which allows sequence similarity searches for both nucleotide and peptide to be run on npinter entries . importantly , for a noncoding rna with no recognized name or identified function , it is also possible to search its potential interactions with other molecules in npinter simply based on its sequence . additionally , npinter also offers a graph - based visualization of interactions ( figure 2 ) . in the visualization graph , each molecule is a node and interactions between molecules are designated as edges . red nodes represent proteins or protein - coding rnas and dna , while blue and pink nodes represent ncrnas and mirnas , respectively . information of each molecule can be checked in a new window by clicking on the corresponding node . the colour of the edges discriminates the source of each interaction , green suggesting the interaction is from npinter and purple suggesting the interaction is from string ( 35 ) . the location of the nodes is determined by the forcedirected layout algorithm as implemented in cytoscape web ( 36 ) . the graph can be opened in the stand - alone webpage to produce high resolution images in which size , colour and location of nodes and edges can be adjusted . to maintain an up - to - date and comprehensive resource , we encourage users to submit newly published ncrna interactions , with pubmed accession numbers required . the initial placement of the nodes is determined by a force - directed layout algorithm that aims to keep the more similar nodes closer together , but the placement may later be adjusted by the user . the initial placement of the nodes is determined by a force - directed layout algorithm that aims to keep the more similar nodes closer together , but the placement may later be adjusted by the user . noncoding rnas have emerged as key molecular players in different biological processes ( 37 ) . characterizing ncrna interactions will largely contribute towards the discovery of novel ncrna functions . however , existing databases are not enough to provide a comprehensive resource for such a purpose . for example , starbase ( 38 ) lists all binding sites of several proteins in the transcriptome , but there is no specific annotation for noncoding transcripts . for another example , npidb ( 39 ) focuses on nucleic acid protein complexes from pdb , thus , most rnas included are either rrnas or trnas . in contrast , npinter is more informative on ncrna interactions than other databases , with aims on integrating interactions of the other types of ncrnas that serve a variety of functions . in other words , npinter is one of the most comprehensive databases of interactions between ncrnas and other biomolecules . compared with the previous version of npinter , the new version is a step towards a more integrated knowledge database . the total number of functional interactions of ncrnas has been expanded . npinter v2.0 is also developed to present graphical molecular interaction networks that will enable biological scientists to explore their data in a more systems - oriented manner . the authors plan to update npinter whenever there is an accumulation of novel ncrna interactions reported in literature or other sources . it is worth mentioning that npinter is included in our systematic platform for noncoding rnas . our platform consists of ncrna resources such as noncode and npinter , as well as online tools and web servers ( 4043 ) including cnci ( 40 ) and ncfans ( 43 ) for analysis of ncrnas . as a member of the union of specific databases and tools for noncoding rnas , npinter is expected to remain an informative and valuable data source on the biological roles of ncrnas for the scientific community . funding for open access charge : national high - tech research and development projects 863 [ 2012aa020402 ] ; training program of the major research plan of the national natural science foundation of china ; national natural science foundation of china ; chinese academy of science strategic project of leading science and technology [ xda01020402 ] ; national key basic research and development program 973 [ 2009cb825401 ] ; the national center for mathematics and interdisciplinary sciences .

npinter ( http://www.bioinfo.org/npinter ) is a database that integrates experimentally verified functional interactions between noncoding rnas ( excluding trnas and rrnas ) and other biomolecules ( proteins , rnas and genomic dnas ) . extensive studies on ncrna interactions have shown that ncrnas could act as part of enzymatic or structural complexes , gene regulators or other functional elements . with the development of high - throughput biotechnology , such as cross - linking immunoprecipitation and high - throughput sequencing ( clip - seq ) , the number of known ncrna interactions , especially those formed by protein binding , has grown rapidly in recent years . in this work , we updated npinter to version 2.0 by collecting ncrna interactions from recent literature and related databases , expanding the number of entries to 201 107 covering 18 species . in addition , npinter v2.0 incorporated a service for the blast alignment search as well as visualization of interactions .

INTRODUCTION DATA COLLECTION AND ANNOTATION DATABASE CONTENT AND STRUCTURE DATA ACCESS AND VISUALIZATION CONCLUSION SUPPLEMENTARY DATA FUNDING

fecundity is reduced in male patients with congenital adrenal hyperplasia ( cah ) due to 21-hydroxylase deficiency . recent studies suggested that development of testicular adrenal rest tumors ( tarts ) , which cause an obstruction of the seminiferous tubules , may play a major role [ 1 , 2 ] . in addition , suppression of the gonadal axis due to adrenal androgen excess might also cause reduced fertility . both pathomechanisms are thought to be a consequence of insufficient hormonal control . besides these somatic causes of impaired fertility in cah males , there might be aspects of psychosocial adaption and sexual well - being which may be additional factors for impaired fertility . however , up to now there are no studies investigating sexual well - being in male cah patients . sexual function is best measured by patient self - report avoiding interviewer bias and only patients can report on issues such as sexual interest and the extent to which sexual dysfunction has an adverse effect on their quality of life . the brief sexual function inventory ( bsfi ) provides an excellent tool to assess a self - reported measure of current sexual functioning . the aims of our two - year prospective study in adult male patients with congenital adrenal hyperplasia wereto investigate changes in hypothalamic - pituitary - testicular regulation by gnrh testing , to evaluate changes in sexual functioning and quality of life . to investigate changes in hypothalamic - pituitary - testicular regulation by gnrh testing , to evaluate changes in sexual functioning and quality of life . the subjects were adult male patients with confirmed classical cah due to 21-hydroxylase deficiency with regular hormonal follow - up at the outpatient clinic of the department of endocrinology of the charit campus mitte hospital , berlin . the study was approved by the ethics committee of the charit campus mitte berlin ( permit no . exclusion criteria were other diseases with impairment of gonadal capacity and other general and psychiatric diseases . all patients were seen at the outpatient clinic by two experienced endocrinologists ( m.q . ; m.v . ) on a regular basis every six months . in each patient physical examination , blood drawings ( between 0800 and 1000 h , 2 h after morning medication ) , questionnaires , and testicular ultrasounds were performed at study start ( baseline ) and two years later ( follow - up ) . the treating physicians tried to optimize treatment during the study period according to the endocrine practice guidelines . the standard medications for the treatment of 21-ohd deficiency are hydrocortisone ( hc ) , prednisolone ( pr ) , and dexamethasone ( dx ) [ 6 , 7 ] . since these glucocorticoids have different biological strengths , dosage for pr and dx were converted into hydrocortisone equivalent ( pr was converted 1 to 5 to hc , dx 1 to 70 to hc ) [ 8 , 9 ] . after conversion to the hydrocortisone equivalent dose , the daily total amount of hydrocortisone equivalent in milligrams was calculated as well as the total daily dose per body surface area ( mg / m ) . grey - scale and color doppler ultrasonography of the testes was obtained in longitudinal and transverse sections . circulating concentrations of 4-androstenedione ( ad ) ( beckmanncoulter , krefeld , germany ) , testosterone , dheas ( dpc biermann gmbh ; bad nauheim , germany ) , lh , fsh , renin concentrations , acth , and 17-hydroxy - progesterone ( 17-ohp ) ( mp biomedicals gmbh ; eschwege , germany ) were measured by commercially available assays . the gnrh stimulation test was performed by administering 100 g gnrh ( aventis pharma gmbh , frankfurt , germany ) as an i.v.bolus . serum fsh and lh levels were measured at 0 and 30 min after gnrh dose . we used the differences between peak and basal lh and fsh concentrations , referred to as max , as response variables to eliminate the additive effect of basal lh or fsh level on the peak . a normal lh response in gnrh testing was assumed if lh levels rise was at least 3-fold ; normal fsh response if fsh rises was at least 50% or above 3 psychometric evaluation of patients was performed using three validated self - assessment subjective health status ( shs ) questionnaires : the sf-36 , the brief form of the giessen complaint list ( gbb-24 ) , and the hospital anxiety and depression scale ( hads ) . in addition , the sexual functioning was assessed by the male brief sexual function inventory ( bsfi ) . all four questionnaires were presented as self - explanatory , multiple - choice self - assessments . the sf-36 questionnaire is the most widely used generic instrument to assess quality of life ( qol ) . it consists of eight multi - item domains representing physical functioning ( pf ) , role functioning physical ( rp ) , bodily pain ( bp ) , general health perception ( gh ) , vitality ( vt ) , social functioning ( sf ) , role functioning emotional ( re ) , and mental health ( mh ) . the domain scores range from 0 to 100 with higher values indicating better qol [ 12 , 13 ] . each item is scored as a number , with a maximum score of 21 for each subscale . higher scores indicate higher levels of anxiety or depression . a cut - off value of 8 is regarded as indicating mild impairment , and a cut - off value of 11 is indicative of severe impairment . the short form of the gbb-24 questionnaire consists of 24 items defining four subscales ( exhaustion tendency , gastric symptoms , pain in the limbs , and heart complaints ) , each including six items with ratings from 0 to 4 . in addition , a global score of discomfort ( gsd ) is calculated by adding the four subscale scores . the maximum value for each subscale is 24 , and for the global score 96 . higher scores indicate greater impairment of well - being . regarding control group data we calculated the z - scores by using reference data for sf-36 scores obtained from the german national health survey ( bundesgesundheits - survey 1998 , robert koch institut , berlin 2000 , public use file bgs 98 ) comprising a representative random sample of 7124 subjects from the german population aged between 18 and 79 yr . reference data for the hads ( n = 4410 ) and the gbb-24 ( n = 2076 ) were obtained from previously performed surveys [ 1517 ] . the brief sexual function inventory ( bsfi ) was used to assess perceived problems associated with sexual drive ( two items ) , erection ( three items ) , problem assessment ( three items ) , ejaculation ( two items ) , or overall satisfaction ( one item ) . each question was scored on a 5-point scale , ranging from 0 to 4 , with lower scores indicating worse sexual function . regarding control group data we calculated the z - scores by using normative data for the bsfi obtained from a representative random sample of 1185 subjects from the norwegian population aged between 20 and 79 yr . results are expressed as mean standard deviation ( sd ) if not stated otherwise . the significance of data was determined by students t test in normally distributed and in not normally distributed data by mann - whitney - wilcoxon test where appropriate . eight patients were excluded due to testicular operations or other exclusion criteria . finally 20 patients were enrolled into the study . three patients did not participate in the 2-year follow - up visit and were not included into the 2-year follow - up analysis . clinical and genetic characteristics are shown in table 1 : 14 patients had salt - wasting cah and 6 patients had simple - virilizing cah . patients with salt - wasting cah were diagnosed within the first week after birth ; patients with simple - virilizing form were diagnosed in the first 7 years after birth . biochemical and hormonal parameters of the 17 patients at baseline and at the 2-year follow - up visit are presented in table 2 . over the study period bmi , systolic blood pressure , lipids , androgens , and androgen precursors did not change significantly in the whole cohort or in the sw and sv subgroups . of these , two were fathers ( 12.5% ) , one of one child and the other of two children . during the study period three adrenal crises occurred resulting in a calculated incidence of 8.8 adrenal crises per 100 patients / year , which is higher than the recently reported frequency in cah patients ( 4.8 crises per 100 patients / year ) and resembles more the frequency in patients with primary adrenal insufficiency ( 6.6 crises per 100 patients / year ) . decreased dheas levels were measured in 15 patients ( 88.2% ) at baseline and in all patients at follow - up ( 100% ) . nmol / l ) was observed in no patients at baseline ( 0% ) and in only one patient ( 5.9% ) at follow - up , whereas elevated levels of 17-ohp in serum ( > 36 nmol / l ) were present in 4 patients ( 23.5% ) at baseline and 2 patients at follow - up ( 11.8% ) . the androstenedione to testosterone ( ad / t ) ratio as indicator of testicular testosterone production was normal ( < 0.2 ) in 11 patients ( 64.7% ) at baseline and follow - up ; three patients ( 17.6% ) had an ad / t ratio > 1 suggesting testosterone from predominantly adrenal origin ( table 2 ) . estradiol levels were within the normal male range ruling out any suppression of the hypothalamus - pituitary - gonadal axis by estradiol . total testosterone levels were decreased in 4 patients ( 23.5% ) at baseline and in 2 patients ( 11.8% ) at follow - up ; calculated free testosterone index was diminished in 5 patients ( 29.4% ) at baseline and in 8 patients ( 47.1% ) at follow - up . iu / l ) in all patients at baseline and in all but one patient at follow - up . basal fsh levels were elevated in three patients at baseline ( 17.6% ) and normal in all patients at follow - up . gnrh stimulation induced an adequate increase in lh in all but one patient at baseline ( 5.9% ) and in all but two patients ( 11.8% ) at follow - up . fsh failed to increase sufficiently by gnrh stimulation in two patients at baseline and at follow - up ( 11.8% ) . three patients ( 18% ) showed tart in testicular ultrasound with a size of 611 mm . in one patient tart regressed and was not detected after 2 years . in a subset of patients ( 6 of the 17 patients ) patients with an ad / t ratio below 0.2 , indicating sufficient adrenal suppression and a testosterone of testicular origin , showed significant lower 17-ohp and ad levels than patients with an ad / t ratio > 0.2 ( table 3 ) . significantly more patients with an ad / t ratio < 0.2 received dexamethasone . basal lh and fsh levels as well as testosterone levels were not different between the groups . however , the max increase in lh in gnrh testing was significantly higher in the patients with an ad / t ratio < 0.2 than those with an ad / t ratio > 0.2 ( table 3 ) . analysis of the qol questionnaires ( gbb-24 , hads , and sf-36 ) revealed no significant changes in z - scores during the 2-year study period in our adult male cah patient cohort ( figure 1 ) . however , all dimensions of the gbb-24 showed a trend to increased z - scores indicating an impairment of qol ( figure 1(a ) ) . similar results were found for the anxiety and depression z - scores of the hads questionnaires ( figure 1(b ) ) . z - scores of the sf-36 questionnaire showed a trend to impairments especially in the dimensions physical functioning , general health perception , and emotional role functioning ( figure 1(c ) ) . the dimensions role physical functioning and the analysis of the participants ' z - scores revealed that male cah patients exhibited a slightly reduced sexual drive . no significant differences in bsfi z - scores were found between patients with ad / t ratio < 0.2 or > 0.2 . further analysis showed that ad levels significantly negatively correlated with z - scores of the dimension sexual drive ( p < 0.05 ; figure 3 ) with higher ad levels associated with lower z - scores ( = impaired sexual drive ) . decreases in qol and sexual well - being were not correlated with the presence of tarts . development of tarts and suppression of the gonadal axis are possible factors that might cause reduced fertility in male cah patients [ 1 , 2 , 21 ] . it is suggested that adrenal - derived androgen excess due to insufficient hormonal control might be the underlying cause [ 2 , 3 ] . a recent study in adult male cah patients revealed a high prevalence of impaired leydig cell function and impaired spermatogenesis . however , the authors found no correlation between semen parameters , hormonal control , and tart prevalence or size . in our current study , the majority of patients showed basal testosterone and lh within the normal range of young healthy men suggesting normal leydig cell function in most of the patients . after two years no significant differences were observed in our patients indicating stable therapeutic regimens . however , lh and fsh showed a more pronounced increase ( max ) after gnrh stimulation than reported in healthy normal males . we further subdivided our cohort into a group with good hormonal control and only testicular testosterone production indicated by an androstenedione / testosterone ( ad / t ) ratio < 0.2 and a group with poorer disease control and mixed adrenal and testicular testosterone production indicated by an ad / t ratio > 0.2 . the group with an ad / t ratio > 0.2 showed a normal lh and fsh response ( max ) to gnrh compared to healthy young men . however , the group with an ad / t ratio < 0.2 presented a significant higher lh response to gnrh testing . this resembled a prepubertal response in gnrh testing but might be also due to a suppressed hypothalamic - pituitary axis with a decreased release of gnrh from the hypothalamus . this might be caused by abundant adrenal androgens , which seems not to be the case in this group with ad / t ratio < 0.2 . interestingly , the percentage of dexamethasone treated patients was significantly higher in the group with an ad / t ratio < 0.2 compared to the group with a ratio > 0.2 , but we did not find a significant difference in total daily glucocorticoid equivalent dose per body surface between the two groups . in addition , the amount of glucocorticoid used was approximately similar to that used in other recent studies with male cah patients [ 1 , 23 ] . we assume that total glucocorticoid doses were not too high because our patients showed still normal and not suppressed lh levels . in summary , this suggests that dexamethasone has a profound effect on the hypothalamic - pituitary feedback regulation . this is in accordance with previous reports that changing glucocorticoid medication from hydrocortisone to dexamethasone resulted in an increased fertility . besides these somatic causes of impaired fertility in cah males , there might be aspects of psychosocial adaption and sexual well - being which might be additional factors for impaired fertility . we performed this in a prospective fashion and used also quality of life questionnaires to detect possible other changes or general influences during the 2-year study period . during the study period our cah patients showed unchanged bmi , unchanged metabolic and hormonal parameters , and unchanged impaired z - scores in qol questionnaires . impaired qol in male cah patients has been shown in previous studies [ 7 , 2527 ] ; however , these had only cross - sectional and not a longitudinal design . we did not find differences in qol z - scores in patients that were on dexamethasone and prednisolone treatment compared to hydrocortisone only as previously reported . sexual drive , erections , and ejaculations were impaired in our cohort . it is important to point out that overall satisfaction should not be confused with the mean score of the functional domains of the bsfi , and additional factors might be involved not covered by the questions . it is known that patients with low scores on functional domains , for example , ejaculatory impairment as a side - effect of an anti - depressant drug , do not necessarily report reduced overall sexual satisfaction . we are the first to describe a clearly impaired sexual well - being in male cah patients by using an established sexual function questionnaire . interestingly , we observed that poor disease control , according to elevated androstenedione levels , was associated with a reduced sexual drive . therefore , we believe that aspects of psychosocial adaption and sexual well - being might be important additional factors for impaired fertility in our male cah patients first , cah patients have had a chronic disease since their childhood , as well as having been exposed to exogenous glucocorticoids also during pubertal development . secondly , male cah patients have still a lower height than the average male population and this might cause problems in psychosocial adaptation . however , a recent hungarian study showed that sexual activity was not clearly related to other anthropometric parameters such as height . ( 1 ) there is no normative data for germany for calculating z - scores for the bsfi , and we had to rely on normative data from norway . however , no significant differences in functional bsfi scores were found between the norwegian data and american data from the olmsted county . ( 2 ) there is increasingly reduced sexual function concerning drive , erection , ejaculation , and problem assessment with age with most of these age - related effects starting at > 50 years old . however , our patients were all below the age of 50 y. ( 3 ) our study is a rather small cohort of male cah patients ; however , this is the first longitudinal study in adult male cah patients . in conclusion , we showed that male cah patients with a normal ad / t ratio showed an increased lh and fsh response in gnrh testing indicating possible decreased hypothalamic gnrh release by glucocorticoid therapy . secondly , we found that male cah patients had impaired sexual well - being , especially regarding erections , ejaculations , and sexual drive .

introduction . men with congenital adrenal hyperplasia ( cah ) due to 21-hydroxylase deficiency show impaired fecundity due to testicular adrenal rest tumors and/or suppression of the gonadal axis . sexual well - being might be an additional factor ; however , no data exists . patients and methods . prospective longitudinal monocentric study included 20 male cah patients ( 14 salt wasting , 6 simple virilizing ; age 1849 yr ) . clinical assessment , testicular ultrasound , biochemical and hormonal parameters , three validated self - assessment questionnaires ( sf-36 , gbb-24 , and hads ) , and male brief sexual function inventory ( bsfi ) were analyzed at baseline and after two years . results . basal lh and testosterone levels suggested normal testicular function . lh and fsh responses to gnrh were more pronounced in patients with a good therapy control according to androstenedione / testosterone ratio < 0.2 . this group had significant higher percentage of patients on dexamethasone medication . gbb-24 , hads , and sf-36 showed impaired z - scores and no changes at follow - up . bsfi revealed impairments in dimensions sexual drive , erections , and ejaculations , whereas problem assessment and overall satisfaction revealed normal z - scores . androstenedione levels correlated ( p = 0.036 ) inversely with z - scores for sexual drive with higher levels associated with impaired sexual drive . conclusion . male cah patients showed a partly impaired sexual well - being which might be an additional factor for reduced fecundity .

