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How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

An ideal delivery agent should protect siRNAs from enzymatic degradation, facilitate cellular uptake, and promote endosomal escape inside the cells with negligible toxicity. Multiple approaches for the delivery of siRNAs have been reported, ranging from the relatively simple direct administration of saline-formulated siRNAs to lipid-based and polymer-based nanoparticle approaches and siRNA conjugation and complexation approaches . The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

The siRNA, ALN-RSV01, is directed against the mRNA encoding the N-protein of RSV that exhibits specific in vitro and in vivo anti-RSV activity. It is delivered without a delivery vector as a nasal spray and targets the upper respiratory tract instead of the lower lung area. ALN-RSV01 has undergone complete phase I intranasal and inhalation studies in healthy adults and has been found to be generally well tolerated .

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

This successful evidence can be because that naked siRNAs for clinical applications are highly chemically modified to prevent nuclease-induced degradation and presumably minimize immune stimulatory effects. Although it is unclear how the naked siRNAs cross the cell membrane, gain access to the cytoplasm, and remain intact to perform their biological action, both animal and human trials have been conducted successfully, showing the efficacy of naked siRNAs ALN-RSV01 that were administered intranasally. This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

It is important to maintain a clear view of the trachea during the procedure. Intranasal delivery is another common method of pulmonary drug application in animal studies. In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 .

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs. Therefore, it is important to consider the route of administration in animal studies when assessing the delivery and therapeutic efficacy of a formulation for pulmonary delivery. Careful choice of efficient delivery in response to the condition of lung diseases is necessary.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

Many commercial siRNA transfection agents are lipid-based delivery system, some of which are also employed for pulmonary delivery-DharmFECT , Oligofectamine , Lipofectamine and TransIT-TKO . Similarly, cationic polymers have also been assessed for siRNA delivery to lung cells. Cationic polymer polyethylenimine PEI is widely used for siRNA delivery .

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.

How are siRNAs typically delivered for systemic effect?

The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation. Although drugs are commonly delivered to the lungs by inhalation, most in vivo studies using siRNAs have relied on intratracheal or intranasal delivery. The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process.

Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 . A remarkable feature of the respiratory tract is its high degree of branching. Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

The anatomical feature of the respiratory tract is its high degree of branching. The mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles. The deposited particles on the ciliated epithelial cells are rapidly cleared by the mucociliary clearance actions. Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 .

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases. Figure 2 . Extracellular barriers to pulmonary siRNA delivery. The anatomical feature of the respiratory tract is its high degree of branching.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance . Moreover, asthma and COPD are both chronic inflammatory conditions of the lung associated with structural "remodeling" that is inappropriate to the maintenance of normal lung function . The airway wall thickness, the high viscosity, and the composition of the mucus layer might be altered in patients who have inflammatory lung diseases.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient. At the active stage of lung disease, the physiological conditions of the airways might change and have significant impact on the efficiency of the pulmonary delivery system. During infection, inflammation, and allergic reaction, there is an increase in mucus secretion along with the impaired mucociliary clearance .

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

These related diseases include asthma, pulmonary fibrosis, and chronic bronchitis. They are influenced by a combination of environmental, genetic, and epigenetic components . COPD is a chronic inflammatory disease of the airways. This disease is hallmarked by airflow that is not fully reversible.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract.

What structures form the human airway?

