Hepatitis B is a potentially deadly liver disease transmitted by the virus of hepatitis B (HBV). Globally, 2 billion people have contracted hepatitis B, according to WHO estimates, and approximately 1.5 million people get hepatitis B each year; the combined figure equals 800,000 deaths annually, largely from liver cirrhosis and hepatocellular carcinoma that occurs later in hepatitis B. The WHO estimates that some 4.5 million premature deaths could be prevented with the vaccine against hepatitis B by 2030. Preventive hepatitis B vaccines do much to avert HBV infection. Therapy vaccines are the best immunotherapies for hepatitis B. The effective hepatitis B vaccines have been created so far in recombinant protein vaccines, DNA vaccines, lipopeptide vaccines and adenovirus-vector vaccines. But no therapeutic hepatitis B vaccine exists for clinical use because the vast majority of recombinant protein and lipopeptide vaccines in use today target only HBsAg as a single target, and cannot induce multi-antigen immunity, so activating hyporeactive specific T cells left in the body. DNA vaccines have a short action time in the body and require the help of effective delivery vehicles to function. It is difficult for a single DNA vaccine to inhibit HBV DNA replication and induce a specific immune response. Although adenovirus-vector vaccines can produce potent levels of CD8⁺ T cells, it’s unclear how bad this vaccine is for the human body. The vaccine may become less immune-effective if the body produces specific antibodies to adenovirus upon vaccination. Therefore, the search for alternative technical pathways for therapeutic hepatitis B vaccines remains urgent.

While current evidence-based applications of preventive hepatitis B vaccine in HBsAg-cleared populations has demonstrated its safety and efficacy, larger, multi-center trials are far from being completed. When it comes to HBV, scientists have developed companion in vivo and in vitro models of HBV that meet the needs of their respective research objectives. Although flawed in many ways, these models have helped individuals to gain a better understanding of HBV and assisted in the creation and testing of novel medications to prevent and treat HBV.

HBV Animal Models

Non-human primate model

Non-human primates, including chimpanzees, gibbons and baboons, are susceptible to HBV infection. These vulnerable animals can be used to investigate the viability, natural infection and pathogenesis of HBV and the body’s immune clearance of the virus. However, there are limiting factors such as mild degree of virus infection, animal ethics and species endangerment. In order to establish animal models similar to HBV natural infection in humans, researchers have never stopped exploratory research on the macaques. These exploratory animal models play an important role in studying the mechanism of HBV infection and the host’s immune response during infection, but the scale is extremely limited and there is a lack of more confirmatory experiments on HBV infection.

Tree shrew model

Tree shrews are an animal model discovered after the above-mentioned primates that can be easily infected by HBV. After infection with HBV, they will experience temporary viremia, virus clearance process, and HBsAg seroconversion. The infection process is similar to that of humans, with a short infection cycle (about 40 days). In this type of model, tree shrews are mostly captured in the wild. The infection situation is related to the animal’s age, breed, HBV genotype, etc., and there is currently a lack of specific detection of hepatitis B antibodies for it, so the reliability, controllability, repeatability of this model and the test results are still controversial.

Mice model

Currently, the most commonly used hepatitis B animal model is mice, but HBV cannot directly infect mice. It’s easy to classify mature mouse models of HBV infection into two main types: the chimeric mouse model, and the most common genetically deficient mouse model. These mouse models are susceptible to HBV infection and are mostly used to study the immune response, virus replication, virus pathogenesis, etc. HBV infection) and to screen and test anti-HBV medications. However, they have shortcomings such as low reproduction rate and high mortality rate, slow process of HBV infection and certain immune tolerance to the virus. The other type is a transgenic mouse model, which includes two types: HBV expressing and NTCP (Na⁺-Taurocholate cotransporting polypeptide) deficiency transgenic mouse, with the former being the most used. This type of model plays an important role in studying HBV replication and evaluating anti-HBV drugs and vaccines. However, there are defects that HBV is not naturally infected and viral RNA comes from the integrated HBV genome. Therefore, it cannot be used to study early viral infection and viral infection. Mechanism, and is not suitable for studying mechanisms that affect cccDNA regulation and transcription activity and evaluating the pharmacodynamic effects of drugs on cccDNA production.

Woodchuck hepatitis virus (WHV) infection model

HBV is part of the Hepadnavirus family, and is subdivided into the orthohepadnaviruses and avihepadnaviruses. Beyond human HBV (receptive to human and non-human primates), the most identifiable, and widely distributed, member of the orthohevivirus genus is woodchuck hepatitis B virus (WHV). The adult woodchuck model established using WHV is mainly used to study the process of clearing infected cells by the body during acute virus infection, the mechanism of chronic infection and cancer, and to evaluate anti-HBV drugs and vaccines. However, due to differences in genetic background, the conclusions drawn from this model cannot fully reflect the status of HBV in the human body. In addition, insufficient purification of the WHV strain, incomplete antibody sets to study immune responses, and high animal prices remain bottlenecks that restrict the widespread use of this model.