1. Introduction 2. Methods 3. Results 4. Discussion

conventional laparoscopic surgery requires the placement of multiple ports through the abdominal wall , with the aims of maintaining adequate internal spacing of instruments to reduce clashing and facilitating tissue manipulation for dissection . these multiple transabdominal punctures are associated with morbidity and risks such as herniation , bleeding , and damage to internal organs , as well as decreased cosmesis . laparoendoscopic single - site ( less ) surgery has been developed to overcome the port - related complications of laparoscopic surgery , to minimize morbidity , and to maximize cosmetic outcome . less surgery is performed through a single keyhole incision , typically at the umbilicus , allowing the completion of several urologic procedures with the use of familiar laparoscopic instruments and skills . since raman et al . first reported less nephrectomy ( less - n ) in 2007 , subsequent studies have demonstrated that less - n is safe and feasible with outcomes equivalent to those of conventional laparoscopic nephrectomy for both benign and malignant kidney diseases . the skill of surgeons at high - volume surgery centers has now reached a sufficient level such that less partial nephrectomy of small selected renal masses yields results comparable to those of conventional partial nephrectomy . however , the passage of all of the instruments through a single access point promotes instrument clashing and maneuverability problems , loss of triangulation , and unfamiliar working angles , even for surgeons skilled at less surgeries , and these limitations have prevented these procedures from entering mainstream clinical practice . furthermore , when the need for bleeding control , increased traction , or a suture arises , it can become necessary to apply an additional port or convert to conventional laparoscopy . these limitations are encountered not only by novices but also by experienced surgeons during complicated or difficult cases . to facilitate less techniques and overcome the learning curve for novices , magnetic anchoring and guidance systems ( magss ) these devices harness magnetic forces to steer and operate completely insertable intracorporeal instruments via externally controlled magnets . the devices are typically inserted through an already established entry site into the peritoneal cavity and are then coupled via magnetic attraction across the body wall to a handheld external component . by moving the external component around on the patient 's abdominal wall the aim of this study was to investigate the feasibility and safety of mags for surgeons performing less - n for the first time in a porcine model . in this prospective study we compared the perioperative outcomes of simple less - n with or without the use of a magnetic anchoring device . a total of 10 less - n procedures were performed on five 23-month - old female pigs weighing approximately 50 kg . all animal experiments were approved by the institutional review board of samsung medical center ( seoul , korea ) and were conducted in accordance with the national institutes of health guide for the care and use of laboratory animals . the surgeons were 6 urologists working in the samsung medical center as fellowship trainees , including five participants with no prior experience with conventional laparoscopic and less surgery and one experienced laparoscopic surgeon with no prior less surgery experience ( table 1 ) . four of the surgeons performed simple less - n with and without the magnetic anchoring devices in the nonsurvival porcine model with procedures occurring at least 2 weeks apart . the pig was placed under general anesthesia and positioned obliquely on the table ( semilateral position ) . the alex wound retractor was inserted through the 2- to 2.5-cm incision site made at the midline . the homemade single - port device was constructed by first cutting the tips off the 1st , 3rd , and 5th fingers of a size 6 surgical glove . two 10-mm trocars were placed in the 3rd and 5th finger openings , respectively , and one 5-mm trocar was placed in the 1st finger opening . the glove was then fixed to the outer ring of the wound retractor . after establishing pneumoperitoneum ( controlled at less than 14 mmhg ) , long , rigid 5-mm laparoscopes were used to obtain a view of the operative field through the 3rd finger 10-mm trocar of the homemade port . six pieces of a cylindrical shaped neodymium internal magnet with a central hole ( outer diameter , 5 mm ; thickness , 5 mm ; weight , 0.69 g ; diameter of central hole , 1 mm ) were then fixed to the renal parenchyma with a 1 - 0 vicryl suture ( ethicon , somerville , nj , usa ) ( fig . a cube - shaped external handheld magnet ( width , 74 mm ; length , 74 mm ; height , 38 mm ; weight , 1,543 g ) was applied to the flank of the pig ( fig . the position of the kidney could then be changed depending on the position of the external magnet ( fig . 3 ; supplementary video clip ) . specialized articulating instruments ( laparo angle ; cambridge endo , framingham , ma , usa ) were used for most of the dissection and traction maneuvers . standard laparoscopic instruments such as a hook cautery , straight dissector , and straight scissors were also used . the surgical procedure was similar to that of a simple conventional laparoscopic transperitoneal nephrectomy in humans . after division of the renal artery and vein , the artery was clipped with a hem - o - lok clip ( teleflex medical , wayne , pa , usa ) and then transected . the renal vein was subsequently transected with a vascular endo - gia stapler ( medtronic , minneapolis , mn , usa ) . the ureter was also clipped with a hem - o - lok clip and divided . procedure duration was regarded as the intracorporeal time , not including the time required for port placement , magnetic anchoring device placement , specimen retrieval , and wound closure . all operations were observed by a urologist who recorded the procedure duration and any intraoperative complications . the mean time to complete the operation ( standard deviation ) with the magnetic anchoring device ( the m - less - n group ) was 6320.8 minutes ( range , 4595 minutes ) , which was shorter than that for surgery without the magnetic device ( the c - less - n group ; mean , 8240.7 minutes ; range , 36114 minutes ) ( table 1 ) . among four surgeons who performed two simple less - n procedures with and without the aid of mags , 3 surgeons who had no prior laparoscopic experience first performed the surgery without mags and then used mags in subsequent procedures ( table 1 ) . the other operator , who was an experienced laparoscopic surgeon but had no prior less surgery experience , initially used mags but did not use it in the subsequent surgery ( table 1 ) . a remarkable decrease in the duration of the procedure was observed with the aid of mags for the 2 surgeons who were novices in laparoscopic surgeries , whereas there was no significant improvement in procedure duration for the surgeon who was an expert in laparoscopic surgeries ( fig . all m - less - n procedures were uneventful and were completed with no need to convert to open surgery and no need to add a second port . intraoperative complications were observed in the c - less - n group only , in which there were 2 cases of renal vein injury during dissection of the renal hilum . the surgeons were unable to control the bleeding because the blood loss was rapid and they lacked the required skills . in these cases it was so difficult for the operators to continue the procedure that they decided to transect the renal artery and renal vein together without dissection by using a vascular endo - gia stapler . interestingly , 1 surgeon who experienced renal hilar injury during a c - less - n procedure completed the operation successfully using mags ( table 1 ) . since the first laparoscopic nephrectomy was performed by clayman et al . in 1991 , variations of this surgery have been performed with laparoscopic techniques . recently high - volume surgery centers have reported on their experiences performing nephrectomies , adrenalectomies , pyeloplasties , and nephroureterectomies with the less technique . even though these surgeons had experience with laparoscopic surgery , some cases of less surgery needed an additional port . these additional ports can cause bleeding , internal organ damage , or postoperative pain and hernia , thereby diminishing the cosmetic benefit and minimizing the value of the less surgery . therefore , an improved modality is required for this surgical technique to be universally applied . to this end , magnetically anchored technology is a way to minimize the number of incisions needed to operate while increasing the degree of triangulation compared to standard single - incision surgeries . the cadeddu group demonstrated the feasibility of mags for less - n in the porcine model . in addition , they reported a redesigned mags camera that provides improved optics and easy maneuverability during less porcine nephrectomy . the mags camera allows the use of standard rigid , straight laparoscopic instruments , rather than the articulating or curved ones commonly used during complex less operations . the initial results of human trials using a magnetically guided intra - abdominal camera in conjunction with less - n and less appendectomy were reported to be successful . mags camera technology significantly decreases the surgeon workload and improves ergonomics , although suturing and knot - tying during less surgery remain challenging tasks that require training . additionally , the use of magnetic forceps resulted in no intraoperative complications in 40 reported cases of human less cholecystectomy . in addition to the development of novel devices , mags is also being utilized in broader applications and in a greater number of surgical fields , including natural orifice transluminal endoscopic surgery . our study showed shorter procedure durations with use of the magnetic anchoring device compared with surgery without the device . complications requiring early clipping of the pedicle without dissection occurred in the group without the magnetic anchoring device . with use of the magnetic anchoring device , the kidney could be dragged to the abdominal wall and the pedicle could be straightened for easy division . the shorter procedure duration and fewer hilar complications observed in the group using the magnetic anchoring device support adoption of this methodology for nephrectomies . these findings are consistent with findings from previous reports of the use of magnetic devices in less - n in which surgeries were completed successfully without any intraoperative complications , although the authors of one study noted that they experienced decoupling in one human case . first , the surgeons in this study had no previous experience with performing less surgery . second , the magnetic device used was different . in our study , we simply used the magnetic force between the internal and external magnets to pull the kidney , whereas the device used in the previous porcine or human model procedures contained retractors , a magnetic camera , and a robotic cauterizer . we dissected the pedicle and clipped the renal artery and vein separately , whereas in other studies the artery and vein were transected together with the vascular endo - gia stapler . although hilar complications were observed in this study , they would not have happened without hilar dissection , which was unique to this study . despite these differences , the results of our study are consistent with those of previous studies and indicate that use of magnetic devices in these types of operations is feasible with minimal complications . in our study , 2 surgeons who initially operated without the magnetic device exhibited a remarkable reduction in procedure duration for the second surgery using mags . these surgeons may have benefitted from the assistance of the magnetic anchoring device in the second surgery . additionally , because they struggled harder and took a longer time to complete the first less - n , the long intraoperative time may have allowed them to practice and learn the skills required to shorten the second operation time . one surgeon exhibited the opposite trend : the duration of the second procedure was longer than that of the first . in this case , the pedicle was ligated early with the endo - gia stapler because of renal vein injury inflicted during the hilar dissection in the first operation . therefore , the surgeon had a shorter time to practice and the initial operative time was underestimated . as a result , it took longer to complete the second surgery with the magnetic anchoring device . in contrast , a reduction in the time to perform less - n using mags was not observed in the procedures performed by the experienced laparoscopic surgeon . there were some limitations to this study . because it was a pilot study , the number of surgeries was too small to yield statistically reliable data . second , postoperative complications were not evaluated because we sacrificed the porcine models after surgery . we did not evaluate postoperative complications because the primary focus of the experimental design was to compare the intraoperative variables of surgery aided by the magnetic devices versus the conventional less surgery . third , 4 surgeons performed less - n both with and without the magnetic anchoring device . the purpose of this study was to compare less - n with our magnetic anchoring system versus the conventional procedure . however , surgical skills could be improved by any prior less surgery and might have influenced the duration of the second procedure . therefore , comparisons between the group that used the magnetic anchoring device and the group that did not might not be reliable , even though an interval of at least 2 weeks between the surgeries was implemented to minimize this factor . in conclusion , less - n using a magnetic anchoring device is feasible and can be safely performed by surgeons with no previous less surgery experience . judging from 2 cases of renal vein injury and the slightly longer procedure duration in the group without the magnetic anchoring device , it can be suggested that the device may help novices performing less surgery dissect the renal hilum more easily . accompanying videos can be found in the ' urology in motion ' section of the journal homepage ( www.icurology.org ) . the supplementary video clips can also be accessed by scanning a qr code located on the fig .

purposemagnetic anchoring devices may reduce the number of port sites needed in laparoscopic surgery . in this study , we prospectively assessed the feasibility of using a magnetic anchoring and guidance system ( mags ) in laparoendoscopic single - site ( less ) surgery performed by novices.materials and methodsa total of 10 less simple nephrectomies were performed with or without mags in a nonsurvival porcine model by 6 operators with no previous less surgery experience . after installation of the homemade single port , an intra - abdominal magnet was fixed to the renal parenchyma with suturing and stabilized by an external magnet placed on the flank so that the position of the kidney could be easily changed by moving the external handheld magnet . the length of the procedure and any intraoperative complications were evaluated.resultsoperative time ( meanstandard deviation ) was shorter in the group using the magnetic anchoring device ( m - less - n ) than in the group with conventional less nephrectomy ( c - less - n ) ( 6320.8 minutes vs. 8240.7 minutes , respectively ) . although all nephrectomies were completed uneventfully in the m - less - n group , renal vein injury occurred during dissection of the renal hilum in two cases of c - less - n and was resolved by simultaneous transection of the renal artery and vein with an endo - gia stapler.conclusionsless-n using mags is a feasible technique for surgeons with no less surgery experience . taking into account the 2 cases of renal vein injury in the c - less - n group , the application of mags may be beneficial for overcoming the learning curve of less surgery .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS SUPPLEMENTARY MATERIALS

multiple sclerosis ( ms ) is a neurological disease characterised by the multifocal nature of neurological lesions in the central nervous system , which results in demyelination , white and grey matter injuries and/or atrophy . as a consequence of the cooccurrence of lesions , a wide range of symptoms deficits on cognitive domains as anterograde memory , executive functions , attentional processes , or information processing speed have been described and a number of clinical studies have also developed different cognitive rehabilitation programmes for ms patients . however , very few studies have been carried out in ms to investigate autobiographical memory ( abm ) . briefly stated , abm is the capacity to relive detailed events , evoking the spatiotemporal context , in which they were encountered , as they are remembered . abm deficit in ms has been debated in the literature despite the comparatively modest number of works on the topic . paul et al . found personal semantics impairment in ms patients , while kenealy et al . observed episodic personal memory deficits , but these studies did not control for the illness subtypes . more recently , mller et al . reported impaired abm in ms patients in secondary progressive ms patients and no deficit in relapsing - remitting multiple sclerosis patients ( rr - ms ) , while ernst et al . demonstrated abm deficit in rr - ms patients . although , no direct comparison can be drawn between these two recent studies ( mller et al . and ernst et al . ) , due to several methodological discrepancies , the contradictory findings are probably only apparent ( see the discussion section ) . our previous clinical study has shown that abm impairment in rr - ms patients was very likely caused by a deficit of retrieval strategies , and that abm deficit in ms could be alleviated with a cognitive facilitation programme . this programme was based on the cueing role of mental visual imagery ( mvi ) in abm , which is involved in the reconstructive process of past events and allows to cue visual and other sensory modality information about the event [ 10 , 11 ] . the clinical conclusions of our previous work led us to consider the cerebral substrates of the documented improvement of abm functioning following the facilitation programme . several fmri studies in ms have been reported , demonstrating cerebral activations during different cognitive tasks , particularly attentional processes and working memory tasks [ 1214 ] . nevertheless , only a few studies have explored the possibility of an induced cerebral plasticity in ms patients following cognitive rehabilitation . these investigations on training - induced brain plasticity in ms have effectively shown cerebral activations changes after cognitive rehabilitation for attentional deficit , attentional , dysexecutive and information processing impairments , or anterograde memory impairment . no such study has been realised on abm in ms patients , to our knowledge . therefore , the aim of the present preliminary study is to better document the mechanisms sustaining the efficacy of our mvi - based facilitation programme on abm retrieval . to this end two sets of studies on neurocognitive relationships are relevant in the present work . in the first place , the studies showing that the abm cerebral network is widespread and recruits predominantly the left and medial cerebral regions . among those regions , structures are the prefrontal cortex , medial and lateral temporal cortices , parieto - occipital regions , temporoparietal junctions , and cerebellum . concerning prefrontal regions , several authors have pointed out their central role in the retrieval process [ 19 , 20 ] . the further group of relevant studies in the present work are those reporting that mvi relies mostly on posterior cerebral regions and that mvi implication in abm is also supported by the partial overlapping of cerebral activations observed during abm and mvi tasks . on those bases , we hypothesised that the patients showing an improvement of abm performance documented by clinical changes measured after cognitive training , would also show cerebral activation changes during the remembering processes of personal past events . more specifically , we expected an increased activation of posterior cerebral regions in the postfacilitation fmri session given that our programme was constructed to enhance and optimise the role of mvi in abm . eight patients with definite ms according to the mac donald criteria were recruited at the neurology units of two french hospitals ( strasbourg and colmar ) . inclusion criteria were the diagnosis of a relapsing - remitting disease course , a mild functional disability corresponding to an expanded disability status scale ( edss ) score 4 , an absence of major signs of depression according to the montgomery and asberg depression rating scale ( madrs ; significant clinical threshold score 15 ) , no recent exacerbation of ms symptoms and right - handedness for patients who underwent fmri sessions . fifteen healthy controls were also recruited to constitute a normative database for the abm test . exclusion criteria for all the participants included documented psychiatric illness , neurological disorder ( other than ms for the patient group ) , and poor knowledge of french . demographic and clinical data for the groups of ms patients are summarized in table 1 . all subjects signed prior informed consent , we complied with the apa ethical standards , and the research was approved by the committee for protection of persons verbal iq was assessed with the short form of the wais - iii verbal scale and nonverbal reasoning with the advanced progressive matrices set 1 . anterograde memory was examined using the rey auditory verbal learning test ( ravlt ) for the verbal modality and the rey - osterrieth complex figure ( rocf ) [ 29 , 30 ] was used to assess the visual modality . executive functions were assessed using the phonological and categorical fluency tests ( national hospital , london ) , the brixton spatial anticipation test , the tower of london and the cognitive estimation task . concerning attentional and information processing , the information processing speed test from the adult memory information processing battery ( amipb ) , the stroop test and the months back test ( national hospital , london ) were also administered . since the experimental test assessing abm is language - dependent , language was also examined , by means of the dno 100 test . the visuoperceptual and visuospatial abilities were tested using the silhouettes and cube analysis subtests from the visual object and space perception battery ( vosp ) . finally , the short version of the echelle de mesure de l'impact de la fatigue ( emif - sep ) was proposed to evaluate the impact of fatigue in daily life . the autobiographical interview ( ai ; kindly communicated by the author , brian levine to one of us , lm . ) was translated into french and adapted following addis and colleagues . three cue - words were presented to the participants to probe past events for each of the four or five life periods depending on subject 's age ; 011 years , 1220 years , 21 to ( current age 1 ) or 2135 years , 36 to ( current age 1 ) and the previous year . we therefore obtained either 12 or 15 past events per participant , which differs from the usual ai procedure in which only one memory per life period is required . however , if only one single event per period was to be found , the participant was more likely to provide the most accessible detailed recollection [ 41 , 42 ] . consequently , we departed from the standard procedure to test a greater number of recollections in order both to probe more comprehensively the retrieval process and assess the benefits of our facilitation programme . participants were asked to retrieve unique past events temporally and contextually specific , having occurred over minutes or hours ( but not more than one day ) and to generate freely as much details as possible about this event . they were informed that the cue - words , as stimuli triggering personal recollections , were intended to be used flexibly rather than literally . no time limit was set to avoid the potential influence of decreased cognitive processing speed on abm performance . general probes were used to clarify instructions if necessary and to encourage the recall of additional details . the next step consisted in administering the specific probe phase , that is , a structured interview to test further details about a given event . only two phases of recall were distinguished , that is , the free recall and specific probe phases , since the free recall and the general probe phases were analysed as a whole considering the minimal effect of general probes on recall . however , scores were analysed cumulatively across the two levels of recall ( i.e. , free recall and specific probes ) . the first step of scoring was the identification of the main event , this permitting to classify each detail as internal ( i.e. , an episodic detail related to the main event ) or external ( i.e. , nonepisodic information as semantic detail , repetition , metacognitive statement or episodic details but not related to the main event ) . a qualitative assessment of the episodic re - experiencing was also provided by ratings for episodic richness , time , space , perception and emotion / thought composites for each memory . after an extensive training in the scoring method ( also supplied by brian levine to one of us , l. manning ) , scoring for all the participants was done by one scorer ( a. ernst ) and 10% of the memories were analysed by a trained second scorer ( five trained students ) , following levine et al . coefficients for all measures showed a high interrater reliability ( between 0.82 and 0.99 ) . we used a semistructured interview to obtain a qualitative evaluation by the patients of their abm performance . the aim of this interview was twofold to characterise more precisely the patient 's abm difficulties as well as the potential effects of those difficulties in real life and to evaluate the benefits of the facilitation programme . this interview was adapted from the memory experiences questionnaire , from which we selected four dimensions ( vividness , accessibility , sensory details and emotional intensity ) based on our previous study . a semi - structured interview was deemed to be better adapted than a questionnaire to explore everyday life . the cognitive facilitation programme was constructed to alleviate abm retrieval difficulties by means of a memory facilitation technique based on mvi . this technique was presented to the patients as a tool to facilitate access to a given recollection and the details associated to it . therefore , during the mvi training sessions , patients found it increasingly easier to pay close attention to details in the mind 's eye , which was used spontaneously when evoking personal recollections . moreover , they were encouraged to try and use this technique in their everyday life and if needed , to ask questions to the neuropsychologist about the use of this tool . treatment receipt , that is , the extent to which the patient understands the strategies or techniques taught , and demonstrates the capacity to use them ( p. 836 ) was verified at different times during the training process , by means of direct questions . the programme consisted in 2-hour individual sessions once a week for at least six weeks . the patients performed mvi tasks of increasing difficulty , the content of which was divided into four steps . the rate of progression through these steps was flexible and could be adapted to each patient . completion of the entire programme required at least six sessions but additional sessions were proposed if necessary to complete the programme at the patient 's rhythm of training . all over the duration of the training programme , the role of the neuropsychologist was to teach how to construct visual scenes , and to provide continuous guidance throughout the training sessions.the screening test was based on three sub - tests of the imagery and perception battery : the mental representation of physical detail test , the morphological discrimination test , and the colour comparison test . we used a shortened version of each test with normative data established with a group of 15 healthy controls ( different from the healthy control group who participated in the present study ; unpublished data ) . we used these tests to probe basic visual imaging abilities , which enabled us to exclude patients presenting with severe mvi impairment ( incompatible with the realisation of the facilitation programme ) . thus , patients who presented scores below the normal range for all the three sub - tests were excluded.the external visualization exercise included 10 verbal items that the patient had to imagine and describe . ( we called this step external visualisation because the purpose of the exercise was to imagine an object and not the participant him / herself ) . the instruction ran as follows : i will tell you the name of something ( e.g. , an onion ) , imagine it and describe its colour , shape , size , consistency and every detail that you see . each item description was checked for accuracy using a list of typical traits . for each item , the patient had to describe static aspects ( e.g. , colour , shape ) and an action carried out with the item ( e.g. , slicing an onion ) . the action was imagined to be performed either by him / herself or someone else ( no instruction was given as to who was supposed to act , since the patient was asked to concentrate on an object having changed in its physical appearance owing to an action).the construction exercise of the programme consisted in figuring out complex scenes bringing into play several characters . the goal was twofold : to increase the complexity of the scene by comparison with the previous step and to introduce several characters performing different complex actions . five items were proposed with , for each one , a first training step and a subsequent scene construction step . during the training phase , a neuropsychologist guided the patient , starting from a general idea ( e.g. , a cook prepares a meal ) , to more precise subheadings ( e.g. , the cook is in front of a metallic table ) . within subheadings , detailed items were distinguished ( e.g. , on the table , you can see butter , some onions and mushrooms ) . the present step was considered to be external since the participant was asked to imagine some characters and focus primarily on them , their actions and the context of the scene rather than the patient him / herself . responses were checked for accuracy in terms of their relation to each subheading , but no list of typical or expected responses was used . the number of headings and subheadings , as well as details produced by the patients was recorded . for the scene construction phase , an item similar to the one used in the training section was proposed ( e.g. , can you imagine the job you would most enjoy ? will you describe it , as we just did with the example of the cook?).the self - visualisation exercise was the training phase that shared the most similarities with abm since the patients were asked to visualise themselves within a given scenario , to imagine it as though they were actually living the scene and to describe whatever details , sensations or feelings came to mind . the presentation was similar to that of the previous exercise , comprising a training phase ( e.g. , you are heading towards a hotel reception desk ) and a scene construction phase ( e.g. , your room is not ready ; imagine yourself in the hotel bar ) . the difference was that the participant was asked to focus his / her attention on him / herself , that is , using internal , personal knowledge . as for the previous step , we recorded the number of headings , sub - headings and details produced by the patients . the screening test was based on three sub - tests of the imagery and perception battery : the mental representation of physical detail test , the morphological discrimination test , and the colour comparison test . we used a shortened version of each test with normative data established with a group of 15 healthy controls ( different from the healthy control group who participated in the present study ; unpublished data ) . we used these tests to probe basic visual imaging abilities , which enabled us to exclude patients presenting with severe mvi impairment ( incompatible with the realisation of the facilitation programme ) . thus , patients who presented scores below the normal range for all the three sub - tests were excluded . the external visualization exercise included 10 verbal items that the patient had to imagine and describe . ( we called this step external visualisation because the purpose of the exercise was to imagine an object and not the participant him / herself ) . the instruction ran as follows : i will tell you the name of something ( e.g. , an onion ) , imagine it and describe its colour , shape , size , consistency and every detail that you see . each item description was checked for accuracy using a list of typical traits . for each item , the patient had to describe static aspects ( e.g. , colour , shape ) and an action carried out with the item ( e.g. , slicing an onion ) . the action was imagined to be performed either by him / herself or someone else ( no instruction was given as to who was supposed to act , since the patient was asked to concentrate on an object having changed in its physical appearance owing to an action ) . the construction exercise of the programme consisted in figuring out complex scenes bringing into play several characters . the goal was twofold : to increase the complexity of the scene by comparison with the previous step and to introduce several characters performing different complex actions . five items were proposed with , for each one , a first training step and a subsequent scene construction step . during the training phase , a neuropsychologist guided the patient , starting from a general idea ( e.g. , a cook prepares a meal ) , to more precise subheadings ( e.g. , the cook is in front of a metallic table ) . within subheadings , detailed items were distinguished ( e.g. , on the table , you can see butter , some onions and mushrooms ) . the present step was considered to be external since the participant was asked to imagine some characters and focus primarily on them , their actions and the context of the scene rather than the patient him / herself . responses were checked for accuracy in terms of their relation to each subheading , but no list of typical or expected responses was used . the number of headings and subheadings , as well as details produced by the patients was recorded . for the scene construction phase , an item similar to the one used in the training section was proposed ( e.g. , can you imagine the job you would most enjoy ? will you describe it , as we just did with the example of the cook ? ) . the self - visualisation exercise was the training phase that shared the most similarities with abm since the patients were asked to visualise themselves within a given scenario , to imagine it as though they were actually living the scene and to describe whatever details , sensations or feelings came to mind . the presentation was similar to that of the previous exercise , comprising a training phase ( e.g. , you are heading towards a hotel reception desk ) and a scene construction phase ( e.g. , your room is not ready ; imagine yourself in the hotel bar ) . the difference was that the participant was asked to focus his / her attention on him / herself , that is , using internal , personal knowledge . as for the previous step , we recorded the number of headings , sub - headings and details produced by the patients . images were acquired using a 3 t mri scanner ( siemens verio ) . the image sequence was a t2 * -weighted echo planar imaging ( epi ) sequence ( tr = 2500 ms , te = 30 ms , matrix = 64 64 voxels , fov = 224 mm , fa = 90 ) . the anterior commissure ( ac ) and posterior commissure ( pc ) were identified in the midsagittal slice , and 45 contiguous slices ( each 4 mm thick ) were prescribed parallel to the ac - pc plane to cover the whole brain . concerning fmri tasks , each of them was developed in two versions , randomly allocated for each patient to before and after facilitation sessions . the experimental task ( i.e. , past events condition ) consisted in the evocation of unique personal past events temporally and contextually specific , occurring over minutes or hours , but no more than one day . thirty - two pairs of words were proposed to elicit memories ( e.g. , purchase - car ; country - walk ) , covering the same life periods than previously mentioned in the ai . based on addis et al . , two phases were distinguished in the evocation of the event : ( i ) the construction phase , which is the search and initial building up of the event , and ( ii ) the elaboration phase , which corresponds to the retrieval of details associated to the event . each trial had a fixed duration of 20 s but the duration of the two phases , respectively , were determined by the patient 's response : once a memory was retrieved , the patient had to press on the button 1 and this ended the construction phase . the remaining time , during which a central fixation cross was presented , was devoted to the elaboration phase . this was followed by a rating phase with two 4-point scales per event , each presented for 4 s , for which the patient had to rate the degree of vividness ( 1 = very poor vividness to 4 = very high vividness ) and of emotional intensity felt during the remembering phase ( 1 = very poor emotional intensity to 4 = very high emotional intensity ) . the control task was a categorical task that included 32 pairs of words , with which patients had to construct a sentence for the construction phase ( e.g. , with leather boots : in the shop , there are a lot of different leather boots ) . once the sentence was constructed , they had to press on the button 1 to pursue with the elaboration phase during which patients had to repeat the same sentence and to replace only the two initial words by words of the same semantic category ( e.g. , with the previous sentence : in the shop , there are a lot of different cloth sneakers ; in the shop , there are a lot of different plastic sandals ; the fixed duration for each trial was also 20 s. two 4-point scales , each for 4 s , followed each trial and patients had to assess the degree of difficulty ( 1 = very few difficulty to 4 = very high difficulty ) and the degree of pleasantness ( 1 = very poor pleasantness to 4 = very high pleasantness ) . in both tasks the last rating was followed by short periods of fixation that were of jittered duration ( mean duration = 1.5 s , range = 1 to 2 s ) . it is to note that in the context of this preliminary study we focused our subsequent analyses on the construction phase of abms , since it reflects the access to abms , which according to our main hypothesis is defective in ms patients . also , due to the small number of participants , we did not use vividness and emotional ratings for further analyses . the experimental design was organised in four sequences of eight stimuli , beginning systematically with the control task and then alternating between past events and control task conditions . at the beginning of each sequence , the name of the condition was displayed on the screen for 6 s. the presentation order of stimuli within each condition was randomised . the programming and responses collection was done with e - prime 2 software ( psychology software tools , inc . ) and responses were given on a mr - compatible four - button response box . words were displayed on a screen in white text with a black background and viewed using a mirror incorporated in the head - coil . prior to scanning , the tasks were explained to patients and they underwent a computerised practice trial for each task in order to be familiar to the experimental design and to the rhythm of presentation of the stimuli . they also received a more specific practice trial for the control task during which they were instructed to avoid self - implication in the sentences that they had to construct ( e.g. , no sentences with i ) and to try and minimise mental visual associations with the words , since these two processes were of interest for the experimental task . immediately following scanning , a postscan interview was realised for the past events condition in order to verify the adequacy of the responses and to exclude potential nonepisodic memories before the data analysis . thus , for each past event , patients had to indicate the type of memory ( unique , repetitive , extensive , semantic , or absent ) and briefly , the spatial and temporal context of the event . the first section of the present study aimed at providing normative data for the ai . the good matching between the group of healthy controls and ms patients was verified for age ( u = 42.00 ; p = 0.24 ) and gender distribution ( = 0.28 ; p = 0.59 ) . mean scores of the number of internal details and total ratings were calculated for each recall phase and used to determine the normative data . the presence of an abm impairment for our ms patients was determined using two measures . ( i ) the scores for the free recall phase owing to the free recall sensitivity to detect abm retrieval deficit , which seems to be the memory process most involved in abm impairment in ms patients . ( ii ) the mean number of internal details , which was 22.12 and total ratings , which was 8.38 . these two measures assess the episodic reexperiencing ability and consequently , our ms patients ' abm performance was considered to be impaired if the mean score for internal details was 22.12 and the mean score for total ratings was 8.38 for the free recall phase . ( i ) the neuropsychological baseline examination ( described above ) to control for the absence of severe cognitive impairment other than abm deficit . to pursue with the second part , patients had to be in the normal range on all tests ( threshold : either z - score 1.65 or the 5th percentile , depending on normative data ) except for attentional and executive functions for which mild impairment was accepted . ( ii ) the abm assessment session was carried out . to be included in the study , patients had to show abm scores below the healthy controls ' mean scores as mentioned above for internal details and total ratings for the free recall phase . the patients included in the study were allocated in two groups : the experimental fmri group and the control group . the allocation was not entirely randomised due to three patients , who were left - handed and could not be included in the experimental fmri group . patients in the experimental fmri group underwent the mvi facilitation programme described above with two fmri sessions following a before / after facilitation design . the effects of the facilitation programme on the behavioural measure were also assessed with the ai at the end of the programme . the control group was included to investigate potential learning effects on the second ai assessment session . therefore , this group carried out the ai twice , with no intervention between the two sessions . however , for ethical reasons , the mvi facilitation programme was proposed to all the patients from the control group following the second assessment ( results not shown here ) . a diagram summarising the study design and progression trough the study phases is presented in figure 1 . the blind allocation of the patients ' group could not be done since the baseline examination , the abm assessments and the facilitation programme were conducted by the same neuropsychologist ( a. ernst ) . nevertheless , to control a potential influence of the investigator awareness of the patients ' group allocation , the second ai scorer was blind of the group membership , in every case . moreover , ai reports were anonymised and memories were not supplied for scoring in the chronological order of assessment ( i.e. , after facilitation ai from a patient was not systematically given for scoring after the before facilitation ai ) . potential differences between the two groups of ms patients for all tests of the neuropsychological baseline assessment were analysed with the mann - whitney test . concerning the ai data , the mann - whitney test was used to detect between - group differences for each measure ( i.e. , mean number of internal details and the mean total ratings ) for the free recall and specific probe phases . these comparisons were carried out for the two assessment sessions . moreover , for each measure and recall phases mentioned above , a descriptive comparison between the patients ' mean scores and the normative data from the healthy controls was carried out for the two ai assessments . a within - group analysis was also realised , by means of the wilcoxon matched pairs test , to measure the degree of abm improvement / stability between the two assessment sessions for the experimental fmri and the control group . this procedure was used for the number of internal details and the total ratings for the free recall and specific probe phases . preprocessing and statistical analyses were conducted using spm5 software ( wellcome department of cognitive neurology , london , uk ) . time - series were realigned to the first volume to correct for motion artefacts , spatially normalised to a standard epi template based on the montreal neurological institute ( mni ) reference brain in talairach space , and then spatially smoothed using an 8 mm full - width at half - maximum isotropic gaussian kernel . for both conditions and fmri sessions , evoked hemodynamic responses time locked to the onset of the cue presentation ( construction phase ) were modelled with a canonical hemodynamic response function , and hemodynamic activity related to elaboration was modelled with a boxcar function of 10 sec - duration . although all trials were modelled , only memory trials corresponding to unique past events ( based on participants ' responses during scanning and post - scan interviews ) and correct control trials were included in regressors of interest . statistical parametric maps were generated for the comparison between the construction phase of past and control conditions ( past versus control ) , for each subject , in the context of the general linear model . the two sets of contrast images ( before and after facilitation ) were then subjected to a second level of analysis to perform a within - subject comparison between before and after facilitation data ( paired t - test ) . for all these analyses , due to the small sample of patients , the significance threshold was set at p = 0.01 , uncorrected for multiple comparisons , with a minimum extent threshold of 20 contiguously activated voxels . no significant difference was observed between the two groups of patients for the neuropsychological baseline examination , except for the madrs , for which a higher mean score was observed for the control group ( table 2 ) . however , the madrs mean score for the two groups remained noticeably under the significant clinical threshold ( score 15 to consider the presence of mild depression ) , consequently , this difference between the two groups was not taken into account in further analyses . neuropsychological baseline 's results showed impaired performances in only one attentional test for one patient in each group and low - average scores for one or two tests exploring executive functions for 3 patients of the experimental fmri group and 2 patients of the control group . turning to abm performances , for the first ai assessment , the mean number of internal details and the mean total ratings obtained during the free recall phase were 18.07 and 5.58 , respectively , for the experimental fmri group and 19.22 and 5.72 , respectively , for the control group . for both groups , the scores were below the mean scores established with the healthy controls ( see above ) . in regard to the first ai assessment , no significant difference for the mean number of internal details ( u = 8.00 ; p = 1.00 ) nor the mean total ratings ( u = 5.00 ; p = 0.38 ) during the free recall phase were observed between the two groups of patients . the same results were obtained for the specific probe phase ( mean number of internal details : u = 7.00 ; p = 0.77 and mean total ratings : u = 6.00 ; p = 0.56 ) . likewise , the patients ' mean scores for the specific probe phase ( 39.33 internal details for the experimental fmri group and 39.00 internal details for the control group , with a mean total ratings of 11.12 for the experimental fmri group and 10.58 for the control group ) , were below those obtained by the healthy controls ( 40.2 for the mean number of internal details and 12.47 for the mean total ratings ) . in contrast , the scores on the second ai assessment showed significant differences between the experimental fmri and the control groups for the free recall phase ( mean number of internal details : u = 0.00 ; p = 0.02 and mean total ratings : u = 0.00 ; p = 0.02 ) and for the specific probe phase ( mean number of internal details : u = 0.00 ; p = 0.02 and mean total ratings : u = 0.00 ; p = 0.02 ) . in every case , patients included in the experimental fmri group performed significantly better than those in the control group during the second ai assessment , which corresponded to the post - facilitation examination for the experimental fmri group . with regard to the experimental fmri group , the within - group analysis for before and after facilitation ai assessment highlighted a significant improvement of the patients ' scores on the ai for all the measures of the free recall phase ( number of internal details : t = 104.50 ; p = 0.00 and total ratings : t = 95.00 ; p = 0.00 ) and of the specific probe phase ( number of internal details : t = 81.00 ; p = 0.00 and total ratings : t = 155.50 ; p = 0.00 ) . improvement of ai scores for the experimental fmri group after facilitation condition was supported by the comparisons with those of the healthy controls . indeed , the patient group showed a normalisation of their mean score for all the free recall phase measures ( mean number of internal details = 36.9 versus 22.12 and mean total ratings = 9.39 versus 8.38 ) and for the specific probe phase ( patients ' mean number of internal details = 68.85 versus 40.2 and mean total ratings = 14.79 versus 12.47 ) . conversely , the comparison of the two sessions of ai assessment for the control group showed no significant differences during the free recall phase for the number of internal details ( t = 622.50 ; p = 0.70 ) nor the total ratings ( t = 557.00 ; p = 0.94 ) . also during the specific probe phase , no significant differences were observed for the number of internal details ( t = 571.50 ; p = 0.39 ) nor the total ratings ( t = 545.00 ; p = 0.65 ) . for the second ai assessment of the control group , the results of the comparisons with the healthy controls ' mean scores were parallel to their first ai examination , including mean scores below those of the healthy controls for the free recall phase ( mean number of internal details = 20.6 versus 22.12 and mean total ratings = 5.83 versus 8.38 ) . for the specific probe phase , an apparent normalisation was observed for the mean number of internal details ( patients , 42.12 versus 40.2 ) . qualitative analysis revealed a punctual variation that resulted in an increased score ( namely , one recollection ) . this observation together with the absence of a significant difference for the mean number of internal details between the two ai assessments for the control group , led us to rule out a normalisation process in this case . moreover , no improvement was observed for the mean total ratings relative to normal controls ( 11.08 versus 12.47 ) . based on the semi - structured interview , the patients ' comments confirmed the positive effects of the facilitation programme during the second ai testing as well as in everyday life . the perceived benefits concerned a greater easiness of retrieval , a greater amount of details and better vividness during memory evocation . however , no change was mentioned concerning the emotional intensity . an effective treatment receipt seemed to be obtained since the patients acknowledged an easy use and transfer of this technique in their daily life functioning . additionally , we also had the opportunity to get spontaneous comments by some patients ' relatives who confirmed the benefits of the facilitation programme . the paired t - test comparison we performed , for the contrast between past and control conditions , revealed that brain regions exhibiting a greater difference in activity between after and before facilitation fmri sessions were primarily located in posterior regions . more precisely , after the facilitation programme , our patients elicited greater recruitment of the right cuneus ( ba 19 ) , the left inferior and superior occipital gyri ( ba 18 , 19 ) , the left precuneus ( ba 19 ) as well as of parts of the lateral temporal cortex , primarily on the left ( ba 22 , 38 , 39 ) ( see table 3 and figure 2 ) . the reverse comparison indicated that regions showing a greater difference in activity before the facilitation programme were exclusively located in anterior parts of the brain : the two larger clusters of differential activity were indeed located in the prefrontal cortex ( pfc ) , the first encompassing both dorsolateral and medial aspects of the pfc , the second corresponding to the dorsolateral pfc ( ba 45 , 9 ) ( see table 4 and figure 3 ) . in the present preliminary study , we reported abm performance improvement following a facilitation programme in four rr - ms patients and provided evidence of changes at the neural level . the clinical improvement was verified on the ai , which entailed obtaining french normative data . based on these normative data , we showed a normalisation of patients ' abm free recall performances following the facilitation programme , which suggested a compensation of the initial retrieval deficit . we controlled for the potential learning effects of the abm test by means of comparisons between an experimental fmri group and a control group comprising also four rr - ms patients , who received no cognitive intervention but underwent the ai test twice . besides our previous study , only three further works , to our knowledge , and they are contrary to those obtained by paul et al . and mller et al . these two latter studies concluded that episodic personal memory was preserved in their ms patients either with no specification of the illness subtypes or in defined rr - ms subtype . the test used in these two works was the autobiographical memory interview ( ami ) , which has been said to have a good sensitivity for the personal semantics section , but a poor sensitivity for the episodic incident section [ 50 , 51 ] . interestingly , mller et al . also found that patients with secondary progressive ms exhibited graded loss of personal incident memory on the ami . however , the absence of such abm deficit for rr - ms in mller et al . 's study could suggest that the abm impairment in rr - ms is moderate and subtle and consequently , not readily detected by the ami . these results and what they suggest are coherent with our results using a more stringent abm test in rr - ms patients , which show mild to moderate abm deficit . although this is the first study , to our knowledge , to use the ai in ms , it has already been used in different neurological conditions like temporal lobe epilepsy , alzheimer disease or semantic dementia , showing the effectiveness of this autobiographical test in clinical settings . moreover , our preliminary fmri findings are coherent with our retrieval - deficit hypotheses , involving a frontal lobe dysfunction [ 19 , 20 ] , in the abm deficit in ms . indeed , the enhanced cerebral activation during the construction phase of past recollections was observed in the bilateral prefrontal regions , only during the before facilitation session . this observation suggests a bi - lateralisation of cerebral activations since the abm core cerebral network involves predominantly left cerebral regions . furthermore , it could reflect a compensation mechanism attempt , although insufficient to result in normal abm performance . this suggestion is supported by studies exploring spontaneous functional reorganisation in ms , which show compensation mechanisms during cognitive task , but which are not always powerful enough to result in a complete preservation of cognitive functioning and consequently give rise to mild cognitive impairment [ 13 , 54 ] . with regard to our fmri results following the facilitation programme , they are coherent with the published reports dealing with the effects of various cognitive rehabilitation programmes on neural substrates in ms patients , that is , the presence of cerebral activation changes after cognitive intervention [ 1517 ] . the present fmri findings are coherent with our hypothesis since they showed that the improved abm performance was accompanied by cerebral activation changes during the construction processes of personal past recollections . importantly , changes in activation were observed in posterior cerebral regions , which are known to be associated with the mental processes we trained in our ms patients , that is , mvi . moreover , these changes were observed during the construction phase , which indicates the effective use of mvi to retrieve memories . this latter finding is consistent , not only with the global improvement of abm scores after the facilitation programme but also , and above all , with the normalisation of abm scores for all the patients during the free recall phase , which is a sensitive indicator of retrieval deficit . the posterior cerebral regions showing activation changes were , probably , under activated secondary to an insufficient pfc activation ( see above ) . this suggestion is based on research using hemodynamic response function ( hrf ) analyses during abm construction processes . indeed , an earlier peak in the pfc relative to peaks reached in more posterior regions has been shown . in cases in which the initial pfc hrf is subnormal , the hrf curve of the following ( more posterior ) regions could be affected and present under activation . this would also imply a possibility of return to normal either by a normalised initial hrf peak or by compensation mechanisms induced by cognitive training . beside these quantitative changes , the differences between the before and after facilitation semi structured interviews of patients showed positive effects in terms of a greater easiness of retrieval , a greater amount of details , a better vividness and an effective use of this technique in their daily life functioning . in addition , some patients ' relatives confirmed the benefits of the facilitation programme in daily life . these last points are of importance considering that the primary goal of cognitive rehabilitation is to improve everyday life functioning in patients [ 55 , 56 ] . while these preliminary findings are of interest , we are aware of some methodological limitations that restrict the conclusions that can be drawn from this study . despite the statistical evidence obtained for behavioural and fmri data , our sample of patients is small and replication of these results with a larger sample would be required . additionally , a larger sample could also give the opportunity to explore more precisely the link between the changes observed for clinical and fmri measures . with regard to the facilitation programme , the presence of a nursing effect can not be ruled out until a placebo group of patients is included . furthermore , long - term follow - up measures would be valuable to examine the robustness of the treatment effects . notwithstanding these limitations , our preliminary findings seem to be promising and encourage further investigations , all the more so that our results are in agreement with findings from previous studies which have explored the effect of cognitive rehabilitation on behavioural and neuroimaging measures in ms in diverse cognitive functions ( attention , executive functions and information processing or anterograde memory ) . finally , this preliminary study is , to the best of our knowledge , the first neuroimaging study focusing on abm cognitive facilitation in ms patients . expanding this type of intervention for ms patients seems necessary due to the deleterious impact of cognitive deficit in everyday life . this is particularly the case with abm , considering its importance in everyday life functioning [ 57 , 58 ] .