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

This explanation has not been confirmed, but the physiological damage of respiratory epithelial cells caused by viral infection may have possibly influenced the mystery. The active change in airway epithelial cell membrance caused by infectious disease might affect cellular internalization. Naked siRNA delivery has some advantages, such as simple formation and the absence of toxicity or inflammatory responses that are usually associated with delivery vectors.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

DPIs are devices that deliver medication to the lungs in the form of dry powder. The use of DPIs has already shown promise for the in vivo delivery of therapeutic macromolecules such as insulin and low-molecular-weight heparin ; thus, it could be a better device for delivering siRNAs to the lungs. The advantages of DPIs are improved stability and sterility of biomolecules over liquid aerosols and propellant-free formation.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency. In addition, the macrophages located in the alveoli rapidly engulf the foreign particles by phagocytosis. The particles taken up into the macrophages are subsequently degraded inside the cells. These factors present major barriers to targeted pulmonary delivery.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

Intranasal entry has long been used to administer small molecules, such as proteins, for systemic delivery. Because the nasal mucosa is highly vascularized, delivery of a thin epithelium of medication across the surface area can result in rapid absorption of the medication into the blood. Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

Mucus and mucociliary clearance of mucus-trapped particles is a pulmonary defense mechanism as a physiological barrier. In the alveolar, clara cells and type II alveolar cells secrete on the surface of the alveolar epithelium, forming a thin layer of pulmonary surfactants. The surfactants act as the main barrier for siRNA delivery because they reduce the transfection efficiency.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

In the long term, we anticipate that there will be more sophisticated devices for clinical use and that those currently being developed will be more suitable. There are two main barriers to efficient pulmonary siRNA delivery to the cells of the lung. The first is the complex, branched anatomy of the lungs and biomechanical barriers, such as the mucus layer covering the airway cells Figure 2 .

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

In many studies, in vivo success has been demonstrated in delivering siRNAs to the lungs intranasally 22, 35, 36 . An experimental setup of intranasal delivery by spray or droplet is simple and painless for the animal. Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy , this approach has potential for the clinical application of siRNAs.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

Careful choice of efficient delivery in response to the condition of lung diseases is necessary. The use of aerosols to deliver medication to the lungs has a long history. Administration by inhalation is a popular and non-invasive method of delivering agents into the lungs. There are several inhalation devices available for the delivery of drugs into the lungs.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

There are several inhalation devices available for the delivery of drugs into the lungs. Metered dose inhalers MDIs and dry powder inhalers DPIs are the most common modes of inhaled delivery. MDIs are the most commonly used inhalers for several lung diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease COPD , and a spacer is an external device that is attached to an MDI to allow for better drug delivery by enhanced actuation and inhalation coordination.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

Therefore, siRNAs administered intranasally might be deposited in the nose, and some of them may be unable to reach the lower respiratory tract. In fact, it has been reported that intranasal application of unformulated siRNAs resulted in lower delivery efficiency and homogeneous pulmonary distribution than that achieved with intratracheal application . The intranasal method is suitable for some lung diseases, such as upper respiratory infection by RSV, and it also has potential for systemic delivery rather than pulmonary delivery of siRNAs.

What size of particle has been shown to be most effective in the delivery to the lower airway?

Deposition in the airway is affected by the particle size and patient's pulmonary function. A particle size between 1-5 μm is found to be the most appropriate for deposition at the lower respiratory tract . In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery.

For efficient endocytosis to occur, particles should be under 150 nm in size. Particles within this size range could also avoid macrophage uptake and delayed lung clearance . The physicochemical properties of siRNAs also play a significant role in crossing the biological membrane.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

These factors present major barriers to targeted pulmonary delivery. The second is the airway cell membrance and its intracellular barriers Figure 3 . For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density. For efficient pulmonary siRNA delivery, the particles must be deposited in the lower respiratory tract. Deposition in the airway is affected by the particle size and patient's pulmonary function.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

Thus, some studies of systemic delivery of naked siRNAs have failed to achieve the downregulation of the targeted gene . In contrast, there have also been some successes of locally delivering naked siRNAs to the lungs . A few of them reported that the use of delivery vectors showed no significant difference in gene silencing efficiency compared to that of naked siRNAs .