this preliminary study tackles the assessment and treatment of autobiographical memory ( abm ) in relapsing - remitting multiple sclerosis ( rr - ms ) patients . our aim was to investigate cerebral activation changes , following clinical improvement of abm due to a cognitive training based on mental visual imagery ( mvi ) . we assessed abm using the autobiographical interview ( ai ) in eight patients and 15 controls . the latter subjects established normative data . the eight patients showed selective defective performance on the ai . four patients were trained cognitively and underwent pre- and post - ai and fmri . the remaining four patients took a second ai , at the same interval , but with no intervention in between . results showed a significant improvement of abm performance after the facilitation programme that could not be explained by learning effects since the ai scores remained stable between the two assessments in the second group of patients . as expected , abm improvement was accompanied by an increased cerebral activity in posterior cerebral regions in post - facilitation fmri examination . we interpret this activation changes in terms of reflecting the emphasis made on the role of mvi in memory retrieval through the facilitation programme . these preliminary significant clinical and neuroimaging changes suggest the beneficial effects of this technique to alleviate abm retrieval deficit in ms patients .

1. Introduction 2. Materials and Methods 3. Imaging 4. Procedure 5. Statistical Analyses 6. Results 7. Neuroimaging Results 8. Discussion

restrictive cardiomyopathies are defined as restrictive ventricular physiology in the presence of normal or reduced diastolic volumes ( of one or both ventricles ) , normal or reduced systolic volumes , and normal ventricular wall thickness . usually , patients develop severe symptoms of heart failure over a short period , and the majority die within a few years following diagnosis unless they receive a cardiac transplant . genetic investigations have shown that the condition may be caused by mutations in sarcomeric disease genes or genes for desmin and even may coexist with hypertrophic cardiomyopathy in the same family as seen in our case . a 25-year - old woman , gravida 4 , para 1 , living 1 , abortion 2 , presented to us at 34 weeks of gestation with antepartum hemorrhage . her past pregnancies were uneventful . on examination , pulse was 112/min and blood pressure was 120/80 mm hg . respiratory system showed bilaterally equal , normal vesicular breath sounds with no rhonchus or crepitations . obstetric scan showed a single live intrauterine gestation without any evidence of placenta previa / abruption . her electrocardiogram ( ecg ) showed biatrial enlargement and partial right bundle branch block pattern . echocardiography showed dilated left and right atria , concentric left ventricular hypertrophy with mild mitral and tricuspid regurgitation , normal left ventricle systolic function , ejection fraction 60% , pulmonary artery hypertension ( pulmonary artery systolic pressure-45 mmhg ) with small pericardial effusion [ figure 1 ] . doppler showed increased early diastolic filling ( e ) to atrial filling ( a ) ( e / a ) ratio ( 2.4 ) of the mitral inflow signal , mitral flow ( e-0.85 , a-0.35 ) , and pulmonary flow ( 0.90 m / s ) [ figure 2 ] . in light of these results , genetic counseling of the family was done and genetic testing was advised , but the patient declined testing . connective tissue disorders were ruled out ( antinuclear antibody and antiphospholipid antibody were negative ) . metabolic disorders were ruled out by serum creatine phosphokinase ( cpk ) ( 112.8 u / l ) and serum lactate ( 1.4 mmol / l ) which were found to be normal . she went into spontaneous labor 10 days later , and she had a preterm vaginal delivery of a live female baby weighing 2 kg . the patient is on diuretics and she continues to be on regular clinical follow - up . echocardiography reveals biatrial enlargement with normal systolic function doppler shows increased early diastolic filling to atrial filling ratio ( 1.6 ) common etiologies of rcm include infiltrative ( amyloidosis , sarcoidosis ) , storage ( hemochromatosis ) , noninfiltrative ( scleroderma , idiopathic ) , and endomyocardial processes ( endomyocardial fibrosis , hypereosinophilic syndrome , carcinoid , radiation , malignancy , anthracycline toxicity ) . feld and caspi reported a cardiomyopathy with variable hypertrophic and restrictive features that affected three generations of a family with a shared human leukocyte antigens haplotype . in our case , the sibling had hypertrophic cardiomyopathy . depending on the stage of the disease , the fourth heart sound or third heart sound may be present . in our patient , ecg may show progressive atrioventricular ( av ) conduction delay due to disease of the av node , or his - purkinje system is common in many genetic diseases that affect the myocardium , and in this case , ecg showed biatrial enlargement and partial right bundle branch block pattern . certain signs and symptoms that could raise the suspicion of specific diagnoses grouped according to the main echocardiographic phenotype such as learning difficulties and mental retardation ( noonan syndrome ) , paresthesia / sensory abnormalities / neuropathic pain with bilateral carpal tunnel syndrome ( amyloidosis ) and muscle weakness generally distal progressing to proximal ( desminopathies ) . in our patient the classical doppler finding is the restrictive pattern - mitral inflow pattern e / a > 1.5 as seen in our case . increased creatine kinase is seen in desminopathies , and increased proteinuria is seen in amyloidosis . in our case , rectal biopsy was done and it did not show any deposits of amyloidosis and cpk was normal . cardiac transplantation can be considered in patients with refractory symptoms in idiopathic or familial restrictive cardiomyopathies . the present case report shows the importance of careful evaluation of patients so that risk stratification can be done to avoid adverse disease complications and diminish the rate of sudden death .