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells. Some viral vectors, such as retroviruses and adenoviruses, have been demonstrated to mediate gene silencing in an in vitro lung model and to induce RNAi in a range of animal tissues . Recently, Guo et al.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

In addition, the presence of mucus and surfactant proteins, the mucociliary clearance actions, and phagocytosis by macrophages present major barriers to targeted pulmonary delivery. Therefore, delivery systems usually require delivery vectors, and these vectors need to be designed in order to maximize the siRNA deposition to the diseased area of the respiratory tract. Besides, the extracellular barriers to siRNA delivery also depend on physiological features of the respiratory tract, which may change with the disease stage and characteristics of the patient.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

The negative charge and chemical degradability of siRNAs under physiologically relevant conditions make its delivery a major challenge. Accordingly, the delivery of siRNAs usually requires a vector or carriers for their transfection into the target cells. In general, both viral and non-viral vectors are being assessed for siRNA delivery to lung cells.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

To direct target-gene silencing, the siRNAs need to escape from the endosome into the cytoplasm, where they associate with the Ago2/RNA-induced silencing complex RISC to direct the cleavage of mRNAs bearing complementary binding sites. As an alternative to viral vectors, non-viral vectors, including lipid and polymer-based vectors, have been generally used for the delivery of siRNAs to the lungs due to their reduced toxicity . Ongoing research into the transfection of primary cells and whole organisms with siRNA using non-viral transfection agents has produced some promising results.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

The difficulty associated with miRNA delivery is mainly equal to that of siRNAs. The critical problems for the development of this therapy are effective delivery into target sites, potency of the therapy, and elimination of off-target effects . There are two strategies as the therapeutic applications of miRNAs for lung cancer .

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

The advancement of pulmonary siRNA delivery to the clinic illustrates that RNAi-based therapy holds a central place in the future treatment of lung diseases. On the other hand, miRNAs have the opportunity to target multiple genes in a fine-tuned manner, and the miRNA-based therapy will provide an attractive anti-tumor and anti-inflammatory approach for various lung diseases. In particular, anti-miRNA therapy by chemically modified antimiR oligonucleotides has become a potential therapy for lung diseases because the oligonucleotides can be successfully delivered without delivery vectors.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

The reason could be the difficulty in formulating inhalable siRNAs and maintaining the stability during the delivery process. A suitable carrier is also needed to protect nucleic acids from degradation due to shear force and increased temperature during the drying process. The use of spray-drying as a technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue, has been demonstrated .

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

In addition, some viral vectors may insert their genome at random positions in the host chromosome, which eventually restrict the gene function . . Intracellular barriers to pulmonary siRNA delivery. Barriers to cellular internalization are dependent on the surface properties of siRNA and carriers e.g., charge and size .

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

Some of these studies have successfully shown the efficacy of RNAi-based therapy through intrapulmonary administration of siRNAs with non-viral vectors. Although strategies to minimize off-target and nonspecific immune stimulatory effects must be devised, these data suggest that the silencing of the target gene with siRNAs is an attractive strategy for the prevention and treatment of primary and metastatic lung cancer. There are currently some clinical trials in progress estimating the safety and efficacy of siRNA-based drugs for cancer treatment.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis.

What are the essential conditions in siRNA delivery to effectively produce gene silencing in the lungs?

For efficient gene silencing in the lungs, siRNAs must be delivered to their site of action, be stable, enter the target cells, and be present in the cytoplasm at sufficient concentration. Once the siRNAs reach the target cells, they must be trafficked into the cytoplasm and taken up by Argonaute Ago 2/RNA-induced silencing complex RISC , which degrades mRNAs and, subsequently, suppresses the sequence-specific gene expression. For efficient endocytosis to occur, particles should be under 150 nm in size.

Airway consists of respiratory bronchioles, alveolar ducts, and alveolar sacs. All of these structures bear alveoli, the tiny air sacs in which the gas exchange takes place. It is generally acknowledged that the critical factor for efficient siRNA delivery depends on the properties of RNAi drug particles in terms of size, charge, shape, velocity and density.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Cotton rats are an important animal model to study infectious diseases. They have demonstrated higher susceptibility to a wider variety of human pathogens than other rodents and are also the animal model of choice for pre-clinical evaluations of some vaccine candidates. However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research . Historically in biomedical research, the mouse has been exploited as the default animal model. This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus . This model has been valuable for adenovirus-based gene replacement therapy research , and was also proven to be indispensable in pre-clinical evaluation of the prophylactic antibodies RespiGam 1 , and Synagis 1 . Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials .

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Indeed, the cotton rat model was found to be valuable in terms of its biological and immunological relevance, it was deemed unnecessary to test the adenovirus-based gene therapy and the Synagis 1 prophylactic treatment against RSV disease in non-human primate prior to the human trials . A number of methods and reagents have been developed for the analysis of immune responses in cotton rats over the last decade. Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production. weeks old cotton rats were obtained from an inbred colony maintained at Envigo USA . All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression. The availability of CD40L gene identity could greatly facilitate mechanistic research on pathogen-induced-immunopathogenesis and vaccine-elicited immune responses. Text: The cotton rat Sigmodon hispidus was first used in polio research in the 1930s , and throughout the last century, it has proven to be an excellent model for biomedical research .

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Up to date, more than 200 genes encoding cytokines, chemokines, cell surface markers and regulatory molecules have been cloned, with various related research reagents being commercially available. As a result, the use of cotton rats in pathogenesis studies addressing mechanistic questions has significantly increased. Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

All animal experiments were conducted in accordance with Institutional Care and Use Committee IACUC of Health Canada Ottawa Animal Care Committee which approved this study. The rats were housed 3 animals per cage in Allentown NexGen individually ventilated cages with free access to food and water. These cages provided a floor space of 142 in 2 / 916 cm 2 .

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

To isolate splenocytes from the animals, isoflourane was used to put the animals to sleep via inhalation with oxygen for euthanasia. The spleens from three naïve cotton rats were removed aseptically and snap frozen in liquid nitrogen. The spleens were homogenized individually with a TissueLyser II Qiagen and total RNA extracted using the RNeasy Mini kit Qiagen with on-column DNase digestion according to the user's manual.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein. The recombinant cotton rat CD40L was recognized by an Ab against mouse CD40L in direct ELISA, and showed biological activity by upregulating maturation markers CD40, CD54, CD80, and CD86 as well as I-A d on immature bone marrow murine DCs and moreover, inducing upregulation of IL-6 gene and cytokine expression in these cells. The isolation of the cotton rat CD40L sequence and availability of CD40L has the potential to positively impact basic immunological research and vaccine development, given the critical importance of this protein in orchestrating immune responses .

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

The avidity of the mouse CD40L antibody to the cotton rat CD40L protein was evaluated in the presence of 6M urea. The difference between the untreated and 6M urea treated for each group was calculated using students t-test ÃÃÃ p<0.001, ÃÃÃÃ p<0.0001 n = 2 . Data shown is a representative experiment of three separate experiments where two n = 2 technical replicates are conducted in each experiment.

Why are cotton rats considered a strong animal model for biomedical research?

This is in part due to its well defined immunological and genetic information, costeffectiveness, and abundant inbred strains and research reagents. However, the use of mice as models to study infectious diseases has its limitation since mice are not naturally infected by most human pathogens. On the other hand, cotton rat is susceptible to many human pathogens and is the ideal model of choice for measles paramyxovirus , herpes simplex oral a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 and ophthalmic , influenza orthomyxovirus , HIV-1 , RSV respiratory syncytial virus , adenovirus , human parainfluenza , and human metapneumovirus .

Protein was detected with Tetra-HIS Ab Qiagen and goat anti-mouse IRDye-800CW LiCor . Membranes were developed using the Odyssey system LiCor . The vaccinia virus carrying the crCD40L gene was propagated in BHK21 cells.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites . TNF superfamily members include TNF TNF-alpha , LT lymphotoxin-alpha, TNF-beta , CD40 ligand, Apo2L TRAIL , Fas ligand, and osteoprotegerin OPG ligand, among others . The TNF superfamily is composed of 19 ligands and 29 receptors, in which each has vastly diversified roles in the body and exhibit pro-inflammatory activity, partly via activation of NF-kB .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

We next examined the functional domains in crCD40L in comparison with other known CD40L. As shown in Fig 3a, crCD40L has a putative tumor necrosis factor TNF superfamily Using EZmol software , we predicted folding of the protein as shown in Fig 3b. The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

The cotton rat CD40L cDNA that we have isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783bp which coded for a 260 aa protein. The homology of cotton rat CD40L, at both the amino acid and nucleic acid level, is closer to members of the Cricetidae family hamster and deer mouse than to those of the Muridae family rat and mouse as shown in Fig 2b. As with other known CD40L proteins, there is a putative TNF superfamily domain, a transmembrane domain, trimerization sites, and receptor binding sites .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein. The recombinant cotton rat CD40L protein was recognized by an antibody against mouse CD40L. Moreover, it demonstrated functional activities on immature bone marrow dendritic cells by upregulating surface maturation markers CD40, CD54, CD80, and CD86 , and increasing IL-6 gene and protein expression.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

To investigate IL-6 secretion by murine bone marrow DCs, supernatant from forty hour stimulated cultures were collected and assayed using the Mouse IL-6 DuoSet ELISA Kit R & D Systems following the manufacturer's protocol. The complete mRNA sequence of CD40L was obtained in two steps Fig 1 . A sequence corresponding to nucleotides 535 through to the poly-A tail was obtained using the 3' RACE kit and mRNA as starting material, which was isolated from cotton rat splenocytes and a rodent consensus sequence as a primer.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

The protein was eluted off the column with buffer B + 250 mM imidazole pH 7.8 . The resulting protein was dialysed against PBS pH 7.5 and then confirmed by western blot. 96-well plates were coated with either recombinant mouse CD40L R&D Systems or the recombinant crCD40L protein 2ug/ml in 100μl PBS.

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

What is the structure of the CD40 Ligand?

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival . We tested if the recombinant crCD40L could induce IL-6 gene expression Fig 6a and production of the cytokine Fig 6b by immature murine bone marrow DCs. The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells . It is a member of the TNF superfamily consisting of a sandwiched extracellular structure composed of a β-sheet, α-helix loop, and a β-sheet, allowing for the trimerization of CD40L, an additional feature of the TNF family of ligands . Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers. CD40-induced activation of cytokine gene expression in DCs by CD40L is an important process in the initiation of primary immune responses and is critical for DC maturation and the generation of antigen-specific T cell responses . IL-6 is a highly pleiotropic cytokine in that it stimulates the activation, proliferation, and survival of T cells, and furthermore, modifies DC function and survival .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

Nevertheless, the gene encoding CD154 and CD40 ligand CD40L , remains elusive. CD40L plays a critical role in orchestrating immune responses against pathogens. Depending on the post-translational modification, the murine CD40L is a 32-39 kDa type II membrane glycoprotein that was initially identified as a surface marker exclusive to activated CD4 + T cells .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

We speculate this to be due to the species incompatibility since we are stimulating mouse bone marrow cells with cotton rat CD40L. Nevertheless, the crCD40L was able to promote up-regulation of key co-stimulatory markers on immature DCs promoting DC maturation. The gating strategy used for the flow cytometry analysis is provided in S3 Fig along with overlapping histograms of the intracellular adhesion marker and co-stimulatory markers.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L . Moreover, mouse I-A d major histocompatibility complex is also up-regulated upon stimulation with CD40L . When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 .

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

addition of urea treatment would substantially weaken the interaction between the antibody and crCD40L. Since the cotton rat CD40L protein sequence shared 82% identity with the mouse CD40L protein sequence with similar functional domains, we evaluated the biological activity of the recombinant crCD40L on immature murine bone marrow DCs. We conducted experiments based on known functional activities of CD40L in other animal species.