restrictive cardiomyopathy ( rcm ) is a relatively rare form of cardiomyopathy with high mortality which is characterized by impaired filling of the ventricles in the presence of normal wall thickness and systolic function . rcm is generally seen in association with infiltrative , storage , or systemic diseases . we present a rare case of familial rcm with pregnancy which was successfully managed .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

hypothyroidism is known to be associated with premature atherosclerosis and increased frequency of cardiovascular events [ 13 ] . the increased cardiovascular risk in hypothyroid patients is mainly associated with hypercholesterolemia and an increased low - density lipoprotein cholesterol ( ldl - c ) level , caused by a decrease in the number of ldl - c receptors in the liver [ 46 ] . however , in hypothyroid patients increased high - density lipoprotein cholesterol ( hdl - c ) also has been observed , as well as changes in size and composition of plasma hdl - c [ 79 ] . lately , it has been suggested that subclinical hyperthyroidism may be not associated with cardiovascular mortality or disorders , except for development of atrial fibrillation . regardless of possible impact on atherosclerosis progression , hypothyroidism is associated with changes in lipoprotein metabolism , including not only lipid disturbances , but also abnormal plasma protein levels . an increase in apolipoprotein b and apolipoprotein a and changes in lipid transfer protein activity in hypothyreotic patients were observed . lipid transfer proteins such as cholesteryl ester transfer protein ( cetp ) and phospholipid transfer protein ( pltp ) are responsible for the formation of mature forms of hdl - c and regulate the concentration , size , and composition of the circulating hdl particles . moreover , in some studies cetp activity was positively correlated with total cholesterol ( tc ) , ldl - c , and non - hdl - c levels , whereas pltp activity was related to body mass index ( bmi ) and serum glucose concentration . increased cetp or pltp mass / activity is considered to be a pro - atherosclerotic factor , mainly related to the decrease in hdl - c level , but also to increased pro - oxidant and pro - inflammatory potential [ 1618 ] . a decrease in cetp or pltp activity is intended to have anti - atherosclerotic properties , mainly by causing an increase in circulating , small , dense hdl3 particles with increased anti - oxidative activity and increased reverse cholesterol transport efficiency . in some observations cetp activity less is known about the pltp activity in patients with dysfunction of the thyroid gland . the aim of this study was to evaluate simultaneously serum cetp and pltp activity in patients diagnosed with hypothyroidism ( studied group ) . the selection criteria for control group members ( without thyroid dysfunction ) in this case to case study were levels of tc , ldl - c , hdl - c , and triglycerides ( tgs ) similar to those in study group patients . the next criteria were the following : the same gender , similar bmis , smoking habits , and alcohol consumption . the differences in lipid transfer proteins between the studied and control groups were related to the level of thyroid - stimulating hormone ( tsh ) . the study was performed in the group of 202 residents of lower silesian region in poland : 166 women aged 59.610.4 years and 36 men aged 57.910.1 years . there were 101 patients ( 83 women and 18 men ) diagnosed with hypothyroidism ( ht group ) and 101 healthy volunteers ( 83 women and 18 men ) constituting the control group ( euthyreotic [ et ] group ) . in all subjects , anthropometric data ( body weight , height ) bmi was estimated as the ratio of weight to height squared ( kg / m ) . in the overall population , about 23% of women and 33% of men were tobacco smokers , and about 58% of women and 77% men reported moderate alcohol consumption ( women : 1 alcohol unit per day ; men : 14 alcohol units per day ) . because the selection criteria for control group members matched to study patients included the same sex and similar age , bmi , tc , ldl - c , hdl - c , and tgs , these parameters did not differ significantly between ht and et groups . also , the percentages of smokers or alcohol drinkers among females and males were similar in the studied and control groups . the tsh level was higher in the study group in comparison with controls ( table 1 ) . only 10 hypothyreotic women were taking l - thyroxine substitution therapy ; the remaining patients began treatment after this study . venous blood was taken from subjects after 12 hours of fasting and centrifuged at 1000 g for 20 minutes at 4c . the thyrotropin in serum was determined using the roche test by electrochemiluminescence method ( normal value : 0.54.0 iu / ml ) . serum tc , tgs , and hdl - c were measured using the spinreact ( santesteve de bas , girona , spain ) enzymatic assay . mmol / l ( 400 mg / dl ) by means of the friedewald formula . the quantolip hdl ( technoclone gmbh , vienna , austria ) precipitation test was used to measure hdl2 and hdl3 cholesterol ( hdl2-c and hdl3-c ) . the non - hdl - c was calculated as a difference between tc and hdl - c concentrations . serum cetp and pltp activities were determined using the cetp activity assay kit and the pltp activity assay kit ( biovision research products , 2455-d old middlefield way , mountain view , california , usa ) with the fluorescence spectrophotometer hitachi f-2500 . the cetp assay uses a synthetic fluorescent ce donor particle and apo - b - containing lipoprotein acceptor particles . cetp - mediated transfer was determined by an increase in fluorescent intensity in the acceptor . the serum pltp assay uses a fluorescent phospholipid donor and a synthetic acceptor , and again , pltp - mediated transfer was measured by an increase in fluorescent intensity . for both assays , the intra - assay and inter - assay coefficients of variation ranged from 11% to 15% , similar to fluorometric assay procedures that are described by others . results were presented as mean standard deviation or median and interquartile range . in the case of normal distribution , t - tests were applied , and a statistical significance between means was calculated using analysis of variance and post hoc tukey s ( checked by least significant difference ) tests . in case of qualitative variables , nonparametric tests were used . the association between presence of hypothyroidism and lipid metabolism was analyzed in the multivariable linear regression model . the independent variable was defined as diagnosis of hypothyroidism , and was individually analyzed in relation to lipid transfer protein and lipid parameters . the following variables were included as potential confounders : age , bmi ( bmi 25 kg / m was treated as normal , bmi > 25 kg / m as overweight , bmi > 30 kg / m as obesity ) , tobacco use ( smokers or nonsmokers ) , alcohol consumption ( moderate drinkers or nondrinkers ) , and coexistence of coronary heart disease or diabetes mellitus ( present or absent ) . three - way analysis of variance ( using presence of hypothyroidism , smoking habits , and alcohol consumption as independent factors ) was also applied with one - dimensional or multidimensional significance tests . all analyses were conducted using the stat statistical package , version 12.0 ( statistica 12 pl . the study was performed in the group of 202 residents of lower silesian region in poland : 166 women aged 59.610.4 years and 36 men aged 57.910.1 years . there were 101 patients ( 83 women and 18 men ) diagnosed with hypothyroidism ( ht group ) and 101 healthy volunteers ( 83 women and 18 men ) constituting the control group ( euthyreotic [ et ] group ) . in all subjects , anthropometric data ( body weight , height ) bmi was estimated as the ratio of weight to height squared ( kg / m ) . in the overall population , about 23% of women and 33% of men were tobacco smokers , and about 58% of women and 77% men reported moderate alcohol consumption ( women : 1 alcohol unit per day ; men : 14 alcohol units per day ) . because the selection criteria for control group members matched to study patients included the same sex and similar age , bmi , tc , ldl - c , hdl - c , and tgs , these parameters did not differ significantly between ht and et groups . also , the percentages of smokers or alcohol drinkers among females and males were similar in the studied and control groups . the tsh level was higher in the study group in comparison with controls ( table 1 ) . only 10 hypothyreotic women were taking l - thyroxine substitution therapy ; the remaining patients began treatment after this study . venous blood was taken from subjects after 12 hours of fasting and centrifuged at 1000 g for 20 minutes at 4c . the thyrotropin in serum was determined using the roche test by electrochemiluminescence method ( normal value : 0.54.0 iu / ml ) . serum tc , tgs , and hdl - c were measured using the spinreact ( santesteve de bas , girona , spain ) enzymatic assay . ldl - c was estimated among patients with a tg concentration lower than 4.52 mmol / l ( 400 mg / dl ) by means of the friedewald formula . the quantolip hdl ( technoclone gmbh , vienna , austria ) precipitation test was used to measure hdl2 and hdl3 cholesterol ( hdl2-c and hdl3-c ) . the non - hdl - c was calculated as a difference between tc and hdl - c concentrations . serum cetp and pltp activities were determined using the cetp activity assay kit and the pltp activity assay kit ( biovision research products , 2455-d old middlefield way , mountain view , california , usa ) with the fluorescence spectrophotometer hitachi f-2500 . the cetp assay uses a synthetic fluorescent ce donor particle and apo - b - containing lipoprotein acceptor particles . cetp - mediated transfer was determined by an increase in fluorescent intensity in the acceptor . the serum pltp assay uses a fluorescent phospholipid donor and a synthetic acceptor , and again , pltp - mediated transfer was measured by an increase in fluorescent intensity . for both assays , the intra - assay and inter - assay coefficients of variation ranged from 11% to 15% , similar to fluorometric assay procedures that are described by others . results were presented as mean standard deviation or median and interquartile range . in the case of normal distribution , t - tests were applied , and a statistical significance between means was calculated using analysis of variance and post hoc tukey s ( checked by least significant difference ) tests . in case of qualitative variables , nonparametric tests were used . the association between presence of hypothyroidism and lipid metabolism was analyzed in the multivariable linear regression model . the independent variable was defined as diagnosis of hypothyroidism , and was individually analyzed in relation to lipid transfer protein and lipid parameters . the following variables were included as potential confounders : age , bmi ( bmi 25 kg / m was treated as normal , bmi > 25 kg / m as overweight , bmi > 30 kg / m as obesity ) , tobacco use ( smokers or nonsmokers ) , alcohol consumption ( moderate drinkers or nondrinkers ) , and coexistence of coronary heart disease or diabetes mellitus ( present or absent ) . three - way analysis of variance ( using presence of hypothyroidism , smoking habits , and alcohol consumption as independent factors ) was also applied with one - dimensional or multidimensional significance tests . all analyses were conducted using the stat statistical package , version 12.0 ( statistica 12 pl . in the total population of 202 people , the linear , positive correlations between tsh and tc ( r=0.21 ; p<0.01 ) , ldl - c ( r=0.16 ; p<0.05 ) , non - hdl - c ( r=0.21 ; p<0.01 ) , and tgs ( r=0.18 ; p<0.05 ) were shown . there were no linear correlations between tsh and hdl - c ( p=0.9796 ) , hdl2-c ( p=0.4574 ) , or hdl3-c ( p = 0.3391 ) . on the other hand , at similar concentrations of serum lipids ( tg , tc , ldl - c , non - hdl - c , and hdl - c ) , the differences between hypothyreotic and control groups ( among females and males ) occurred in hdl subclasses ( table 1 ) . in hypothyreotic patients , the hdl2-c concentrations were lower and the hdl3-c concentrations were higher compared with those observed in healthy subjects ( table 2 ) . serum cetp and pltp activities in hypothyreotic patients were lower ( p<0.001 ) in comparison with those in healthy subjects . this lowering in the proteins activities was associated with a significant decrease in the hdl2-c level and an increase in the hdl3-c level ; however , in men the increase in hdl3-c was not statistically significant ( table 2 ) . in linear regression analysis a positive correlation between cetp and hdl2-c ( r=0.27 ; p<0.001 ) and between pltp and hdl2-c ( r=0.37 ; p<0.001 ) as well as a negative correlation between cetp and hdl3-c ( r=0.22 ; p<0.01 ) and between pltp and hdl3-c ( r=0.24 ; p<0.001 ) has been demonstrated ( figures 14 ) . multiple linear regression analyses adjusted for age , sex , bmi , smoking habits , and alcohol drinking showed an association between hypothyroidism and activity of lipid transfer proteins . the significant beta coefficients ( 95% confidence intervals ) for cetp ( p<0.0003 ) and pltp ( p<0.0000001 ) were estimated . simultaneously , no statistically significant association between thyroid function and lipid classes was observed ( table 3 ) . at the same time the linear inverse relationship between tsh and cetp ( r = 0.21 ; p<0.01 ) and between tsh and pltp ( r=0.24 ; p<0.001 ) was shown ( figures 5 , 6 ) . the high values of beta coefficients ( until 0.82 ) estimated for impact of smoking on lipids and the statistically significant impact of alcohol consumption on tgs and hdl3-c ( beta coefficient=0.17 ; p<0.05 ) were the reason for exclusion of smoking and alcohol drinking as confounders and treating them , together with a thyroid dysfunction , as independent variables . three - way analysis of variance showed that activities of cetp and pltp , being dependent on thyroid function , were not influenced by single confounders such as smoking or alcohol drinking . however , cetp activity was influenced by the interaction between smoking and moderate alcohol drinking ( table 4 ) . hypothyroidism is considered to be a condition associated with an increased cardiovascular risk because of changes in lipid metabolism , plasma viscosity , and pro - oxidative and inflammatory potential . the linear positive correlations observed in this study between tsh and tc , ldl - c , non - hdl - c , and tgs are consistent with the proatherogenic potential of hypothyroidism . in fact , even within the normal range of tsh values , a linear increase in tc , ldl - c , and tgs has been observed with increasing tsh . in this study , the tsh concentration in the studied group was moderately elevated , reaching a maximal value of 7 iu / ml . because of absence of clinical symptoms , subhypothyroidism rather than hypothyroidism could be diagnosed . the main novelty of this study is an observation that moderate hypothyroidism is associated with simultaneous decrease in cetp and pltp activities . this association does not depend on lipid pattern , because the ht patients displayed lower lipid transfer proteins compared with healthy subjects , whereas tc , ldl - c , hdl - c , and tgs were similar in both groups . it can be hypothesized that the reduction of pltp and cetp activities is related directly to the thyroid function . it was confirmed in our study by linear negative correlations between tsh and cetp , and also between tsh and pltp , but first of all , by a multiple linear regression analysis adjusted for age , sex , bmi , smoking habits , and alcohol drinking . the association between increased tsh and lipid transfer proteins was strong for cetp ( beta coefficient was significant at the level of 0.0003 ) and very strong for pltp ( p=0.0000001 ; 95% ci ) . decreased cetp activity in clinically manifesting hypothyroidism in adults was observed in some studies [ 68,21 ] . on the contrary , however , there are no published data about concomitant changes in pltp mass or activity in patients with hypothyroidism , neither in clinical nor in experimental studies . in the present study , more important than the demonstration of a reduction in cetp activity alone was the demonstration of simultaneous reduction in pltp and cetp activities in patients with subclinical hypothyroidism . the extent of the reduction of pltp activity in patients with hypothyroidism was found to be similar to the reduction of cetp activity . the role of pltp in lipid metabolism is still under investigation , and most observations have been made on pltp changes in patients diagnosed with diabetes or metabolic syndrome . it seems that reduced pltp activity , as well as reduced cetp activity , has a protective effect on the cardiovascular system . the main common mechanism for these effects could be the increase of total hdl - c fraction . in other studies in ht patients , reduced cetp activity was associated with a decrease in hepatic lipase activity . both proteins take part in hdl - c metabolism , increasing this fraction through reduction of cholesteryl ester transport from hdl - c to vldl - c and ldl - c . however , the increase in hdl - c levels does not necessarily indicate an increased antiatherogenic potential , because hypothyroidism can induce dysfunctional hdl - c with increased pro - oxidative and pro - inflammatory properties . thyroid hormones can influence hdl - c metabolism by increasing the activity of cetp , which transfers cholesteryl esters from hdl2-c to vldl particles , and tgs from vldl particles to hdl2-c . thyroid hormones also stimulate the lipoprotein lipase , which catabolizes the tg - rich lipoproteins , and the hepatic lipase , which hydrolyzes hdl2-c to hdl3-c and contributes to the conversion of intermediate - density lipoproteins to ldl - c , and the next ldl - c to small , dense ldl particles . in our study , the total hdl - c concentration was similar in subjects with hypothyroidism and euthyreosis . however , significant changes in hdl - c subfractions in patients with hypothyroidism was observed . levels were lower and hdl3-c concentrations were higher in the group with hypothyroidism compared with the group with euthyreosis , both in women and , to a lesser extent , in men . in some other studies , clinically manifesting or clinically silent hypothyroidism was connected with an increase in hdl2-c subfractions , which was explained by a reduction in cetp activity and in consequence , attenuated transfer of cholesteryl esters from hdl2-c to hdl3-c . in our study , the hdl2-c subfraction decreased , whereas the hdl3-c subfraction increased ( the ratio of hdl2/hdl3 significantly decreased ) . it is possible that the cause of this discrepancy in relation to observations from other authors is another degree of thyroid gland dysfunction in studied patients , i.e. , subclinical hypothyroidism in our study versus clinical hypothyroidism in other studies . if so , changes in cetp , pltp , and hdl - c subfractions constitute very sensitive indicators of thyroid function disorders . moreover , changes in hdl - c subfractions in patients with subclinical hypothyroidism were directly caused by reduction in cetp and pltp activity , which was demonstrated in a linear regression analysis by a positive correlation between cetp ( pltp ) and hdl2-c , as well as a negative correlation between cetp ( pltp ) and hdl3-c . simultaneously , there was no effect of subclinical hypothyroidism on hdl - c subfractions ( table 3 ) , as was observed in other studies . there were no significant differences between smokers and nonsmokers or between alcohol drinkers and nondrinkers in cetp or pltp activity . on the other hand , the impact of the interaction between smoking and drinking on cetp activity was observed . in the study , neither coexistence of chronic diseases ( such as diabetes type 2 or coronary heart disease ) nor chronic treatment with pharmacological drugs had any significant effect on lipid transfer proteins . this may be attributed to a relatively small number of patients diagnosed with these diseases ( fewer than 10% ) and a small number of patients treated with pharmacological drugs ( only 10 women were treated with l - thyroxine ) . reduction in cetp activity with the aim of reducing cardiovascular risk is the subject of continued research using cetp inhibitors [ 3032 ] . the increase in hdl - c levels reduces the risk of complications of atherosclerosis through various mechanisms , among which the most important one is the increased reverse cholesterol transport . increased concentration of hdl3-c increases the antioxidant and anti - inflammatory potential . therefore , patients with a moderate increase in tsh reveal simultaneously pro - atherosclerotic changes ( increase in ldl - c , tgs , apolipoprotein b ) as well as anti - atherosclerotic changes ( reduced cetp and pltp activities , increased tc and hdl3-c levels ) in lipid metabolism . hypothyroidism is associated with decreased activity of cetp and pltp , as well as changes in serum hdl - c subclasses . decreased hdl2-c and increased hdl3-c levels in subjects with hypothyroidism are consequences of decreased activity of lipid transfer proteins . these changes are early symptoms of lipid disturbances in hypothyroidism because they appear in hypothyreotic patients with normal levels of tc , ldl - c , hdl - c , and tgs .

backgroundplasma cholesteryl ester transfer protein ( cetp ) activity is often decreased in patients with hypothyroidism , whereas less is known about the phospholipid transfer protein ( pltp ) . we aimed to evaluate simultaneously serum cetp and pltp activity in patients diagnosed with hypothyroidism.material/methodsthe selection criteria for control group members ( without thyroid dysfunction ) in this case to case study were levels of total cholesterol , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides similar to those in study group patients ( 101 patients diagnosed with hypothyroidism ) . serum cetp and pltp activities were measured by homogenous fluorometric assays using synthetic donor particle substrates.resultsserum cetp and pltp activities in hypothyreotic patients were lower ( p<0.001 ) compared with those in healthy subjects . this lowering was associated with significant changes in hdl - c subclasses : decrease in hdl2- and increase in hdl3 cholesterol levels . multiple linear regression analyses adjusted for age , sex , body mass index , smoking habits , and alcohol drinking showed a strong association between hypothyroidism and activity of lipid transfer proteins . a linear inverse relationship between thyroid - stimulating hormone ( tsh ) and cetp ( r=0.21 ; p<0.01 ) and between tsh and pltp ( r=0.24 ; p<0.001 ) was shown . there also was a positive correlation ( p<0.001 ) between cetp and hdl2 cholesterol ( r=0.27 ) and between pltp and hdl2 cholesterol ( r=0.37 ) . a negative correlation between cetp and hdl3 cholesterol ( r=0.22 : p<0.01 ) and between pltp and hdl3 cholesterol ( r=0.24 ; p<0.001 ) has been demonstrated as well.conclusionsthe decreased hdl2 and increased hdl3 cholesterol levels in subjects with hypothyroidism are consequences of decreased activity of lipid transfer proteins . these changes are early symptoms of lipid disturbances in hypothyroidism .

Background Material and Methods Subjects Biochemical measurements Statistical analysis Results Discussion Conclusions

malignant hyperpyrexia syndrome ( mhs ) is a rare life - threatening entity characterized by rapid rise in body temperature , heart rate and muscle rigidity triggered by an exposure to certain anesthetic agents like succinylcholine and volatile anaesthetics . we present a case of mhs in a 10-year - old girl emphasizing that a prompt identification , rational symptomatic and supportive therapy until the availability ofthe dantrolene sodium can be lifesaving . a 10-year - old girl ( 18 kg ) was scheduled for an open reduction internal fixation of the fracture shaft of right humerus . a detailed preanaesthetic checkup revealed no significant past or family history . after obtaining written informed consent from the parents , the patient was wheeled into the operation theatre followed by an application of a pulse oximeter ( spo2 ) . the inhalational induction was conducted with 100% oxygen ( o2 ) and 8% sevoflurane using jackson rees ayre t piece ( jrm - atp ) circuit . the sevoflurane was reduced to 4% after initial 4 - 5 breaths and then to 2% . after the loss of consciousness , a 22-gauge intravenous line was secured and a non - invasive blood pressure and electrocardiography ( ecg ) leads were applied . injections fentanyl 40 g , propofol 40 mg and vecuronium bromide 2 mg were administered intravenously ( iv ) followed by an intubation with a 5.5 cuffed endotracheal tube . a circle system with volume control mode of ventilation was used with a tidal volume , respiratory rate and inspiration : expiration of 150 milliliters , 18/min and 1:2 , respectively . after induction , the end tidal co2 ( etco2 ) and nasopharyngeal temperature were also monitored . the sevoflurane was replaced with halothane after 20 min of anaesthesia and intraoperative vitals were maintained within normal limits throughout the surgical time of 45 minutes . at the end of surgery , the patient bit the tube while the peak airway pressures and etco2 increased suddenly . considering the possibility of a light plane of anesthesia , propofol 20 mg and succinylcholine 5 mg iv were administered . this was however followed by an increased jaw tone : rise in the etco2 to a maximum of 190 mmhg over 10 min and core temperature to 107f ( 41.67c ) . these with hypertonicity of the limbs and an unexplained tachycardia of 200 - 210 beats per minute ( bpm ) suggested a diagnosis of mhs . the iv dantrolene sodium was neither available in the hospital nor in any nearby pharmacy . the supportive management was started and iv lidocaine 1.5 mg / kg bolus was administered which decreased the heart rate ( hr ) to 170 - 180 bpm . iv amiodarone was given in a loading dose of 3 mg / kg followed by an infusion @ 1 mg / kg / hour . the whole body cold sponging was done and the operation theatre ( ot ) temperature was decreased to 20c . the circuit was replaced with a new jrm - atp circuit ; the vaporizer and sodalime were bypassed and 100% o2 was used . the ecg started showing tall t waves and an arterial blood gas ( abg ) revealed a combined respiratory and metabolic acidosis [ ph-6.8 , pco2 - 169 mmhg , po2 - 200 mmhg , potassium-7 milliequivalents / liter ( meq / l ) and sodium- 135 a glucose insulin drip was started with 10% dextrose plus 10 unit insulin @ 50 ml / h and sodium bicarbonate was administered in a dose of 2 meq / kg . the patient 's hr settled down to 100 - 110 beats per minute while the temperature , etco2 and ecg returned to normal . as the patient started having respiratory effort the neuromuscular blockade was reversed with neostigmine and glycopyrrolate . the trachea was extubated and the patient was shifted to the pediatric intensive care unit ( picu ) for further observation and management . meanwhile , serum creatine phosphokinase ( cpk ) was checked which was found to be markedly elevated ( 25000 u ) . the thyroid function tests and a repeat abg and serum electrolyte were in the normal range . the iv dantrolene sodium could not be arranged but oral dantrolene sodium capsules were made available after 6 h of long search at various pharmacists . the oral dantrolene was administered in a dose of 25 mg three times a day orally for 3 days . the patient was shifted to the ward after 3 days and finally discharged home with a note indicating that she suffered from an episode of mhs during general anaesthesia and her parents were sensitized to inform the concerned anesthesiologist about this event , next time whenever she is posted for a surgery . mhsis an autosomal dominant disorder triggered by an exposure to certain anaesthetics agents in susceptible individuals with a mutation at the ryanodine receptor gene ryr1 . this leads to a greatly increased intracellular calcium release due to a lowered activation and heightened deactivation threshold . the increased intracellular calcium activates the myosin atpase resulting in an increased atp consumption , o2 consumption , co2 production , hyperthermia and rigidity . there are no simple signs , symptoms or tests demonstrating a susceptibility to the mhs , which can occur even hours following an exposure to a triggering agent . dantrolene sodium , a hydantoin derivative and muscle relaxant act directly on the ryr1 to prevent calcium release from the sarcoplasmic reticulum and is the drug of choice for the treatment of mhs . it is used in doses of 2 mg / kg every 5 min to a maximum of 10 mg / kg . our patient was a 10-year - old girl from a rural background with no personal or family history suggestive of a susceptibility to the mhs . all the classical signs such as masseter spasm ( biting of the tube ) , increased rigidity of the limbs , a rapid and sudden rise in the body temperature ( 107f , 41.67c ) , tachycardia ( 200 - 210 bpm ) , hypercapnia ( 190 mmhg ) , respiratory acidosis ( ph 6.8 ) and hyperkalemia ( 7 meq / l ) were in accordance with the established diagnostic criteria for the mhs . later , ecg changes suggestive of hyperkalemia appeared followed by the ventricular tachycardia ( vt ) for which iv lignocaine was given and an amiodarone infusion was started in view of persistent vt . the temperature was brought down by surface cooling and decreasing the ot temperature to 20c . an early control of temperature and hyperkalemia is of vital importance but prolonged cooling after achieving normal body temperature must be avoided , otherwise dangerous hypothermia with loss of central temperature control can occur . the etco2 returned to normal after some time . to compensate for an increased o2 consumption and co2 production hyperventilation with 100% the muscle cell damage due to atp depletion and increased temperature can lead to hyperkalemia , myoglobinemia , increased creatine phosphate and creatine kinase . the patient was investigated for the cpk , abg , serum electrolytes and thyroid function tests . the caffeine halothane contracture test could not be conducted due to its unavailability in our hospital and poor economic condition of the patient . this case report highlights that the clinical judgment , an ongoing supportive treatment , discontinuation of all the anesthetic agents and stringent perioperative monitoring along with oral dantrolene may provide an answer to the mhs crisis in the face of an unavailability of diagnostic tests and timely availability of the iv dantrolene ; as may be the case in many rural and developing set - ups . however , this is not to undermine that the dantrolene should be available as a standard of care wherever anesthesia is practiced . a markedly high level of cpk indicated that this child has a high tendency for developing mhs next time whenever she is exposed to the triggering agent . therefore , the plan for removal of k - wire would be preoperative dantrolene prophylaxis , total intravenous anesthesia ( tiva ) , use of a new anesthetic circuit along with the bypass of the vaporizer and sodalime .