What is the effect of CD40L on Dendritic Cells?

When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells . In addition, CD11c is a DC integrin marker and upon stimulation, is down-regulated . Intracellular adhesion marker CD54, along with co-stimulatory markers CD40, CD80, and CD86 are all upregulated upon stimulation with CD40L .

However, the genome of cotton rats remains to be fully sequenced, with much fewer genes cloned and characterised compared to other rodent species. Here we report the cloning and characterization of CD40 ligand, whose human and murine counterparts are known to be expressed on a range of cell types including activated T cells and B cells, dendritic cells, granulocytes, macrophages and platelets and exerts a broad array of immune responses. The cDNA for cotton rat CD40L we isolated is comprised of 1104 nucleotides with an open reading frame ORF of 783bp coding for a 260 amino acid protein.

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

Since its initial discovery, CD40L has been shown to be not only expressed on CD4+ T cells, but on dendritic cells DCs , B cells , and platelets . It has been shown that upon interacting with its receptor, CD40, CD40L induces profound effects on T cells, DCs, B cells, endothelial cells, as well as many cells of the hematopoietic and non-hematopoietic systems. Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells.

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes . Here we report the successful cloning of the gene encoding cotton rat CD40L crCD40L ; we also expressed and purified the CD40L produced in mammalian cells. Further characterisation of the recombinant cotton rat CD40L revealed its functional activities in promoting DC maturation and cytokine production.

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

The results indicate that a significant increase in both IL-6 gene expression and cytokine production in immature murine bone marrow DCs was observed forty hours after stimulation with the crCD40L. Collectively, the observation that both the upregulation of immature DC cell surface maturation markers and increased IL-6 gene expression and cytokine production provide strong evidence of the biological activity of crCD40L. In summary, the cotton rat CD40L cDNA that we isolated was a 1104 nucleotide sequence with a poly-A tail containing an ORF of 783 bp which coded for a 260 aa protein.

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

The BHK21 cells expressing the crCD40L construct were collected and lysed with 6 M guanidine hydrochloride with reduced glutathione and sonication. The lysate was loaded on the nickel column and the washed with denaturing buffer as described in materials and methods. The bound proteins were refolded on the column with gradient buffer exchange, to allow slow refold the protein, given that CD40L biological activity is dependent on a homo-trimer configuration .

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

We conducted experiments based on known functional activities of CD40L in other animal species. Specifically, maturation of immature DCs after exposure to antigen is known to play a crucial role in their immunity-stimulating function , while trimeric recombinant CD40L has been shown to stimulate DC immunomodulating functions . When CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells .

What is the effect of CD40L on B Cells?

Moreover, when CD40L engages CD40 on the surface of DCs, it promotes cytokine production, the induction of cell surface co-stimulatory molecules, and facilitates the cross-presentation of antigen by these cells , enabling DCs to mature and effectively induce the activation and differentiation of T cells. When CD40L engages CD40 on the surface of B cells, it promotes germinal center formation, immunoglobulin Ig isotype switching, somatic hypermutation to enhance antigen affinity, and lastly, the formation of long-lived plasma cells and memory B cells .Various studies have been conducted to utilize gene delivery of CD40L to DCs and tumor cells for tumor immunotherapy. It was found that expression of CD40L in a small proportion of tumor cells was sufficient to generate a long-lasting systemic anti-tumor immune response in mice that was shown to be dependent on cytotoxic T lymphocytes .

When our recombinant crCD40L was used to stimulate immature murine bone marrow DCs, we observed similar results to that when murine CD40L is used Tables 1 and 2 . CD11c was down regulated in both median flouresence intensity Table 1 and the percentage of positive cells Table 2 . The co-stimulatory molecules CD54, CD40, CD80, and CD86 were all up-regulated in both median fluorescence intensity Table 1 and the percentage of positive cells Table 2 .