malignant hyperpyrexia syndrome ( mhs ) is a rare entity and may not be encountered by the anaesthesiologists throughout their professional career . whenever it manifests can be a challenging task to manage and prove to be fatal when a timely diagnosis and required therapeutic measures are not taken . althoughthe dantrolene should be available wherever anaesthesia is practiced , considering the rarity of the syndrome this may not be the scenario always . we are reporting a case of mhs in a pediatric patient to highlight the facts that prompt clinical diagnosis , ongoing supportive treatment , discontinuation of all the anaesthetic agents and and stringent perioperative monitoring along with postoperative oral dantrolene may provide an answer to the mhs crisis in the face of an unavailability of the iv dantrolene ; as may be the case in many rural and developing set - ups .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

severe obesity is defined as a body mass index ( bmi ) > 40 kg / m which is associated with comorbidities such as insulin resistance , diabetes mellitus , systemic hypertension , dyslipidemia , and cancer . furthermore , severely obese subjects have an increased total mortality with a concomitant increased risk of sudden death , which may be caused by fatal arrhythmias . heart rate variability ( hrv ) which is the fluctuation of heart rate around mean heart rate that may be assessed with a 24-hour cardiac holter monitoring provides valuable information on the activity of the cardiac autonomic nervous system ( ans ) . the ans is an important contributor to the regulation of both the cardiovascular system and energy expenditure and it is assumed to play a role in the pathophysiology of obesity and related complications [ 1 , 2 ] . in obese subjects , many studies have observed abnormalities in the sympathetic and the parasympathetic ans activity , which could partly explain the relation between obesity , comorbidities , sudden death , and arrhythmias [ 1 , 3 ] . available data regarding the metabolic and ans impacts of weight loss in severely obese subjects by other methods than gastric bypass [ 4 , 5 ] and hypocaloric diet [ 3 , 69 ] are sparse . obesity is associated with decreased left ventricular ( lv ) systolic function and impaired lv diastolic function [ 1 , 10 ] . whereas some studies using surgical procedures have reported that substantial weight loss induces significant improvements in lv diastolic function [ 1014 ] , the effect of modest weight loss on lv diastolic dysfunction in obese subjects has been less extensively investigated but has never been reported in severe obesity . diet and exercise programs are associated with disappointing long - term results on weight loss in severely obese subjects . weight loss medication is recommended for subjects with a bmi > 30 kg / m or with a bmi > 27 kg / m associated with 1 risk factor of cardiovascular disease . orlistat is a gastrointestinal lipase inhibitor , reducing fat absorption , which may result in weight loss of approximately 5%10% of the initial weight after one year . it may be relevant to investigate the impact of modest weight loss on lv diastolic function and on hrv since these parameters may be associated with increased cardiovascular risk [ 18 , 19 ] . this pilot study aimed to determine the impact of weight loss induced by orlistat on hrv and on lv diastolic function in severely obese patients with type 2 diabetes . a total of 38 severely obese patients with type 2 diabetes were recruited from the waiting list of bariatric surgery in our institution . the experimental protocol was approved by the ethics committee of iucpq and we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research . subjects were excluded if they used medication known to influence hrv or if they had documented chronic heart failure and/or kidney failure . subjects were randomized to a control group or to a treatment group , which consisted of 120 mg of orlistat ( xenical roche , nutley , nj , usa ) thrice a day , 30 minutes before meal , for 12 weeks . the medication was provided at no cost . for both treated and control groups , no reinforced specific nutritional intervention was recommended during this study other than the usual nutritional advice regarding the medication , reflecting a real clinical context . follow - up was systematically conducted by phone for all subjects at 4 and 8 weeks into the study , in order to ensure subject 's compliance with the medication and to note any significant changes in lifestyle . ( 1 ) one underwent bariatric surgery , ( 2 ) one had medication change within follow - up period , and ( 3 ) five did not attend their follow - up visit . in the treated group , ( 1 ) one had clinically significant side effects on the medication , and ( 2 ) one did not attend the follow - up visit . total body mass , lean and fat mass , height , and bmi have been determined with an electrical bioimpedance balance ( tanita tbf-350 , tokyo , japan ) . blood pressure and resting heart rate were measured following a 30-minute resting period while subjects were lying on their side during the echocardiogram study . blood pressure was measured using an adapted size blood pressure cuff and an electronic sphygmomanometer ( welch - allyn , 5200 series , arden , nc , usa ) . glycemia was determined using an enzymatic method ( hitachi , 717 auto analyzer ; roche , laval , canada ) and glycated hemoglobin ( hba1c ) was measured by binding affinity ( abbot imx , mississauga , canada ) . plasma cholesterol and triglycerides concentrations were measured using previously described methods ( hitachi , 717 auto analyzer ; roche , laval , canada ) [ 20 , 21 ] . serum hdl - cholesterol was analysed with an enzymatic precipitation of ldl - cholesterol and vldl - cholesterol , using phosphotungstate and mgcl2 . echocardiographic measures were performed with a commercial ultrasound system ( sonos 5500 ; hewlet packard , andover , massachusetts ) . standard parasternal , short - axis , and apical views were performed in accordance with the recommendations of the american society of echocardiography and the same observer , who was blinded to randomisation , obtained all recordings and measurements . left ventricular diastolic dysfunction ( lvdd ) , using transmitral and pulmonary veins recordings , was evaluated using well - standardized criteria as previously reported [ 20 , 21 ] . left ventricular mass ( lvm ) was calculated according to the following formula : lvm ( g ) = 0.8 1.04[(lvedd + ivst + pwt ) ( lvedd ) ] + 0.6 , where lvedd represents the left ventricle end diastolic dimension , ivst the interventricular septal thickness , and pwt the posterior wall thickness . heart rate variability ( hrv ) was derived from a 24-hour holter monitoring system ( marquette electronics inc . , numerous indices in the frequency and the time domains were determined [ 7 , 21 ] : mean value of all rr intervals ( mean nn ) , standard deviation of the rr intervals ( sdnn ) , standard deviation of the mean nn intervals for each 5 minutes period ( sdann ) , square root of the mean squared difference of successive rr intervals ( rmssd ) , percentage of differences between adjacent normal rr intervals exceeding 50 milliseconds ( pnn50 ) , high frequency ( hf ) , and low frequency ( lf ) . within the 24-hour evaluation , three periods were assessed : ( 1 ) 24 hours , ( 2 ) daytime period defined as 8:00 am to 8:00 pm , and ( 3 ) night time period defined as 12:00 am to 6:00 am . comparisons between normal , spontaneous , and pseudonormal pattern of lv filling pre and posttreatment were conducted with a mixed anova design . this analysis allowed us to first compare groups ( treatment versus control ) in regards to baseline data and to determine the treatment effect as well as interactions between groups and treatment . the dependent variable associated to lv diastolic function was analysed using a cumulative multinomial distribution with an independent covariance structure . thereafter , if significant differences occurred , a posteriori tukey 's comparison technique was performed to determine differences . relationships among variables were measured using pearson 's correlation coefficients . in order to test the normality distribution of the data , brown and forsythe 's variation of levene 's statistics test was used to verify the homogeneity of variances . data were analysed using the statistical packages sigma stat ( chicago , il , usa ) and sas ( sas institute inc . , cary , nc , usa ) . table 1 presents clinical characteristics of patients in both groups before and after treatment . there were no significant statistical differences between the two groups for all parameters at the beginning of the study ( table 1 ) . after 3 months of treatment with orlistat , there was a significant reduction in body weight ( p < .001 ) , bmi ( p = .003 ) , and waist circumference ( p = .005 ) . when compared to the control group , mean weight loss ( 3.7 3.0 versus 0.5 2.2 kg ; p = .003 ) , corresponding to a percentage of weight loss of 2.9 2.5 versus 0.3 1.5% ( p = .003 ) , was statistically different . weight loss in the treatment group was associated with improvements in fasting glycemia , which decreased only in the group treated with orlistat ( p = .03 ) . however , there was a significant decrease in hba1c ( controls : p < .001 and treated : p = .02 ) as well as in the ldl - cholesterol levels in both groups ( controls : p = .05 and treated : p = .03 ) . in the treatment group , no significant difference was observed between the two groups after treatment for the other anthropometric and metabolic parameters ( table 1 ) . in the 24-hour hrv indices , there was a significant increase in rmssd ( p = .02 ) as well as in pnn50 ( p = .03 ) in the treated group following weight loss ( table 2 ) . while there was no significant change in the hrv daytime indices , night time measures showed a significant improvement in rmssd in the treated group only ( p = .023 ) . following weight loss , high - frequency ( hf ) power measured during the 24-hour period increased significantly ( p = .05 ) , but during daytime and night time , increments did not reach statistical significance ( daytime : p = .056 and night time : p = .1 ) ( table 2 ) . the lf / hf ratio decreased significantly in the treated group in the 24-hour assessment ( p = .038 ) as well as during daytime ( p = .006 ) . echocardiographic parameters measured in both groups as well as normal values are shown in table 3 . there were no statistically significant differences in heart dimensions or parameters of lv diastolic function between the control group and the orlistat - treated group at the beginning of the study . despite normal baseline values , we observed a nonstatistical increment in ejection fraction after treatment with orlistat ( p = .054 ) , the values after treatment being also significantly greater than the ejection fraction values of the control group ( p = .04 ) . there were 11 out of 16 subjects with impaired lv diastolic function ( 7 spontaneous and 4 pseudonormal lvdd ) in the treatment group while there were 7 out of 13 subjects ( 5 spontaneous and 2 pseudonormal lvdd ) in the control group ( figure 1 ) . isovolumetric relaxation time , a wave velocity , e / a ratio , deceleration time , as well as the a wave duration were not statistically different following treatment . however , e wave velocity significantly increased in the treatment group ( p = .046 ) . during the valsalva manoeuvre , we observed a significant decrease of the a wave velocity in the treatment group ( p = .007 ) , concomitant to a significant increase in the e / a ratio ( p = .024 ) . this increment was statistically different to the effect observed in the control group ( p = .027 ) . accordingly , for the patients with a pseudonormal diastolic function , using the e / a value assessed during the valsalva manoeuvre in order to account for the increased pressure of lv filling , there was a significantly enhanced lv diastolic function in the treatment group ( p = .014 ) . thirty - height percent of the subjects ( 6/16 ) in the treatment group significantly improved their lv diastolic function ( p = .03 ) . in contrast , in the control group , there was no improvement ( p = .35 ) except for one subject while 3 subjects worsen their lv diastolic function . the amelioration of lv diastolic function in the treated group was significantly different to the control group ( p = .026 ) . as expected , a positive correlation was found between body weight and bmi and lv mass ( r = 0.68 and r = 0.44 , resp . ; both p an inverse correlation was also observed between body weight , bmi , and lv ejection fraction ( r = 0.48 and r = 0.52 , resp . ; both p < .001 ) . there was positive correlation between e / a ratio and 24-hour measurement of hf ( r = 0.361 ; p < .01 ) , rmssd ( r = 0.456 ; p < .001 ) , and pnn50 ( r = 0.458 ; p < .001 ) . changes in e / a ratio and delta lf / hf ratio during 24-hour and daytime also showed correlations ( r = 0.377 ; p = .048 and r = 0.458 ; p = .014 , resp . ) . we observed that modest orlistat - induced weight loss enhances sympathovagal balance and cardiac function in severely obese patients with type 2 diabetes . after a 3-month treatment period with orlistat , subjects lost significantly more weight than subjects in the control group . akehi et al . reported that very - low calorie diet - induced weight loss in moderately obese subjects improved nn , sdnn , sdann , rmssd , hf , and lf / hf ratio values . similarly , poirier et al . reported that a 10% diet - induced weight loss in 8 severely obese subjects was associated with significant improvement in autonomic cardiac modulation through enhancement of parasympathetic modulation which translates clinically into decreased heart rate and increased hrv . in our study , rmssd , pnn50 , and hf values of the 24-hour hrv were significantly improved in the treated group . all these indices are considered to reflect the parasympathetic system and being protective from a cardiovascular disease viewpoint . furthermore , another significant improvement was the decrease in the lf / hf ratio assessed during 24 hours and during daytime in the treatment group . accordingly , poirier et al . observed that the improvement in hrv after weight loss occurred mainly during the daytime period . our results show similar improvement in the parasympathetic modulation and in the sympathovagal balance , particularly during daytime . studies [ 10 , 14 , 26 ] reported that , following bariatric surgery which induced important weight loss in severely obese subjects , lv mass was significantly reduced . in our study , weight loss in the treatment group was accompanied with a significant decrease in the posterior wall thickness and in lv mass as also reported by others [ 2729 ] . to our knowledge , there are no data available in severely obese subjects investigating the impact of weight loss on the pseudonormal pattern of lv filling using the valsalva manoeuvre . we observed an improvement of lv diastolic function in over half of the treated subjects compared to only one patient who had an improved lv diastolic function in the control group . of note , the relations between hrv and echocardiographic parameters revealed that rmssd and hf values correlate with e / a ratio . interestingly , we also found a significant positive correlation between changes in e / a ratio and changes in lf / hf ratio during 24-hour and daytime . these associations suggest that improvement in the parasympathetic function is related to improvement in the cardiac lv diastolic function parameters , as we previously reported in overweight subjects with diabetes . of note , there are only few reported studies using pharmacological approach in severely obese patients [ 3032 ] and hrv and diastolic function has not been evaluated within the same study in this population . the magnitude of weight loss induced by the medication is smaller ( 2.9 2.5% ) but may be comparable to findings previously reported , accounting for the time - treatment period . sjstrm et al . have reported a 10% weight lost ( 10 kg ) after a year of treatment with orlistat in a cohort of overweight to severely obese subjects . other studies using orlistat for a longer period ( 12 to 24 months ) have also reported similar results [ 3439 ] . taking into account that our study only lasted three months , we may speculate that subjects of our study could have possibly lost comparable amount of weight if had our study lasted 12 months . indubitably , the definitive treatment in this obesity category is bariatric surgery , but clinicians aim at a 5%10% weight loss in order to achieve better metabolic profile in obese subjects [ 1 , 15 ] . accordingly , fujioka et al . reported that every 4.5 kg reduction in weight should result in a 1.1 mmol / l decrement in fasting glycemia and to a 0.5% reduction in hba1c . however , we also observed a significant decrease in hba1c in the control group , which was not different to the decrease observed in the treated group . we also observed a significant decrease in ldl cholesterol in the treated group as well as in the control group . this is not unusual and it is often encountered in placebo groups of weight loss studies . indeed , similar results have been reported in obese subjects after a 4-week run - in period before treatment with orlistat [ 34 , 35 ] . studies [ 38 , 39 ] in overweight to obese type 2 diabetic patients with longer treatment duration ( 6 to 12 months ) resulting to greater weight loss report greater changes in metabolic profiles , leading after 12 months to significant differences between orlistat - treated group and control group . however , weight loss was significant in the treated group whereas it was not in the control group . also , a few echocardiographic measurements , mainly those regarding the pulmonary veins assessment , were not adequately recorded because of technical reasons due to the corpulence of the subjects . other indices of lv diastolic function , such as tissue doppler - derived indices , may be less load - dependent . however , dumesnil et al . reported that tissue doppler - derived indices are not totally preload independent and should be interpreted in light of the other doppler parameters and the use of valsalva 's manoeuvre . the aim of this study was to determine the impact of orlistat - induced weight loss on the metabolic profile and cardiovascular function in severely obese type 2 diabetes patients . twenty - nine severely obese patients with type 2 diabetes were randomized either to a nonplacebo control group or to a treatment group with orlistat thrice a day . metabolic profile , anthropometric parameters , heart rate variability indices , and echocardiographic variables were measured before and after a 12-week treatment period . our results suggest that a modest weight loss improves fasting glycemia , diastolic function , and sympathovagal balance in severely obese patients with type 2 diabetes . despite modest but significant weight loss , we observed that subjects treated with orlistat showed improvements in fasting glycemia . we also observed improvements in hrv as well as in the lv diastolic function . the improvement in the cardiac parasympathetic ans modulation was associated with enhancement in lv diastolic function . our results emphasize the benefit of even modest weight loss in severely obese patients with diabetes . roche company had no role in the design , collection , analysis , or interpretation of the data nor in the decision to submit the study for publication .

objective . determine the impact of orlistat - induced weight loss on metabolic profile and cardiovascular function in severely obese patients with type 2 diabetes . methods . twenty - nine patients were randomized either to a nonplacebo control group or to a treatment group with orlistat thrice a day . metabolic profile , anthropometric parameters , heart rate variability indices , and echocardiographic variables were measured before and after a 12-week treatment period . results . treatment with orlistat induced a modest but significant weight loss compared to controls ( 3.7 3.0 versus 0.5 2.2 kg , resp . ; p = .003 ) . there was significant decrease in fasting glycemia ( 7.9 3.0 versus 6.7 2.2 mmol / l ; p = .03 ) and significant improvements in left ventricular diastolic function ( p = .03 ) and in the sympathovagal balance ( lf / hf ratio ) ( p = .04 ) in the orlistat group . conclusion . these results suggest that a modest weight loss improves fasting glycemia , left ventricular diastolic function , and sympathovagal balance in severely obese patients with type 2 diabetes .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Summary 6. Conclusions Disclosures

interaction of components of the plasminogen system with fibrin or extracellular matrix promotes plasminogen activation . in a similar way , when components of the plasminogen system are bound to cell surfaces , plasmin generation is increased [ 24 ] . binding of plasminogen to cell surfaces is the most important event in the enhancement of plasmin - mediated pericellular proteolysis . plasminogen binding has two major consequences : ( 1 ) plasminogen activation by either tissue - type plasminogen activator ( tpa ) or urokinase ( upa ) is enhanced when plasminogen is bound to cells [ 5 , 6 ] and ( 2 ) plasmin generated on the cell surface is protected from 2-antiplasmin [ 4 , 7 ] , arming the cell with a more efficient proteolytic activity . the promotion of plasminogen activation by cells can be explained by kinetic interactions with and on the cell surface , conformational effects , and/or receptor occupancy . the amount of plasminogen bound to cell surfaces can be quantified . by using radiolabeled plasminogen , it is possible to detect specific binding of plasminogen to cells and to calculate binding affinities and the number of plasminogen binding sites . with this approach , a wide number of cell types have been analyzed ( reviewed in ) . in humans , platelets and all nucleated cells bind plasminogen with a kd in the 0.3 to 2.8 m range in agreement with the plasminogen concentration in plasma ( 1 - 2 m ) . cells exhibit a high capacity for plasminogen ranging from 1010 binding sites for most nontransformed cells to 1010 for malignant cells and human umbilical vein endothelial cells . in general , plasminogen binding capacity is paralleled by the capacity of the cells to promote plasminogen activation . for example , in studies of plasminogen receptor modulation , it has been demonstrated that upmodulation of plasminogen binding sites , a process that can be induced by several effectors , induces an increase in the promotion of plasmin generation by cells . among malignant cells , the study of fibrinolysis on acute promyelocytic leukemia ( apl ) cells offered new insights in the understanding of this disease and its hemorrhagic complications . apl is due to a clonal proliferation of promyelocytic blast cells carrying the t(15 : 17 ) that fuses the pml gene on chromosome 15 to the rar gene on chromosome 17 [ 10 , 11 ] . in contrast to other leukemic processes , apl onset is frequently associated with life - threatening bleeding complications due to disseminated intravascular coagulation , abnormal fibrinolysis , or both [ 1012 ] . immature promyelocytes secrete high amounts of upa [ 13 , 14 ] that can promote plasmin formation in vivo and cause abnormal bleeding . in addition , a mechanism of promotion of plasminogen activation by cell surfaces has been explored on apl blast cells and on the promyelocytic cell line nb4 . this promyelocytic cell line carries the typical translocation found in apl blast cells and has been used in a wide variety of biological studies on apl . the introduction of all - trans retinoid acid ( atra ) in the treatment of this disease in the nineties has dramatically changed the outcome of apl . in most apl patients atra has several dramatic effects on the hemostatic system on both apl blast cells and on nb4 cells . in this study first , using radiolabeled plasminogen , we analyzed the plasminogen binding capacity of nb4 cells compared with other leukemic cells lines of different lineages . second , we explored the functional consequences of plasminogen activation on nb4 cell surfaces analyzing plasmin generation by these cells . finally , we measured plasminogen bound to this cell line by fluorescence - activated cell sorting analysis using an antiplasminogen monoclonal antibody that specifically interacts with plasminogen bound to cell surfaces and explored the effect of atra treatment of nb4 cells on plasminogen binding . tpa ( actilyse ) and high - molecular - weight upa were obtained from boehringer ingelheim and roger laboratories ( molins de rei - barcelona , spain ) , respectively . the labeled and unlabeled preparations of plasminogen used in this study had the characteristics of previously described preparations from our laboratory [ 1720 ] . fluorescein isothiocyanate ( fitc ) conjugated goat anti - mouse monoclonal antibodies were from sera - lab , ltd . neutrophils , monocytes , and lymphocytes were isolated from blood collected into heparin ( 5 u / ml ) as described . other cell lines were from the american type tissue culture collection ( atcc ) and cultured in rpmi-1640 ( bio - whitakker / ma bioproducts ) containing 1 mm na pyruvate and 510% fetal bovine serum . blast cells from peripheral blood were analyzed from a patient with acute nonlymphoblastic leukemia ( anll ) , categorized according to the fab classification . ligand binding analyses were performed as previously described by separating bound from free ligand by centrifugation over 20% sucrose [ 1720 ] . molecules of ligand bound per cell were calculated based on the specific activities of the radiolabeled ligands . plasminogen activation studies were carried out in microtitre plates in reaction volumes of 100 l as previously described [ 5 , 6 ] . briefly , 20 l of plasminogen activators ( tpa or upa ) ( final concentration 70 and 37 pmol / l , resp . ) were mixed with 40 l of cells ( final concentration 1.5 10 cells / ml ) and 40 l of substrate mix containing glu - plasminogen ( final concentration 100 nmol / l ) and chromogenic substrate s-2251 ( val - leu - lys - p - nitroanilide ; chromogenix ) ( final concentration 0.15 mmol / l ) . reactions were performed in assay buffer consisting of tris - hcl , ph 7.4 , at 37c , and a final ionic strength of 0.12 , containing 1 mg / ml human serum albumin . absorbance was monitored at 405 nm , using a thermomax thermostatted plate reader ( molecular devices corporation , stanford , ca ) . cells were washed with pbs containing 1% bsa and 0.1% sodium azide ( pba ) , incubated with pba containing 10% heat - inactivated normal rabbit serum , washed again , and incubated with mab49 ( 130 nm ) or isotype control , washed , and then stained with fitc - goat anti - mouse igg , which was detected in a flow cytometry analyzer ( coulter 's epics xl - mcl ) . plasminogen binding to cells in whole peripheral blood collected into edta was determined as above with the following exceptions . cells were incubated in 10% heat - inactivated human ab serum in pbs , washed with pba and incubated with anti - mouse igg conjugated to pe , washed and incubated with fitc - conjugated antibodies to specific leukocyte antigens . cells were incubated in ortho - mune lysing reagent ( ortho diagnostic systems inc . ) , centrifuged , and resuspended in pba containing 7-aminoactynomycion d ( molecular probes ) at 1 mg / ml . heparin , tween 80 , tween 20 , -aca , and bovine serum albumin were from sigma ( st . neutrophils , monocytes , lymphocytes , and rbc were isolated from blood collected into heparin ( 5 u / ml ) , theophylline ( 10 mm ) , and prostaglandin e1 ( 10 u / ml ) ( sigma ) as described . to explore the plasminogen binding capacity of this cell line with respect to other leukemic cells lines and peripheral blood cells , radiolabeled plasminogen ( 100 nmol / l ) was added to washed cells ( 25 10/ml ) and incubated for 1 hr at 37c . specific binding was calculated by subtracting counts bound in the presence of 0.15 mol / l -aca . under these conditions , nb4 cells specifically bound 38.0 0.7 10 molecules of plasminogen per cell . for comparison , plasminogen binding was also explored on several leukemic cell lines of distinct lineages and in several types of normal peripheral blood cells . as shown in table 1 , nb4 cells bound plasminogen with a capacity at least one order of magnitude higher than the other cells analyzed . plasminogen binding to leukocytoid cells promotes plasmin formation in the presence of either tpa or upa plasminogen activators [ 46 , 2226 ] . in previous kinetic studies , we have demonstrated that plasmin generation is promoted by leukocytoid cells by 60- to 30-fold when tpa or upa , respectively , was used as plasminogen activators [ 59 ] . with a similar approach , we explored the promotion of plasminogen activation by nb4 cells . upa has a high affinity for upa receptors , and an autocrine mechanism of saturation of upa receptors has been previously described in several cell lines . to assess whether nb4 cells could promote plasmin formation in the absence of extrinsic plasminogen activators , washed cells were incubated with plasminogen and the rate of plasminogen activation was measured as previously described [ 5 , 6 ] under this conditions , nb4 cells generated 0.3 0.01 pmol / l of plasmin sec , whereas 0.07 0.02 and 0.06 0.01 pmol / l of plasmin sec were generated by u937 cells or peripheral blood neutrophils , respectively . these data suggested that a significant fraction of upa secreted by nb4 cells was bound to their cell surface and could activate plasminogen . to better define the role of plasminogen binding sites in the promotion of plasmin formation , nb4 cells were acid treated to remove upa from upa receptors . following acid treatment , < 0.01 pmol / l sec of plasmin was generated when cells were incubated with plasminogen , indicating that the upa was efficiently removed from cells by this treatment . acid - treated nb4 cells were incubated with plasminogen and tpa and the rate of plasminogen activation determined . under these conditions , plasmin generation on acid treated nb4 cells was 0.8 0.04 pmol / l sec while u937 and peripheral blood neutrophils generated 0.3 0.08 and 0.016 0.04 pmol / l sec . taken together , these data suggested that the high number of plasminogen binding sites detected on nb4 cells was paralleled by a high capacity to promote plasminogen activation on their cell surface . to explore whether plasminogen could be detected on the cell surface of nb4 cells , we used an antiplasminogen monoclonal antibody ( mab49 ) that recognizes receptor - induced binding sites ( ribss ) in plasminogen and , therefore , preferentially react with cell - associated plasminogen in the presence of soluble plasminogen . cells were washed and incubated with plasminogen ( 10 mol / l ) for 1 hr at 37c . then , mab49 was added to the system and processed for facs analyses as described in section 2 . the mean fluorescence intensity was 2.2-fold higher in the system containing added plasminogen ( 5.5 units ) than in the negative control without plasminogen ( 2.5 units ) . to assess whether plasminogen binding changes induced by atra treatment of nb4 cells [ 12 , 29 ] could also be detected by mab49 the positive facs signal observed with untreated nb4 cells , preincubated with plasminogen , was markedly decreased after treatment of nb4 cells with atra for 48 hr ( figure 2(a ) ) . for comparison , we explored with a similar approach blast cells from a patient with apl with a large proportion of blast cells ( 80% ) , both prior to and after atra treatment in vivo . a strong facs signal with mab49 was detected prior to atra treatment compared with the isotype control ( figure 2(b ) ) . after both a 4-day and a 5-day of treatment with atra , blast cells exhibited a markedly decreased facs signal , compared with untreated cells ( figure 2(b ) ) . as an additional control , facs analysis of m1 blast cells ( that have not been reported to bind plasminogen ) with mab49 did not show a positive signal compared with isotype control ( figure 2(c ) ) . taken together , these results demonstrate that mab49 can be used to monitor modulation of plasminogen to nb4 cells following atra treatment . changes induced by atra are similar to the ones observed in blast cells of patients with apl . in this study , we have characterized plasminogen binding to the nb4 promyelocytic leukemia cell line by analyzing plasminogen binding to these cells in comparison to other blood cell lines or peripheral blood cells . the functional consequences of the high capacity of plasminogen binding by nb4 cells were explored in kinetic studies measuring plasmin generation . in addition , plasminogen binding to nb4 cells was also explored using an antiplasminogen monoclonal antibody that specifically recognizes plasminogen bound to cells . this antibody allows detection of downmodulation of plasminogen binding induced by atra treatment of these cells . this capacity is one order of magnitude higher than that displayed by other leukemic cells lines of distinct lineages and two order of magnitude higher than normal peripheral nucleated blood cells . thus , the nb4 cell line has the highest capacity for plasminogen among the cell lines tested . this characteristic is also complemented with a high capacity to produce upa and therefore nb4 cells have been used as models to study the bleeding complications of apl patients [ 1015 , 2931 ] . the high plasminogen binding capacity of nb4 cells has been related to the overexpression of plasminogen binding molecules . several proteins have been identified as cell surface binding molecules for plasminogen , including -enolase , annexin ii , tissue factor , and the complex s100a10-annexin ii [ 79 , 12 , 2939 ] . although annexin ii was highly expressed by nb4 and apl blast cells antiannexin ii antibodies reduce plasminogen activation mediated by these cells by 35% , -aminocaproic acid ( eaca ) gave a 71% reduction . because eaca inhibits the interaction of plasminogen with cells , this result implies that other plasminogen receptors in addition to annexin ii could be important for stimulation of plasminogen activation . an antibody to s100a10 molecule fully blocks endothelial cell plasmin production , but no data have been generated using this antibody on nb4 or apl cells . a monoclonal antibody to -enolase ( 11g1 ) blocks the cell surface promotion of plasminogen activation in a wide variety of leukemic cell lines and abrogates nb4 mediated plasminogen activation by 7080% . thus , in addition to the annexin ii - s100a10 complex , -enolase also mediates plasminogen binding to nb4 cells . tissue factor is also a plasminogen - binding molecule but very high concentrations of soluble tf are required to reduce plasminogen binding or cell - dependent promotion of plasminogen activation . thus , the physiological role of tf in apl blast cells as a plasminogen binding molecule should be further studied . nb4 cells secrete high amounts of upa , and therefore we explore the capacity of these cells to promote plasmin generation in the absence of added plasminogen activators . under these conditions , nb4 cells generated plasmin with a 4 - 5-fold higher efficiency that u937 cells or peripheral blood neutrophils . when tpa was added to acid - treated cells , promotion of plasmin generation by nb4 cells was 2026-fold greater than by u937 cells or neutrophils , respectively . these data suggest a parallelism between plasminogen binding capacity and promotion of plasmin formation on nb4 cell surfaces . in flow cytometric and radioimmunometric studies , we have previously demonstrated that a fraction of blood plasminogen is bound to surfaces of peripheral nucleated blood cells and platelets . in these studies , plasminogen bound to cells was detected using a monoclonal antibody to plasminogen which preferentially reacts with plasminogen bound to cell surfaces , suggesting that plasminogen binding to cells induces a conformational change in plasminogen and that latent epitopes in soluble plasminogen become available when plasminogen is bound to cells . these antibodies detect receptor - induced binding sites ( ribs ) in plasminogen induced by its interaction with cells . a practical application of the use of antiplasminogen ribs antibody has been explored on acute promyelocytic leukemia ( apl ) blast cells . this antibody gives a clear positive signal in facs analyses and can be used to explore changes in the amount of plasminogen bound to blast cells in whole blood during all - trans retinoid acid ( atra ) treatment of apl patients . plasminogen was detected on nb4 cells incubated with plasminogen , whereas culture of nb4 cells with atra for 48 hours markedly reduced the antiplasminogen ribs signal . again , these data reinforce the similarity between apl blast cells and the nb4 cell line . in recent studies , atra treatment of nb4 cells induces a downregulation of the plasminogen - binding molecule s100a10 that is paralleled by reduction in fibrinolytic activity . in addition , depletion of s100a10 by rna interference abrogates cell - dependent fibrinolytic activity in nb4 cells . thus , these cells constitute an excellent model to explore the modulation of plasminogen binding to cells using mab49 . taken together , these data suggest that the nb4 cell line constitutes a unique cell model for plasmin generation on cell surfaces . the individual contribution of molecules that bind plasminogen on these cells should be explored in future studies . in addition , the downregulation of plasminogen receptors induced by atra treatment of nb4 cells offers an exciting model to study the modulation of these receptors and their functional consequences .

the nb4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells , including the translocation ( 15 : 17 ) that fuses the pml gene on chromosome 15 to the rar gene on chromosome 17 . these cells have a very high fibrinolytic capacity . in addition to a high secretion of urokinase , nb4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines . when tissue - type plasminogen activator was added to acid - treated cells , plasmin generation was 2026-fold higher than that generated by u937 cells or peripheral blood neutrophils , respectively . we found that plasminogen bound to these cells can be detected by fluorescence - activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces . all - trans retinoid acid treatment of nb4 cells markedly decreased the binding of this monoclonal antibody . this cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all - trans retinoid acid treatment .

1. Introduction 2. Material and Methods 3. Results 4. Discussion 5. Conclusions

maintaining the microsurgical field , free from flooding with irrigation fluid , is important to get good vessel approximation in microsurgery . the commonly followed practice is to keep dry gauze at the periphery of the field so as to siphon off the excessive fluid . a certain amount of the irrigation fluid still remains in the field and causes adhesion of the suture material with the anastomotic clamp or the background material or to the vessel wall and thereby frustrating the surgeon while applying the microknots . we present our technique that avoids the aforementioned , and in addition provides a good platform to perform microvascular anastomosis . this is folded over a dry gauze and then fenestrations are made in the surface and on the two margins [ figure 1 ] of the glove component so as to provide for continuous drainage of the irrigation fluid to the gauze . the tip of the infant feeding tube ( size 4 ) is then passed in to the gauze [ figure 2 ] and the whole assembly is then placed in the microsurgical field deep to the vessels to be anastomosed and thus provides a platform for the microanastomosis . the vessels to be anastomosed are laid over a background on the platform and then the proximal part of the feeding tube is connected to a suction system so as to provide continuous suctioning of the irrigation fluid that keeps the microsurgical field clean and dry and also by keeping away the tip of the feeding tube from the surgical field [ figure 3 ] . the wrist part of the glove with open ends and fenestrations on the surface to facilitate siphoning off of the irrigation fluid the tip of a size 4 infant feeding tube is passed between the layers of gauze the entire assembly is shifted to the neck for anastomosis forming a good platform for anastomosis . the proximal end of the infant feeding tube is connected to the suction tube the wrist part of the glove component has been described in the past to transfer the pedicle of the flap from the oral cavity to the neck for microanastomosis and in this report we present a technique in which the component can be used to keep the operative field clean and dry and also to provide a stable platform to perform a perfect microanastomosis . this is folded over a dry gauze and then fenestrations are made in the surface and on the two margins [ figure 1 ] of the glove component so as to provide for continuous drainage of the irrigation fluid to the gauze . the tip of the infant feeding tube ( size 4 ) is then passed in to the gauze [ figure 2 ] and the whole assembly is then placed in the microsurgical field deep to the vessels to be anastomosed and thus provides a platform for the microanastomosis . the vessels to be anastomosed are laid over a background on the platform and then the proximal part of the feeding tube is connected to a suction system so as to provide continuous suctioning of the irrigation fluid that keeps the microsurgical field clean and dry and also by keeping away the tip of the feeding tube from the surgical field [ figure 3 ] . the wrist part of the glove with open ends and fenestrations on the surface to facilitate siphoning off of the irrigation fluid the tip of a size 4 infant feeding tube is passed between the layers of gauze the entire assembly is shifted to the neck for anastomosis forming a good platform for anastomosis . the proximal end of the infant feeding tube is connected to the suction tube the wrist part of the glove component has been described in the past to transfer the pedicle of the flap from the oral cavity to the neck for microanastomosis and in this report we present a technique in which the component can be used to keep the operative field clean and dry and also to provide a stable platform to perform a perfect microanastomosis .

during microvascular anastomosis , it is important to maintain the microsurgical field irrigated yet dry so as to achieve a good view of the vessels for approximation . in this method , an infant feeding tube ( size 4 ) , with its tip sandwiched between layers of dry gauze and a surgical glove component placed in the anastomotic field and the other end connected to a suction apparatus , is used to maintain the microsurgical field free from flooding . it also has the additional advantage of providing a stable platform for microvascular anastomosis .

INTRODUCTION Technique

kerosene is refined oil belonging to the hydrocarbon group of compounds , obtained from crude petroleum . poisoning is due to inhalation of fumes or ingestion of small amounts accidentally ( children ) or most of the cases reported in the literature were self - inflicted with the intention of committing suicide which constitutes significant emergency admissions . a 34-year - old male patient presented to our out - patient department with a history of injecting about 5 ml of kerosene to his left hand at two sites following an argument with his family members 2 days prior to presentation , with an intention to commit suicide . after 2 days , the pain became intolerable leading to disturbed sleep , with multiple blisters and swelling of the left hand . no other significant medical history was noted . on examination , he was an alert middle - aged man , moderately built and nourished , and well oriented to time , place , and person . multiple vesicles and blisters were present over the left forearm and few over the dorsal aspect of the left hand . black discoloration extending from the hand up to the lower third of the arm was present . local rise of temperature and tenderness was present from the hand up to lower third of arm . refilling could not be elicited [ figures 1 and 2 ] . multiple vesicles and blisters black discoloration extending from the hand up to the lower third of the arm was present . patient was unable to flex the hand few vesicles , blisters , and erosions were present over the dorsal aspect of the left hand there were no other lesions . irritant contact dermatitis the patient was admitted to the ward ; intravenous ( iv ) amoxycillin with clavulinic acid , iv steroid , and iv fluids were started . iv or subcutaneous self - injections of commercial gasoline or kerosene with suicidal intent constitute extraordinary events in psychotic patients . the lethal dose of kerosene for a 70 kg adult is 100 ml . in 1977 , green first described a patient who survived a 3 ml iv injection of energine , a derivate of kerosene . this patient developed lethargy , drowsiness , and a sterile abscess at the injection site without other systemic effects . our patient had injected 5 ml of kerosene and developed lesions over the forearm , as described . neeld and vaziri each reported a case of iv injection of petroleum distillate that caused severe chemical pneumonitis . in both cases , pulmonary edema and hypoxemia developed , and in one , the patient suffered abrupt respiratory arrest immediately after injection . kosa reported an accidental iv injection of 10 ml of gasoline that resulted in a fatal outcome . the case strongly suggested that hydrocarbons , as low - viscosity chemical compounds , have a direct toxic effect on lung tissue , interfering with gas exchange and leading to liquid interstitial pulmonary edema and severe hypoxemia caused by impaired diffusion . other effects of hydrocarbon intoxication include acute renal damage with oliguria , hepatocellular damage , intracellular hemolysis , gastric ulcers , and cardiac toxicity with negative inotropism . a case of self - inflicted acute necrotizing fasciitis with compromised venous return secondary to injection of kerosene was reported . a study on the clinical findings and outcome in suicide attempts by iv injection of kerosene among iv drug abusers was done in iran . although kerosene is a easily available refined oil as it is used for household purposes , poisoning occurs by inhaling fumes and ingestion accidentally in children and with the intention of committing suicide in adults . kerosene injection can produce severe tissue damage , especially if prompt surgical treatment is delayed . therefore , it is important to initiate active and immediate surgical intervention in the form of complete multiple fasciotomies , incision , and surgical excision of non - viable tissue . it should also be followed by active and repeated aggressive wound debridement until healthy granulation tissue is formed .

kerosene is refined oil belonging to the hydrocarbon group of compounds , available for domestic use in developing countries . poisoning is due to inhalation , ingestion . kerosene . we report a rare case of attempted suicide by means of intravenous injection of kerosene . it has a devastating effect and it is important to initiate active and immediate surgical intervention . psychotherapy should also be an integral part of the management .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

mixed urinary incontinence ( mui ) is a troublesome condition for patients and incontinence specialists because for treatment to be successful , both the stress and the urge components of the incontinence need to respond to therapy . the international continence society 's standardized nomenclature defines mixed urinary incontinence as " the complaint of involuntary leakage associated with urgency and also with exertion , effort , sneezing or coughing . " . the most appropriate definition for mixed incontinence is not known . from a pragmatic standpoint , patients with mui and their clinicians want information about the chance of a favorable outcome following their primary incontinence treatment . the clinical consequences of not understanding the role of the stress and urge components in mui include the persistence of urge incontinence after stress urinary incontinence ( sui ) surgery , which the patient then perceives as surgical failure [ 2 - 4 ] . the urgency component in mui may consist of either detrusor instability , urethral relaxation , or an uninhibited premature micturition reflex . others have argued that the " urge incontinence " component of mui appears to be different from that of urge urinary incontinence ( uui ) and have suggested that urge incontinence may be overdiagnosed in patients with sui who misinterpret their fear of leaking ( because of sui ) for urge incontinence . in our study , we tried to identify whether symptoms of urgency increase along with the severity of sui symptoms . in an effort to define more precisely the specific pathophysiology of incontinence in women , we examined whether women suffering from mui exhibit clinical and urodynamic characteristics that are distinct from those of patients with pure sui . two hundred forty - one women with sui and mui from march 2005 to december 2008 were included in this study , and their medical records were retrospectively reviewed . patients with diabetes mellitus , neurologic disease , prior urologic surgery , and urethral abnormalities were excluded . of the total , 164 patients ( 68% ) had sui symptoms only , and 77 ( 32% ) had mui symptoms . urgency was defined as an average of 1 episode per day on a 3-day voiding diary . patients with sui only were defined as group 1 , and those with mui were defined as group 2 . history taking , physical examination , urinalysis , quantitative urine culture , q - tip test , frequency volume chart , uroflow study , postvoiding residual urine measurement , valsalva leak point pressure ( vlpp ) , and urodynamic study that included maximal urethral closure pressure ( mucp ) were performed on all patients . level of incontinence symptoms were categorized into grade i , ii , and iii according to the stamey grade . the cutoff value of vlpp , in which endogenous sphincter dysfunction is suggested by urodynamic study , was defined as less than 60 cmh2o . detrusor overactivity ( do ) was defined as involuntary detrusor contraction of more than 15 cmh2o during filling cystometry with a speed of saline infusion of 30 ml / min . the bristol female lower urinary tract symptom ( bfluts - f2 ) questionnaire was used to evaluate the symptoms of urinary urgency . the grade of urinary urgency symptoms was expressed from 0 to 4 , and higher points represented more severe symptoms . in our study , urgency symptoms were not only urgency but also urge incontinence . baseline clinical characteristics , such as age , body mass index ( bmi ) , parity , duration of urinary incontinence symptoms , previous pelvic surgery , and stamey symptom grade were compared between the 2 groups . degree of urethral hypermobility and cystocele degree from physical examination , residual urine volume , maximal bladder capacity ( mbc ) , bladder volume at first desire , vlpp , and mucp , pdetmax , and presence of do from the urodynamic study were also compared . chicago , il , usa ) and examined continuous variables by use of the independent t - test and non - continuous variables by use of the chi - square test in comparisons of the clinical characteristics and results of the 2 groups . the mean age of the patients was 53.9 years ( range , 35 - 81 years ) , the average parity was 2.5 ( range , 0 - 8 ) , average bmi was 24.7 kg / m ( range , 17.2 - 34.3 kg / m ) , and the average duration of symptoms was 71 months ( range , 1 - 360 months ) . comparing baseline characteristics , age and a positive history of previous pelvic surgery were significantly higher in group 2 than in group 1 ( 56 vs. 53 years old for age , p<0.05 ; 57.1% vs. 44.5% for previous pelvic surgery , p<0.05 ) . in our study , severe degrees of cystocele ( ii - iv ) were more frequently observed in group 2 than in group 1 ( grade 0=69.5% , i=20.1% , ii=7.9% , iii=1.2% , iv=1.2% in group 1 , vs. grade 0=57.1% , i=15.6% , ii=11.7% , iii=9.1% , iv=6.5% in group 2 , p<0.05 ) . the degree of urethral hypermobility was greater for group 1 than for group 2 ( 42.1 vs. 28.6 , p<0.001 ) ( table 1 ) . grade i symptoms were more frequently noted in group 1 than in group 2 ( 45.7% vs. 29.9% ) . however , grade ii or iii symptoms were seen more frequently in group 2 than in group 1 ( 53% vs. 66.2% for grade ii ; 1.2% vs. 3.9% for grade iii ) ( p<0.05 ) . also , a positive linear association ( 6.440 ) was noted between stamey grade and incidence of urgency in the linear by linear association test . that is , the incidence of urgency was proportional to the degree of stamey grade ( 23.5% in grade 1 , 36.9% in grade ii , and 60.0% in grade iii , p<0.05 ) ( table 2 ) . mucp , mbc , and bladder volume at first desire were higher in group 1 than in group 2 ( 54.7 cmh2o vs. 44.1 cmh2o for mucp , p<0.001 ; 356.3 ml vs. 282.0 ml for mbc , p<0.001 ; 144.6 ml vs. 123.2 ml for bladder volume at first desire , p<0.05 , respectively ) . do was more frequently observed in group 2 than in group 1 ( 49.4% vs. 25.9% , p<0.001 ) ( table 1 ) . logistic regression analysis failed to identify any significant risk factors influencing the urgency symptoms accompanied by sui among the clinical and urodynamic parameters . the urgency score measured by the bfluts became higher as the stamey symptom grade advanced ( 2.0 in i , 2.2 in ii , and 3.3 in iii ) , but not significantly so ( r=0.217 , p=0.058 ) ( table 2 ) . in this study , we compared 2 groups of sui patients , those with and without urge incontinence , to determine whether any clinical or urodynamic data could identify differences between them . according to the argument suggested by chou et al , the " urge incontinence " component of mui appears to differ from that of uui , and they suggested that urge incontinence may be overdiagnosed in patients with sui who misinterpret their fear of leaking for their urge incontinence . in our study , we also tried to identify whether the symptoms of urgency increased along with the severity of sui symptoms . in other words , we suspected that the urgency symptoms accompanied by sui may represent overexpression of the patient 's premature voiding by her will to avoid stress - induced urine leakage , although not all . in an effort to define more precisely the specific pathophysiology of incontinence in women , we examined whether women suffering from mui exhibited clinical or urodynamic characteristics that were distinct from those of patients with pure sui . our study showed that the incidence of urgency was proportional to the degree of stamey grade and that women with an mui component had more severe sui symptoms than did those with pure sui . after reviewing epidemiologic and clinical evidence , minassian et al also suggested that stress and urge urinary incontinence ( ui ) are pathophysiologically linked , consistent with the integral theory , which posits that bladder control problems , including stress and urge ui , are consequences of abnormal connective tissue resulting from an insult to the supportive pelvic floor support structures . resolution of urgency symptoms after surgery for sui has been reported to be fair , with percentages of 60.2% , 54.2% , 54% , 63.6% , and 59% [ 8 - 12 ] . these results suggest that in patients with mui , sui may affect the development of urinary urgency symptoms as evidenced by the resolution of urgency following the surgery . mahony et al reported two urethro - detrusor reflexes that increased the excitability of the micturition reflex as urine flowed across the urethral mucosa , modeling the clinical relationship between sui and do . hindmarsh et al proposed that urethral overactivity is often associated with unstable bladder contractions and suggested that bladder overactivity may originate from stimuli in the bladder outlet . also , jung et al demonstrated that fluid passing through the urethra could promote detrusor activity . even with current bladder neck surgery or surgery that places tension on the central urethra , which were conducted before the introduction of the theory of tension - free surgery , not only the treatment of sui but also urgency symptoms were reported to improve . also , fairly high numbers of patients have been reported to show improvement in both the treatment of sui and urinary symptoms with the current tension - free surgery methods . this illustrates the connection between the two diseases of urinary urgency and sui , because such surgery stabilizes the urethra statically or dynamically , thus showing that the urethra is contributing in some part . our study showed that the q - tip test , which assesses the stability of the urethra , has significant meaning between the sui group with no urinary urgency and the group with urinary urgency . also , mucp was verified as a factor with significant meaning between the 2 groups . ku et al reported that mucp is one of the independent prognostic factors that maintains the urinary urgency of urinary incontinence patients after the tension - free vaginal tape ( tvt ) procedure . reports on how duloxetine , which is being researched as a remedy for sui , increases urethral resistance and bladder volume at the same time show that there is an association between urethral pressure and bladder volume . our study also confirmed that the mbc and bladder volume at first desire differed significantly between the 2 groups . others also reported that urinary urgency symptoms and unstable bladder contractions occur as urethral pressure decreases . in our study , age , previous pelvic surgery , and degree of cystocele it could be supposed that with increasing age , previous pelvic surgery , or pelvic organ prolapse , some changes may occur in the bladder and pelvic tissue or detrusor , and that as a result , urinary urgency may have developed . in nguyen and bhatia 's report , 56.7% of patients with pelvic organ prolapse symptoms complained of symptoms of urinary urgency , with pelvic organ prolapse being the factor having a connection with urinary urgency . the presence of do in mui is controversial , because the presence of do may affect the physician 's decision for surgery as well as the surgical outcomes . interestingly , many women with clinically defined mui do not demonstrate do during urodynamic studies . consequently , the relationship between do and clinical symptoms is often conflicting and has not been fully delineated . if the rate of positive do was higher in patients with mui , then we can easily conclude that the cause of urgency symptoms in mui was independent of sui symptoms . however , in real practice , only about 50% at most of women with mui exhibited do during urodynamics , which suggests that the urgency symptoms in mui were closely linked with each other . in a preoperative urodynamic study of women with mui , in our study , it was observed that 49.4% of mui patients exhibited do , which is similar to the report of stoffel et al , but lower than that of lewis et al , who insisted that 56% of sui patients showed do . on the other hand , digesu et al reported 11% of mui patients to have positive do . in a report by chou et al , the rate of do occurrence was less than in the patients with symptoms of urge incontinence in the mui patient group , and he explained this finding as " over diagnosis , " which shows urinary urgency to prevent it in the early stage because patients with severe sui are more aware of urine flows . our results suggested that the urgency in mui was correlated with the severity of sui symptoms , but that the component of urgency may exist independently of the sui symptoms . in our study , it may be controvertible to use the stamey symptom grade in mui , because patients with severe mui symptoms might misunderstand urge incontinence as sui . however , we thought that the stamey grade was a meaningful assessment scale from the point of view of the doctor more than the patient . a more detailed classification between sui and urge incontinence should be considered in patients with severe mui , and more systemic and objective consideration that could analyze sui symptoms will be required . in conclusion , the more serious the symptoms of sui , the higher the incidence of accompanying urinary urgency . mui may represent a more advanced state of sui . according to the considerable rates of do shown in our results , it is also possible that there may be a unique underlying cause that motivates symptoms of mixed ui that is distinct from the factors that stimulate urge ui induced by sui .

purposethis study aimed to determine whether symptoms of urinary urgency increase according to the severity of stress urinary incontinence ( sui ) . for this purpose , we recruited women with symptoms of mixed as well as pure sui and compared the clinical characteristics of each subgroup.materials and methodsa total of 241 female patients who were diagnosed with sui and mixed urinary incontinence ( mui ) were analyzed retrospectively . patients with only sui were categorized as group 1 . patients with mui were categorized as group 2 . clinical and urodynamic differences between the 2 groups were compared.resultsthe proportion of stamey grade was significantly different between the 2 groups : grade 1 sui was higher in group 1 , but grades 2 and 3 sui were higher in group 2 . the incidence of urgency was proportional to the degree of stamey grade ( 23.5% in grade 1 , 36.9% in grade ii , and 60.0% in grade iii ) . in the urodynamic study , the presence of detrusor overactivity was significantly higher in group 2 than in group 1 ( 25.9% vs. 49.4% ) . other clinical parameters were also significantly different between the 2 clinical groups : q - tip angle ( group 1 : 42.1 , group 2 : 28.6 , p<0.05 ) , maximal urethral closure pressure ( group 1 : 54.7 cmh2o , group 2 : 44.1 cmh2o , p<0.05 ) , maximal bladder capacity ( group 1 : 356.3 ml , group 2 : 282.0 ml , p<0.05 ) , and bladder volume at first desire ( group 1 : 144.6 ml , group 2 : 123.2 ml , p<0.05).conclusionsaccording to this analysis , the more serious the symptoms of sui , the higher the incidence of urinary urgency .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

lymphoma is a heterogeneous malignancy of the lymphatic system characterized by proliferation of lymphoid cells or their precursors . they are generally categorized as hodgkin 's and non - hodgkin 's lymphomas ( nhls ) based on the presence and absence of typical hodgkin 's cells . hodgkin 's lymphoma rarely presents as extranodal disease , whereas nhl commonly presents at extranodal sites ( 2545% ) such as gastrointestinal tract , skin and bone . in the head and neck , primary nhl occurs in waldeyer 's ring , oral mucosa , salivary glands , paranasal sinuses , laryngeal tissue and bone . when it does occur , mandibular nhl closely mimics an ameloblastoma , a tumor of odontogenic epithelial origin , both clinically and radiographically . genetic diseases , environmental agents and infectious agents have been attributed to the development of nhl . barr virus , human t - cell lymphotropic virus 1 , human immunodeficiency virus , helicobacter pylori , chlamydia and human herpesvirus-8 are known to be associated with an increased risk of nhl . while the actual role of viruses in lymphomagenesis remains to be understood , some types of nhl have shown insertion of exogenous genes by oncogenic viruses . we present a rare case of rapidly progressing swelling of the left mandible which on routine histopathological examination appeared to be a spindle - cell sarcoma . immunohistochemical analysis enabled a final diagnosis of spindle - cell variant of lymphoma , an occurrence previously reported only once in english literature . a 64-year - old male presented with a rapidly progressive swelling of the left mandible since 6 months for which he had undergone fine needle aspiration cytology and an incisional biopsy elsewhere with a diagnosis of benign mesenchymal neoplasm and chronic inflammatory granulation tissue , respectively . extraoral clinical examination revealed a firm to hard diffuse soft tissue swelling measuring 4 cm 5 cm that extended anteroposteriorly from midline to the left angle of the mandible and inferiorly extended 2 cm below the inferior border of the mandible [ figure 1a and b ] . intraorally , buccal cortical expansion was noted from midline to second molar region obliterating the vestibule with paresthesia of lower lip [ figure 1c ] . overlying skin and mucosa ( a and b ) extraoral examination of left mandibular swelling that was firm to hard diffuse soft tissue swelling extending anteroposteriorly from midline to the left angle of the mandible and inferiorly extended 2 cm below the inferior border of the mandible . ( c ) intraoral view showing the unilateral ( left ) involvement of the lesion cone beam computed tomography ( planmeca promax 3d ; large fov [ 200 mm 100 mm ] ) imaging revealed an ill - defined , moth - eaten radiolucency in the left mandibular premolar / canine region with destruction of buccal and lingual cortical plates [ figure 2a d ] . serum calcium , phosphorous , alkaline phosphatase and parathyroid hormone levels were within normal limits . ( a ) cone beam computed tomography reconstructed panoramic image showing diffuse radiolucency in the left body of mandible extending superiorly to the alveolar crest and inferiorly to lower border ; anteriorly to apex of 33 and posteriorly till the region of 36 . ( b ) cone beam computed tomography coronal section showing radiolucent lesion in the left body of mandible extending through buccal and lingual cortex , minimal mucosal thickening of the right and left maxillary sinus floor is seen . ( c ) cone beam computed tomography sagittal section revealed radiolucency extending from 33 to 38 with erosion of the mandibular nerve canal . ( d ) cone beam computed tomography axial section showing asymmetry on left side with loss of buccal and lingual cortical plates and no evidence of expansion of the cortical plates a clinically aggressive growth in the mandible with radiographic evidence of bone destruction entailed a differential diagnosis of ameloblastoma , malignant salivary gland tumor , aggressive osteomyelitis , malignant connective tissue neoplasm and metastasis from unknown primary , thereby warranting a mandibular resection . gross examination of the resected hemimandible showed attached lesional soft tissue measuring approximately 10.5 cm 8.0 cm 5.0 cm , white to brown in color , with multiple cystic spaces and no crackling upon palpation [ figure 3a ] . microscopic examination showed an encapsulated lesion situated in the connective tissue and separated from the overlying normal epithelium by a grenz zone [ figure 3b ] . morphologically , the neoplastic tissue showed a mixture of proliferating spindle - shaped cells arranged in fascicles and round cells infiltrating into the connective tissue stroma and bone [ figure 3c ] . the neoplastic cells exhibited atypical features such as pleomorphism , hyperchromatism and increased mitotic figures with noncleaved nuclei [ figure 3d ] . ( a ) gross surgical specimen showing the erosion and perforation of the buccal and lingual cortical plates . cut surface of the tumor showing lingual invasion below the inferior border of mandible . ( b ) : photomicrograph showing presence of mixture of spindle- and round - shaped cells arranged in fascicles separated from normal epithelium by grenz zone ( h&e stain , 40 ) . ( c ) photomicrograph showing tumor cells infiltrating the bone ( h&e stain 40 ) . ( d ) : photomicrograph showing round to spindle shaped tumor cells with hyperchromatic nuclei ( h&e stain , 400 ) immunohistochemical staining with desmin [ figure 4a ] , smooth muscle actin ( sma ) [ figure 4b ] , s-100 [ figure 4c ] and cd1a [ figure 4d ] were negative and lymphoid markers leukocyte common antigen ( lca ) and cd20 ( cluster differentiation marker for b - cells ) showed positivity in spindle - shaped cells as well as round cells indicating the tumor to be a lymphoproliferative lesion of b - cell type [ figure 4e and f ] . a final diagnosis of spindle - cell variant of nhl was rendered based on the immunohistochemical profile . round- and spindle - shaped tumor cells showing negativity for ( a ) desmin ( ihc stain , 400 ) ; ( b ) smooth muscle actin ( internal positive control ; vessel wall ) ( ihc stain , 400 ) ; ( c ) s-100 ( internal positive control ; nerve ) ( ihc stain , 400 ) , ( d ) cd1a ( ihc stain , 400 ) . the cells were positive for ( e ) ; leukocyte common antigen ( lymphoid lineage marker ) ( ihc stain , 200 ) ; and ( f ) cd20 ( b - cell marker ) ( ihc stain , 200 ) postsurgical management of the patient was uneventful , and the disease was classified as ann arbor stage iie based on the involvement of the mandible and submandibular lymph nodes . the patient underwent chemotherapy vide the cyclophosphamide , adriamycin , vincristine and prednisolone regimen and has remained disease free for 12 months of follow - up . lymphoma is a malignancy of the lymphatic system characterized by proliferation of lymphoid cells or their precursors with purely a round cell morphology . morphologically , the present case posed a diagnostic challenge by exhibiting proliferating compact spindle and round cells arranged in short fascicles with focal areas showing storiform pattern that was suggestive of spindle - cell sarcoma . the spindle - shaped cells warrant distinction from other neoplasms with spindle - cell morphology . special trichrome stains such as van gieson 's stain and masson 's trichrome stains were performed and supplemented by immunohistochemistry to eliminate the possibility of collagenous tumors . immunohistochemically , the tumor cells were negative for the desmin , sma , s-100 and cd1a but strongly positive for lca and cd20 suggesting a spindle - cell variant of lymphoma . hematolymphoid neoplasms with prominent spindle - cell features , also termed spindle - cell lymphoma , are rare but have been reported to occur in anaplastic large - cell lymphoma , reticulum cell sarcoma , primary bone lymphoma and primary soft tissue and/or skin lymphoma . morphologically , these cases exhibit proliferation of spindle cells arranged in fascicles leading to a diagnosis of spindle - cell neoplasms of nonlymphoid origin . the origin of spindle - cell b - cell lymphomas is uncertain , with previous investigators ( ferrara et al . , 2002 ; lim et al . , 2008 ; yun 2009 ) reporting cases of spindle - cell b - cell lymphomas as variants of follicular lymphoma , whereas others ( wang et al . , 2010 ; kahlifa et al . , 2003 ; ries et al . , 2007 ; 2008 ) have classified them as a variant of diffuse large b - cell lymphoma . while reporting three cases of primary b - cell malignant lymphoma in the maxilla with sarcomatous pattern noted that the spindle cells in these cases did not represent the malignant lymphoid cells , but rather they were a fibrotic stromal response as well as cytoskeleton alterations to the tumor which is seen particularly in tumor invasion of bone and soft tissue . the present case however prominently displayed spindle - cell morphology amidst the lymphoid cell population ( as proven with immunohistochemistry too ) , rather than in the surrounding stromal cells . cerroni ( 2002 ) , in a reported case series of cutaneous spindle - cell b - cell lymphoma , stated that spindled nuclei likely correspond to centrocytes and to a lesser extent centroblasts of follicular center cell origin , whereas somewhat larger and more monomorphous spindle cells are consistent with a spindle - cell variant of diffuse large b - cell lymphoma . a case of cutaneous spindle - cell b - cell lymphoma reported by goodlad , 2001 , exhibited immunohistochemical expression of cd10 and bcl-6 in addition to focal immunostaining of cd23 and cd35 for follicular dendritic cell networks within neoplastic follicles , prompting them to propose a follicular center cell origin . reported five cases of spindle - cell b - cell lymphoma and based on immunohistochemical studies , genetic analysis and clinical presentation , categorized them as a variant of diffuse large b - cell lymphoma . all five cases exhibited immunoreactivity for b - cell markers ( cd20 , cd79a ) and bcl-6 but were negative for t - cell markers ( cd3 and cd5 ) , cd10 and mum-1 . furthermore , the finding of somatic immunoglobulin and bcl-6 mutations favor the origin of this lesion from b - cells in an early phase of intragerminal center maturation . in addition , all cases showed the absence of chromosomal translocation t ( 14;18 ) that is otherwise characteristic of follicular lymphoma . ( 2010 ) reported cases which showed positivity for sma and believed that the spindling probably results from aberrations in the cytoskeleton ; however , the tumor cells were negative for sma in the present case . it has been hypothesized that cytokines such as tumor necrosis factor alpha , platelet - derived growth factor and transforming growth factor beta are produced by tumor cells which induce proliferation of fibroblasts in turn causing spindle - cell deformation within lymphoma cells . the differential diagnosis of spindle - cell tumors does not normally include malignant lymphoma , a neoplasm that commonly has small round - cell morphology . while the spindle - cell b - cell lymphoma has previously been commonly reported to occur within the skin , particularly of the head , neck and back regions , the current case illustrates that on rare occasions , the spindle variant of b - cell nhl may present in the mandible , an occurrence previously only reported by chi et al . , ( 2012 ) . the prognosis of nhl depends mainly on the histology and the stage of the tumor ( whether it is localized or not , stage of evolution , number of extranodal localizations , etc . ) . the location of the tumor ( e.g. presenting in the jaw ) is not found to be a significant prognostic factor as yet .

a 64-year - old male farmer presented with a rapidly progressive swelling of the left mandible since 6 months . the swelling was firm to hard , diffuse , nontender , obliterating the vestibule with paresthesia of lower lip . the cone beam computed tomography imaging revealed an ill - defined , moth - eaten radiolucency with destruction of the buccal and lingual cortical plates . the rapid growth and aggressive behavior of the lesion coupled with guidance from the patient 's previous reports from the incisional biopsy and fine needle aspiration cytology warranted a mandibular resection . microscopic examination showed an encapsulated lesion situated in the connective tissue containing a mixture of proliferating spindle - shaped cells arranged in fascicles and round cells infiltrating into the connective tissue stroma and bone . the neoplastic cells exhibited atypical features such as pleomorphism , hyperchromatism and increased mitotic figures with noncleaved nuclei . a working diagnosis of a spindle - cell sarcoma was arrived at with various differentials provided such as fibrosarcoma , rhabdomyosarcoma , leiomyosarcoma , malignant peripheral nerve sheath tumor , langerhans cell histiocytosis and lymphoma and stating the need for immunohistochemistry to subtype the tumor . the neoplastic cells were negative for van gieson 's stain and masson 's trichrome . immunohistochemical analysis performed using desmin , smooth muscle actin , s-100 and cd1a in a bid to determine the phenotype of the tumor and rule out the previously stated differentials were all negative for the lesion . lymphoid markers such as leukocyte common antigen and cd20 ( cluster differentiation marker for b - cells ) showed positivity in spindle - shaped cells as well as round cells indicating the tumor to be a lymphoproliferative lesion of b - cell type . a final diagnosis of spindle - cell variant of non - hodgkin 's lymphoma was rendered based on the immunohistochemical profile .

INTRODUCTION CASE REPORT DISCUSSION Financial support and sponsorship Conflicts of interest

recently , integration of semiconductor with ferromagnetic function has been focused on in spintronics because of the difficulties associated with the injection of spins from magnetic metal into nonmagnetic semiconductors in conventional spintronic devices . many groups have found rtf in transition or rare earth metal doped compound semiconductors such as tio2 , zno , sno2 , and in2o3 . when groups of people try to explain the fm in transition metal doped semiconducting oxides , an unexpected fm was reported in pure hfo2 thin film , which challenges the understanding of magnetism for the researchers . suggested that oxygen vacancies were key factors in introducing fm to hfo2 , while pemmaraju and sanvito suggested that the origin of rtf in hfo2 was due to defects on hf sites . hu et al . presented that rtf observed in pure mgo results from cation vacancies . . suggested that a small concentration of ca vacancies in cao can also induce fm based on results of the ab initio electronic structure calculation , which could be a path to new ferromagnets . recently , similar fm has been reported in other pure semiconductor materials , such as tio2 , zno , sno2 , in2o3 , al2o3 , and ceo2 , where the origin of fm is believed to be oxygen defects . cuo , as a narrow band gap p - type semiconductor , has been recognized as an industrially important material for a variety of practical applications , such as catalysis , batteries , solar energy conversion , gas sensing , and field emission [ 12 - 14 ] . therefore , the synthesis and study of cuo nanostructures should be of practical and fundamental importance . apart from this , if one can find fm without any magnetic impurity doping , this may bring a new opportunity to the field of spintronics because there will be no issues due to clustering or precipitation of dopants . indeed , punnoose et al . and mishra et al . represented the presence of an exchange interaction between the ferromagnetic surface and the antiferromagnetic core . recently , xiao et al . and shang et al . reported the observation of rtf in cuo nanostructures . however , the fm in cuo remains controversial because most groups suggested that cu atoms have no clustering tendency and cu - based oxides are not ferromagnetic . here , we synthesize cuo nanoparticles by a simple co - precipitation method to avoid the influences of substrate and the interface between film and substrate . we found the cuo nanoparticles show rtf , and the origin of the fm is discussed . cuo nanoparticles were prepared by the precipitation technique with the post - oxidation annealing in air . briefly , 3 g highly pure cu ( no3)26h2o was dissolved in 50 ml de - ionized water , and the nh4 oh solution was added into it gradually until the ph level reached 10 . the mixture was stirred for 4 h at room temperature and then dried at 50c for 6 h. in the end , the precursor was annealed at 800c for 1 h in air . the morphologies of the nanoparticles were obtained by transmission electron microscopy ( tem , jem-2010 ) . x - ray diffraction ( xrd , x pert pro philips with cu k radiation ) was employed to study the structure of the particles . the doping levels and the bonding characteristics were determined by x - ray photoelectron spectroscopy ( xps , vg escalab 210 ) . the compositions of the particles were analyzed by inductively coupled plasma atomic emission spectrometer ( iris , er / s ) . the measurements of magnetic properties were made using quantum design mpms magnetometer based on superconducting quantum interference device ( squid ) and the vibrating sample magnetometer ( vsm , lakeshore 7304 ) . all diffraction peaks can be indexed as the typical monoclinic structure , and no extra diffraction peaks of other phases are observed . refinement gives lattice constants are a = 4.6881(8 ) , b = 3.4293(1 ) , c = 5.1354(1 ) , and = 99.304(9). using the scherrer formula and the full width at half maximum of the main peak , the average crystalline size is estimated to be around 26 nm . figure 1b gives the morphology images of the sample captured by tem , and it can be seen that the particles congregate together and the size of which is about 150 nm . the lattice fringes between the two adjacent planes are 0.253 nm apart , which is equal to the lattice constant of the standard cuo in ( 002 ) plane . a refined xrd pattern b tem and c high - resolution tem images of the cuo nanoparticles annealed at 800c for 1 h the magnetization versus magnetic field ( m - h ) curves for the cuo nanoparticles recorded at 10 and 300 k are shown in fig . 2 , which were investigated using the squid magnetometer , and the paramagnetic ( pm ) signal of the sample and the holder were deducted . the top left inset is the magnification of the central part for the m - h curves . it can be seen that the measured coercivities and the saturated magnetizations ( ms ) of the cuo nanoparticles are 120 oe , 0.018 emu / g , and 57 oe , 0.032 emu / g at 10 and 300 k , respectively . the coercivities are smaller than that of report in reference 18 ( 331.39 and 175.88 oe at 10 and 300 k , respectively ) . the zero - field - cooled ( zfc ) and field - cooled ( fc ) magnetization curves in the temperature range of 2330 k at the field of 100 oe on cuo were shown in below inset of fig . the results indicate there is no blocking temperature in this temperature range , supporting that there is no contamination of ferromagnetic cluster . at the same time , we can conclude that the curie temperature of the sample is above 300 k. figure 3 shows the m - h curves of the cuo nanoparticles annealing at 800c for different annealing time , and the variation of ms is shown in the inset . it can be seen that as the annealing time increasing , the magnetization decreases gradually . and the reason will be discussed later . in addition , it is noticed that magnetic measurements were reproducible for all the cuo samples synthesized repeatedly in different batches under identical experimental conditions . m - h curves of cuo nanoparticles annealed at 800c recorded at 10 and 300 k , in which the paramagnetic signal of the sample and the holder have been deducted . the top left inset shows the magnification of the central part for m - h curves and the bottom right inset shows the fc - zfc curve of cuo nanoparticles annealed at 800c m - h curves for cuo nanoparticles annealed at 800c for different times . the inset shows the variation of ms several publications show contamination is a possible source of the fm in hfo2 , tio2 , sno2 , etc . . in explaining the origin of fm in the cuo nanoparticles , a careful consideration whether the contamination is responsible for the fm has to be undertaken . the capsules used to hold the samples during the magnetic measurements were also checked carefully and showed no ferromagnetic signals . the result of inductively coupled plasma ( icp ) indicates that elements such as fe ( co ) exists in our sample are about 2 10 at.% , which may come from the precursor material of cu(no3)26h2o . however , magnetic measurement indicates that the precursor particles are pm , and it did not influence the magnetic properties of the cuo nanoparticles in agreement with other reports . therefore , we suggest that the observed fm is intrinsic in all samples . so we must reconsider the possibility origin of fm which was previously assumed for the other ferromagnetic undoped oxides : fm due to oxygen vacancies and/or defects [ 5 - 11 ] . figure 4 presents the survey scan and cu 2 p core level binding energy spectra of the cuo nanoparticles . all the indexed peaks are corresponding to c , o , and cu as shown in fig the strong fitting peaks at around 932.1 and 952.3 ev for cu2p3/2 and 2p1/2 peaks and 933.4 ev and 954.4 ev for cu2p3/2 and 2p1/2 peaks are much closer to the other report , which reveals that the chemical valences of cu at the surface of the cuo nanoparticles are mixture valence states of + 1 and + 2 . there are no extrinsic magnetic impurities in the undoped cuo , so the occurrence of fm is predominately due to a complex interplay of two different valence states of the copper ions at the surface of the particles with the oxygen vacancies and the associated defects . the inset shows the cu 2 p core level binding energy spectra in order to experimentally verify this prediction , vacuum and oxygen annealing were done for the cuo nanoparticles as other groups method . figure 5 shows the variation of ms for cuo particles in different annealing conditions . at first , as - prepared cuo nanoparticles were annealed in vacuum atmosphere ( 0.001 pa ) at 400c for 30 min , which is denoted as vacuum in the figure . it is seen that the fm of cuo particles enhances and the ms reaches 0.024 emu / g . then the sample was annealed in the rich oxygen atmosphere ( the mix atmosphere of argon and oxygen ) at 400c for 30 min , which is denoted as oxygen . and the ms decreases to 0.007 emu / g . finally , the fm increases again and the value of ms reaches up to 0.021 emu / g after the sequential repeating annealing in vacuum . the m - h curves for the cuo nanoparticles of each annealing condition are shown in inset of fig . 5 , which were measured by vsm , and the pm signal of the sample and the holder have been deducted . usually , the vacuum annealing effectively introduces oxygen vacancies into oxides , while the rich oxygen annealing reduce oxygen vacancies . it is found in our experiment that the fm of the cuo nanoparticles increases after annealing in vacuum condition and decreases after annealing in the rich oxygen atmosphere , which is corresponding to our previous report in zno nanoparticles .these results prove that the rtf of the undoped cuo nanoparticles originates from oxygen vacancies in our case . theoretically speaking , the oxygen atoms can escape from the bondage of the chemical bond because of heating . then the unpaired electrons show the abnormal spin phenomenon which could cause the magnetism . in addition , as the annealing time increases , the size of particle increases gradually and the particles aggregate together with the decreasing of the ms , which indicate that oxygen vacancies at the surface / or interface of the particles are likely to be responsible for the fm in the cuo nanoparticles . the variation of ms for cuo nanoparticles sequential annealed at different conditions in vacuum and oxygen atmosphere . in summary , cuo nanoparticles were prepared by co - precipitation method with the post - annealing in air . the clear hysteresis loop is observed at room temperature in cuo nanoparticles , and the extrinsic impurity origin is excluded . the results of repeating annealing cuo nanoparticles in different conditions indicate that oxygen vacancies at the surface of the particles are likely to be responsible for the fm . further theoretic investigations into the defects introducing fm are expected and our work is on the way . this work is supported by nsfc ( grant no.50671046 and no.50801033 ) , national science fund for distinguished young scholars ( grant no . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

room temperature ferromagnetism ( rtf ) is observed in pure copper oxide ( cuo ) nanoparticles which were prepared by precipitation method with the post - annealing in air without any ferromagnetic dopant . x - ray photoelectron spectroscopy ( xps ) result indicates that the mixture valence states of cu1 + and cu2 + ions exist at the surface of the particles . vacuum annealing enhances the ferromagnetism ( fm ) of cuo nanoparticles , while oxygen atmosphere annealing reduces it . the origin of fm is suggested to the oxygen vacancies at the surface / or interface of the particles . such a ferromagnet without the presence of any transition metal could be a very good option for a class of spintronics .

Introduction Experiments Results and Discussions Summary Acknowledgments Open Access

simple obesity is currently one of the most common metabolic diseases and its incidence has been increasing every year . this is mainly due to changes in lifestyle and dietary habits of contemporary society as well as the development of inappropriate nutrition and physical activity patterns already in early childhood . obesity increases the risk of hypertension , dyslipidemia and abnormal glucose tolerance , which results in an increased risk of heart and metabolic diseases . obesity as a disease of affluence affects the youngest patients increasingly frequently . over the last decades a gradual increase in the prevalence of excessive body weight and obesity has been observed around the world . this is true not only for the adult population , but for children as well . it is thought that if this trend is not stopped , the present generation of children , despite the considerable progress in medical sciences , will probably be the first generation to live shorter than the parents . the additional tests for children with simple obesity most commonly ordered by primary care physicians include the assessment of thyroid function conducted in order to exclude hormonal imbalances that cause obesity . obese individuals relatively often have elevated levels of the thyroid - stimulating hormone ( tsh ) in the blood serum , called hyperthyrotropinemia , which has a tendency to rise with age . the substance which is responsible for this is leptin , which regulates body weight and hunger , influencing the level of tsh in the blood serum by modifying its synthesis in the pituitary gland . another mechanism accounting for the elevated levels of this hormone in obese patients consists in the possible resistance to thyroid hormones at the level of the pituitary gland and disruption of the negative feedback phenomenon . as a result , the tsh concentration is observed to decrease following the reduction of body weight to a normal level . an attempt at broadening the diagnostic investigation by conducting an imaging examination of the thyroid gland , in addition to tests of free thyroxine , triiodothyronine and antibodies against thyroid peroxidase and thyroglobulin seems to be an obvious course of action . according to them one of the indications for the ultrasound examination of the thyroid gland is a suspected disease based on abnormal laboratory test results , including hyperthyroidism , hypothyroidism and thyroiditis . correct interpretation of a thyroid ultrasound examination in an obese child may become one of the fundamental elements affecting further course of action or therapeutic decisions , especially with borderline values of tsh or thyroid hormones . lesions in the thyroid gland which may be associated with obesity have been reported in medical literature for some time . the most important ones include increased volume of the gland , changed echogenicity and the presence of focal lesions . in a dutch study it was demonstrated that adult obese patients had a larger thyroid gland volume in comparison with a group of individuals with a normal body weight . thyroid volume correlated with lean body mass and tsh and not with body fat mass . in another study it was observed that the thyroid gland volume in healthy euthyroid women may change with the changes in body mass . a larger thyroid volume , correlation between this parameter and body mass , body mass index ( bmi ) and waist circumference was found . the thyroid volume decreased in patients who after a dietary intervention reduced their body weight by at least 10% within 6 months . based on the analysis of a group consisting of 280 obese children stichel et al . found that the volume of the thyroid gland in the studied patients fell within the normal range . the thyroid gland volume in relation to the surface area of the subjects bodies also did not differ from the values found in healthy children from the berlin population . over the last few years a possible link between thyroid cancer and obesity has been reported in medical literature . in large screening studies on adults in seoul a significantly higher rate of concurrent obesity and thyroid cancer in women was observed . a link between the prevalence of thyroid cancer and age , insulin levels or tsh was excluded . one needs to bear in mind , however , that the incidence of thyroid cancer in the youngest patients , however low , seems to be rising . over the last few years papers appeared which suggested that in obese children ultrasound examinations of the thyroid gland often demonstrate changes similar to those observed in the autoimmune hashimoto thyroiditis . however , no antibodies against the thyroid gland were found in the blood of these patients and fine - needle biopsy did not reveal lymphocyte infiltration , which excludes the presence of hashimoto thyroiditis . the authors put forward the hypothesis that the reported lesions in the thyroid gland are linked to a chronic inflammatory process associated with obesity . the adipose tissue , as an endocrine organ , secretes inflammatory cytokines such as the tumor necrosis factor ( tnf- ) and interleukines 1 and 6 ( il-1 and il-6 ) , which may induce a low - intensity inflammatory process in obese patients . cytokines are probably responsible for the dilation and increase in the permeability of the thyroid blood vessels , which may result in an effusion into the thyroid parenchyma that forms a sonography image typical of the hashimoto 's disease . ultrasound examination in children also revealed the return of the thyroid structure to the normal state following weight loss and lifestyle change . this phenomenon is probably associated with the reduction of the intensity of the inflammatory process , which may cause a regression of lesions in the thyroid parenchyma as a result of weight loss . one should bear in mind that the english term echogenicity covers two polish terms : echogeniczno autoimmune inflammation is usually considered to be characterized by both decreased echogenicity and abnormal echostructure . in addition , in the english - language medical literature often the slightest deviation from the norm is believed to be sufficient to identify the pattern suggesting hashimoto 's disease , which leads to numerous overinterpretations . it is worth taking into account the fact that these are relatively general concepts and that the image may not necessarily correspond to the extended criteria for the diagnosis of autoimmune thyroiditis in children described in medical literature . other features which are helpful for the diagnosis of hashimoto 's disease include increased parenchymal flow and the presence of lymph nodes below the lower poles of the gland . it is only the coexistence of these features that provides grounds for an ultrasound diagnosis of autoimmune thyroiditis . one should bear in mind that it is not tantamount to hashimoto 's disease ; it is one of the types of autoimmune thyroiditis and a differential diagnosis is not possible without clinical data . it is also worth remembering that hashimoto thyroiditis is more prevalent in obese children than in the general population . obese children are found to have a higher prevalence of elevated levels of antibodies against the thyroid gland than the general population , especially in the case of the presence of elevated tsh levels . this is probably associated with an increased presentation of the antigen caused by a stronger stimulation of the thyroid . the authors of the present paper were interested in this subject and for this reason they started their own research into the matter , the preliminary results of which were presented in the form of a poster during two congresses : european society for paediatric endocrinology and euroson meetings . this research indicates that the ultrasound image of the thyroid gland in obese children is clearly changed ; however , it is not typical of an autoimmune inflammation . two obese pediatric groups were analyzed : one formed in 20052007 ( 58 children ) and the second one formed in 20112015 ( 109 patients ) . in both groups a characteristic sign of decreased echogenicity of the gland was present ( down to 61% ) , especially in the peripheral areas of the lobes . echostructure disorders are less frequent ( 33% ) , both in combination with changed echogenicity and alone ( 9.2% ) . single cases of hashimoto 's disease were diagnosed . in the vast majority of the remaining patients ( 93% ) neither increased flow nor the presence of lymph nodes in the area of the lower poles were found . a very distinct feature is the abovementioned peripheral distribution of decreased echogenicity , which was found in both groups in 47% and 61% of patients , respectively . it is usually arranged in such a way that the central and medial parts of both lobes have a normal echogenicity in almost every case , while in the remaining parts , especially dorsally , echogenicity is decreased , sometimes heterogeneously . this decrease might partly be explained by the phenomenon of tissue attenuation ; however , it does not account for the changes in echogenicity in the ventral and lateral fragments of the lobes . a possible cause may be the one suggested by radetti a low - intensity inflammatory process or an adipocyte infiltration . such an image , however , is substantially different from the image observed in patients with autoimmune thyroiditis . ultrasound image of the thyroid gland : a. normal b. typical for hashimoto 's disease c. typical for obesity basic differences between the thyroid image in healthy children , obese children and children with autoimmune thyroiditis in summary , the interpretation of this phenomenon is inconclusive and should be based on a very detailed ultrasound examination of the thyroid gland . therefore , when assessing the echogenicity of the thyroid gland in obese children , one should remain very cautious about the interpretation of the changes and in case of doubt the diagnosis should not be exclusively based on an abnormal ultrasound image without proving the presence of antibodies against the thyroid gland . the authors do not report any financial or personal affiliations to persons or organizations that could negatively affect the content of or claim to have rights to this publication .

obesity as a disease of affluence also affects younger children . numerous observations suggest a link between excessive body weight and thyroid function disorders . subclinical hypothyroidism has been diagnosed increasingly frequently in patients with obesity . a growing number of papers also point to morphological changes of the thyroid gland in the ultrasound examination in obese children . these reports mainly concern changes in echogenicity . the present paper discusses the most important aspects of this topic on the basis of the literature as well as containing a brief analysis based on own experiences .

Introduction Thyroid volume Focal lesions Change in echogenicity Conflict of interest

at least 135 people have been infected with a novel influenza a(h7n9 ) virus since february 2013 in eastern china , resulting in a high frequency of severe lower respiratory tract infections and 44 fatalities ( 1 , 2 ) . this novel influenza a virus ( iav ) most likely emerged from a zoonotic reservoir , as closely related viruses were isolated from apparently healthy poultry in those provinces ( 3 ) . genome sequencing indicated that the h7n9 virus derives its genes from at least three different low - pathogenic avian iav strains ( 1 , 4 , 5 ) . affected patients experienced febrile influenza - like illness , rapidly progressing to pneumonia and acute respiratory distress syndrome in many cases , indicating the spread of the virus to the lung ( 1 ) . the capacity to infect the lower respiratory tract was also reproduced in experimental infections of ferrets , pigs , macaques , and mice , which is unusual for an avian influenza virus ( 69 ) . recent analyses detected significantly increased cytokine and chemokine levels in a(h7n9 ) patient serum samples , which may reflect a dysregulation of the immune response contributing to the severity of the disease ( 10 ) . although many a(h7n9 ) patients had underlying chronic conditions , this outbreak challenges the concept that iav with low pathogenicity in birds infect humans very rarely and do not cause severe disease ( 11 ) , raising questions as to the specific properties of this novel zoonotic pathogen in humans . genetic analyses showed that novel h7n9 viruses harbor adaptive changes associated with enhanced fitness of avian iav in human hosts . this includes a glutamine - to - leucine change at position 226 ( h3 numbering ) within the receptor binding site of the viral hemagglutinin , which most likely extends the spectrum of virus receptors by enabling binding to avian ( alpha-2,3-linked sialic acid ) as well as human ( alpha-2,6-linked sialic acid ) receptor determinants ( 8 , 10 , 12 , 13 ) . moreover , the presence of lysine and asparagine at polymorphic amino acid positions 627 and 701 , respectively , in the pb2 protein of patient h7n9 isolates indicates initial adaptation for increased polymerase activity in mammalian cells ( 3 ) . however , virulence of iav is a multigenic trait , and additional genetic changes encoding adaptive amino acids may be present in the novel h7n9 reassortant virus ( 4 ) . an important aspect of iav pathogenicity is the capacity to suppress the innate antiviral response by means of the viral ns1 protein . this multifunctional viral factor limits expression of type i interferon ( ifn - i ) genes via blockade of the signaling pathway governed by retinoic acid - induced gene i ( rig - i ) , silences activation of the kinase pkr , and prevents fast apoptosis of infected host cells ( 14 ) . ns1 proteins of human and avian iav strains have a length of 215 to 237 amino acids and can differ in their primary sequences by more than 30% , indicating specific adaptations to the respective host environment ( 15 ) . we previously showed that a low - pathogenic avian iav ( subtype h12n5 ) was strongly attenuated for growth in explanted human lung tissue but provoked a strong cytokine response in comparison to human - adapted seasonal and pandemic iav ( 16 ) . given the substantial viral loads detected in lower respiratory tract samples of a(h7n9 ) patients ( 3 ) , we herein compared the replicative potential and activation of innate responses by an h7n9 patient isolate and two low - pathogenic h7 subtype viruses of the eurasian lineage in human alveolar tissue . replication analysis in primary human lung tissue showed that the a / anhui/1/2013 ( h7n9 ) virus propagated similarly well as the prototypic human a / panama/2007/1999 ( h3n2 ) strain over a 48-h period , reaching titers in the range of 10 pfu / ml . in contrast , there was hardly any titer increase detectable for two low - pathogenic avian h7 subtype viruses [ a / turkey / italy/472/1999 ( h7n1 ) and a / turkey / germany / r11/2001 ( h7n7 ) ] after inoculation of tissue from identical donor lungs ( fig . the differences between human and avian h7 viruses became highly significant through the course of infection . importantly , lung tissue infected with the two avian h7 viruses released significantly higher levels of antiviral ifn- into the supernatants than the human h7 virus ( fig . the levels of secreted mip-1 , ip-10 , and interleukin 1 ( il-1 ) were also slightly higher for the two avian strains , but those differences were statistically not significant ( fig . differences in viral growth and cytokine induction between avian and human h7 strains might be due to differences in the principal target cells . therefore , we examined the cellular tropism of the h7-subtype viruses by double staining for viral antigen and cell - type - specific markers in the alveolar tissue . this analysis showed that the human and the avian h7 strains were detected in type ii but not in type i pneumocytes ( fig . 1e and data not shown ) , indicating that those viruses target the same host cell type as was previously shown for other avian , porcine , and human iav ( 16 ) . efficient replication , cytokine induction , and cellular tropism of h7n9 virus in human lung tissue infected ex vivo . tumor - free normal lung tissue was stamped into small cylinders ( thickness of ~3 mm , diameter of 8 mm ) and incubated in rpmi 1649 medium ( containing 0.3% bovine serum albumin [ bsa ] , 2 mm glutamine , and antibiotics ) at 37c with 5% co2 as described ( 16 ) . after overnight incubation , lung organ cultures were inoculated with 4 10 ( a ) or 4 10 ( b to e ) pfu of the influenza a viruses a / anhui/1/2013 ( hu - h7 ) , a / turkey - italy/472/1999 ( av - h7-it ) , or a / turkey - germany / r11/2001 ( av - h7-ger ) for 1 h , followed by being washed with phosphate - buffered saline ( pbs ) to remove excess virus and incubation in rpmi 1649 medium at 37c . the epidemic influenza a / panama/2007/1999 virus ( hu - h3 ) was included as a control . for each experiment , tumor - free tissue specimens from at least three donors were analyzed . stocks of the avian h7 viruses were grown in embryonated chicken eggs , whereas the human h3 and h7 viruses were propagated in mdck cells to minimize selection of receptor binding variants . ( a ) aliquots of supernatants taken at 0 , 16 , 24 , and 48 h postinfection ( hpi ) were titrated by standard plaque assay on mdck cells . the relative growth of the strains is depicted as titer increase compared to the start of infection ( 0 hpi ) . the human h7 strain propagated as efficiently as the seasonal human virus , whereas the two avian h7 viruses hardly replicated . mean values and standard errors of the means ( sem ) from 3 independent experiments each done in triplicate are shown . asterisks indicate significant differences between strains ( mann - whitney u test ; * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ) . ( b to d ) aliquots of infected lung culture supernatant taken at 24 or 48 hpi were analyzed for the concentrations of ifn- ( b ) , mip-1 ( c ) , or ip-10 ( d ) by commercial enzyme - linked immunosorbent assay ( elisa ) kits ( fujirebio inc . data points from four independent experiments are shown individually , and mean values are indicated ( line ) . significance values between groups are indicated by asterisks ( mann - whitney u test ; * , p < 0.05 ) . the human h7 virus induced significantly less ifn- than av - h7-it and av - h7-ger . human lung tissue was not infected ( mock ) or was infected with hu - h7 or av - h7-ger for 24 h. the tissue samples were fixed and routinely paraffin embedded as described ( 16 ) . after deparaffinization and antigen retrieval , slices were immunostained with fluorescently ( alexa fluor 488 ) labeled anti - influenza a virus antibody ( serotec ; obt1551 ) ( green channel ) to detect virus - infected cells ( white arrowheads ) . the slices were costained with rabbit antibodies detecting either pro - sp - c ( chemicon ; ab3786 ; top row ) or emp2 ( sigma ; hpa014711 ; bottom row ) to identify type ii or type i pneumocytes ( red channels ) , respectively . an alexa fluor 594-labeled anti - rabbit f(ab)2 fragment was applied as the secondary antibody . immunofluorescence was analyzed by spectral confocal microscopy and linear unmixing of tissue autofluorescence by using a 780 laser - scanning microscope ( objective , 40/1.30 oil differential inference contrast [ dic ] m27 ) . growth curve analyses in mammalian and avian cell lines demonstrated that the human h7n9 strain propagated more efficiently than the avian h7 viruses in human ( a549 ) and canine ( mdck ) cells ( fig . 2a and c ) , whereas there was a more equal growth kinetic in chicken df1 fibroblasts ( fig . this result points again toward an unusually high inherent capacity of the h7n9 virus to propagate in mammalian cells . since the two avian h7 strains provoked significantly higher levels of ifn- in the lung cultures , we next evaluated activation of the human ifn- promoter and the suppression of this response by the ns1 proteins expressed by the different strains . figure 2d illustrates that ns1-deficient human virus strongly activated a stably integrated human ifn- promoter by more than 45-fold , whereas the isogenic wild - type virus stimulated the promoter only by about 3-fold , illustrating the pivotal role of ns1 in ifn suppression ( fig . the three h7 strains induced the ifn- promoter also to a small extent , but the a / anhui/1/2013 ( h7n9 ) strain was the least active one . finally , we tested the capacity of transiently expressed ns1 proteins to block rig - i - dependent upregulation of the ifn- promoter in human cells infected with ns1-deficient virus . the analysis showed that expression of the hu - h7 ns1 gene suppressed viral activation of the ifn- promoter significantly stronger than the avian ns1 proteins , whereas ns1 of the epidemic h3 strain had intermediate activity ( fig . these results suggest that the ns1 protein of the novel h7n9 virus is a potent inhibitor of the antiviral human ifn response and may thereby contribute to its intriguingly strong replication in human alveolar tissue . efficient replication of the h7n9 virus in mammalian cell lines correlates with low activation of the human ifn- promoter . ( a to c ) multicyclic replication of the virus strains a / anhui/1/2013 ( hu - h7 ) , a / turkey - italy/472/1999 ( av - h7-it ) , a / turkey - germany / r11/2001 ( av - h7-ger ) , and a / panama/2007/1999 ( hu - h3 ) was determined in cultures of human a549 lung epithelial cells ( a ) , in chicken df1 fibroblasts ( b ) , and in canine mdck epithelial cells ( c ) . cells were infected with the indicated viruses ( multiplicity of infection [ moi ] = 0.01 ) and were incubated at 37c in the presence of trypsin for 72 h. aliquots taken at the indicated points were titrated on mdck cells by using a standard plaque assay . the graphs show mean values and sem from 3 independent experiments , each done with biological duplicates . hu - h7 replicated as efficiently on a549 and mdck cells as hu - h3 , whereas av - h7 viruses were impaired in growth by about 100-fold . ( d ) mdck cells with a stably integrated human ifn- promoter luciferase reporter were mock treated or infected with hu - h7 , hu - h3 , or two av - h7 viruses for 12 h ( moi = 1 ) . we used an isogenic ns1-deleted h3n2 ( hu - h3 ns1 ) mutant virus to determine reporter activation in the absence of a viral ifn antagonist ( 25 ) . cells were lysed , and equal amounts of protein were analyzed by luciferase assay ( 3 experiments conducted in triplicate each , mean sem ; top ) . expression of viral np and ns1 proteins and cellular actin as the loading control was verified by immunoblotting ( bottom ) . infection with hu - h3 ( ns1 ) caused a strong activation of the ifn- promoter , whereas a modest induction was observed for all other viruses . ( e ) ns1 expression plasmids were constructed by placing viral ns cdna of the indicated strains under the control of the human cytomegalovirus immediate early promoter . human 293 t cells were transfected for 24 h with an ifn- promoter reporter plasmid , flag - rig - i , and prl - tk , expressing a renilla luciferase under the control of the constitutive herpes simplex virus ( hsv ) tk promoter for normalization and an ns1 expression construct from the indicated virus strain as described ( 25 ) . empty vector was used as the control . where indicated , samples were infected for 16 h with ns1-deficient influenza virus ( moi = 1 ) . ifn- induction was determined by dual - luciferase assays ( n = 3 in triplicates , mean sem ; * , p 0.05 ; mann - whitney u test ) . hu - h7 ns reduced ifn- promoter activity significantly stronger than avian h7 strains . the isolation of avian a(h7n9 ) virus from a substantial number of pneumonia patients was unexpected and is a major cause of concern , even if the virus has not yet acquired the ability for sustained transmission among humans . a total of 16 of the 17 subtypes of the iav hemagglutinin are found in feral birds ( 17 ) . among humans , only viruses of the h1 , h2 , and h3 subtypes have circulated to larger extents , rendering most of the human population in principle vulnerable to infection with other subtypes . however , transmission of avian iav to humans occurs rarely , as those viruses usually lack adaptive changes that would enable efficient propagation and spread among humans ( 18 ) . here , we describe an experimental model for the a(h7n9 ) virus in human alveolar lung tissue infected ex vivo that complements recent analyses of this virus in animal models ( 69 ) . as the lung organ culture system revealed clear differences between bona fide avian and human - adapted strains , this approach appears useful to evaluate the zoonotic potential of iav . our initial assessment demonstrated that the human a(h7n9 ) virus is as adapted to efficient growth in human lung tissue as a prototypic epidemic virus strain , whereas this tissue was essentially nonpermissive for two avian h7 isolates . the restriction of the avian viruses is most likely not mediated on the level of virus entry , as infected alveolar cells were easily identified in tissue sections for all strains examined and there was no apparent change in the preference to infect type ii pneumocytes . this finding is consistent with the presence of receptors for both human and avian iav cells in human alveolar tissue and recapitulates that receptor specificity is not a decisive determinant of influenza virus tropism in the lower respiratory tract ( 16 ) . productive iav infection of type ii pneumocytes and resulting cytopathic effects are expected to compromise alveolar structural integrity by reducing the production of surfactant and to diminish the repair capacity of the injured lung ( 19 , 20 ) . the human anhui/1/2013 virus displays an adaptive lysine ( k ) residue at pb2 position 627 , whereas this position is occupied by a glutamic acid ( e ) in the avian h7 strains . notably , the human h7 strain induced significantly lower levels of antiviral ifn than the avian h7 viruses , which in combination with the pb2 e627k change may explain its robust growth . in fact , our analysis suggests that the ns1 protein encoded by the human h7 isolate is a potent inhibitor of rig - i - dependent activation of ifn - i genes . the ns1 protein is well known to contribute to influenza a virus pathogenicity and virulence ( 21 , 22 ) . the primary ns1 sequence of the anhui/1/2013 strain shows conserved residues at positions known to be important for ifn suppression , such as r38 and k41 , but it lacks several other motifs present in ns1 proteins of other iavs which mediate binding to host factors . this includes the c - terminal 4-amino - acid binding motif for cellular pdz domain factors ( 23 ) as well as residues facilitating interaction with cpsf-30 , which is involved in the maturation of cellular transcripts , including ifn- pre - mrna ( 24 ) . clearly , the significance of specific amino acid residues of the h7n9-anhui ns1 protein and other viral polypeptides for replication and cytokine control in human respiratory tissues will need to be examined in future reverse genetic analyses . possibly , only a small number of additional mutations or reassortment events with a human virus strain may increase the potential for enhanced transmission of h7n9-like viruses . considering the unknown extent of h7n9 circulation in domestic and feral birds and the high case fatality rate in vulnerable humans , it appears important to maintain a high level of surveillance and vigilance for these viruses in the human and animal health sectors . this study was approved by the ethics committee at the charit clinic ( projects ea2/050/08 and ea2/023/07 ) . sequence information for the ns and pb2 segments of the av - h7-it and av - h7-ger strains has been deposited at the gisaid sequence database ( http://platform.gisaid.org ; accession no . sequence information for the ns and pb2 segments of the av - h7-it and av - h7-ger strains has been deposited at the gisaid sequence database ( http://platform.gisaid.org ; accession no .

abstracta novel influenza a virus ( iav ) of the h7n9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and , apparently , from healthy poultry in march 2013 in eastern china . we evaluated replication , tropism , and cytokine induction of the a / anhui/1/2013 ( h7n9 ) virus isolated from a fatal human infection and two low - pathogenic avian h7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract . the a(h7n9 ) patient isolate replicated similarly well as a seasonal iav in explanted human lung tissue , whereas avian h7 subtype viruses propagated poorly . interestingly , the avian h7 strains provoked a strong antiviral type i interferon ( ifn - i ) response , whereas the a(h7n9 ) virus induced only low ifn levels . nevertheless , all viruses analyzed were detected predominantly in type ii pneumocytes , indicating that the a(h7n9 ) virus does not differ in its cellular tropism from other avian or human influenza viruses . tissue culture - based studies suggested that the low induction of the ifn- promoter correlated with an efficient suppression by the viral ns1 protein . these findings demonstrate that the zoonotic a(h7n9 ) virus is unusually well adapted to efficient propagation in human alveolar tissue , which most likely contributes to the severity of lower respiratory tract disease seen in many patients .

Observation Nucleotide sequence accession numbers.