Duck hepatitis B virus (DHBV) infection model

Duck hepatitis B virus (DHBV) is more commonly used in the avian hepatitis virus genus. At present, DHBV is mainly used to infect 1-3-day-old ducks for modeling research. This model can be used to evaluate new anti-HBV drugs and study cell surface virus receptors and virus clearance mechanisms. Compared with other types of models, the DHBV animal model has the following advantages: (1) The model is easy to obtain: compared with mammals, ducks have a wider source, simpler breeding conditions, shorter breeding cycles, and DHBV will not infect humans and have safer characteristics; (2) High infection success rate: Although there are differences in the natural infection rates among different duck species, different duck species have no obvious effect on the artificial infection rate. Intravenous injection has a higher infection rate than intraperitoneal injection; (3) The virus replication mechanism is similar: the replication mechanism of DHBV is highly consistent with HBV. Unlike transgenic animal models, the virus in DHBV-infected ducks can self-replicate and can spread vertically; (4) Liver pathological damage can occur: DHBV animal models can continue to express DHBV DNA highly within a certain period of time, and can cause pathological damage to the liver, which is the main difference from HBV full-genome transgenic mice. The flaw with this model is that ducks and humans differ in species, HBV and DHBV differ in structure, and there are many variables which impact the stability of the model, such as duck species, age, source of virus, batch, route of infection etc.

Testing Vaccine Effectiveness in Animal Models

Evaluation of HBV vaccine efficacy in animal models is a complicated process, including animal model selection, immunization routes and doses, and immunogenicity and protection of the vaccine.

Choice of the animal model: First, an animal model should be chosen considering the vaccine target pathogen and the anticipated immune response.

Work out the immunization route and dose: Experiment on the best immunization route and dose. For instance, antibody titters to HBV are analyzed using ELISA to determine the most effective route and dose of immunization.

Immunogeneity measurement: Immunogeneity measurement is done by counting the number of antibodies made in the animal. For instance, a vaccine is judged to be immunogenic by measuring serum antibody concentrations.

Test the vaccine’s safety: The safety of the vaccine is measured using challenge tests. For example, the protective effect of the vaccine is determined by comparing the differences in clinical symptoms, pathological changes, relative daily weight gain, pathogen nucleic acid load, etc. between the vaccinated group and the control group after challenge.

The application of animal models is also reflected in the optimization of vaccine research and development strategies. Through experiments on different animal models, researchers can explore different vaccine formulations, vaccination routes and adjuvants to improve the effectiveness and safety of vaccines. The ideal HBV animal model should be an economical, practical and easy-to-standardize animal model that has close kinship with humans, has a high infection rate for human HBV, has a long maintenance time, and can cause corresponding pathological changes after infection. It is best to have a normal immune system at the same time to facilitate evaluation of immune responses. Although great progress has been made in the research and application of HBV experimental animal models and made outstanding contributions to HBV-related research, so far there is no HBV animal model that meets all the above conditions at the same time. In addition, because the source of human liver tissue samples is very limited and it is difficult to meet relevant experimental research, HBV animal models are still the only effective means to explore and understand the pathogenesis and treatment methods of chronic hepatitis B. At present, researchers can select relatively suitable animal models of HBV infection or replication among the above models for experimental research based on the specific research content.

Our hepatitis virus animal models and related services:

Animal Models

HBV Full-Genome Pseudovirus-Infected Mouse Model

Duck Hepatitis B Virus (DHBV) Infection Liver Necrosis Model

Duck Hepatitis B Virus (DHBV) Infection Model

Woodchuck Hepatitis Virus (WHV) Infection Model

Services

Cell-based ELISA

CC50/IC50 Assay

Cytopathic Effect Inhibition Assay

ELISA Based Assays

Virus Yield Reduction Assay

Microneutralization Assay

Plaque Reduction Assay

Viral Replicon Assay

TCID50 Assay

References

1. Jose-Abrego, Alexis, et al. Host and HBV interactions and their potential impact on clinical outcomes. Pathogens. 12.9 (2023): 1146.
2. Menne, Stephan, and Paul J. Cote. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World journal of gastroenterology: WJG. 13.1 (2007): 104.
3. Hu, Jianming, et al. Cell and animal models for studying hepatitis B virus infection and drug development. Gastroenterology. 156.2 (2019): 338-354.
4. Guo, Wei-Na, et al. Animal models for the study of hepatitis B virus infection. Zoological research. 39.1 (2018): 25.
5. Liu, Yongzhen, Stephanie Maya, and Alexander Ploss. Animal models of hepatitis B virus infection–success, challenges, and future directions. Viruses. 13.5 (2021): 777.

The post Bridging the Gap: Evaluating HBV Vaccine Efficacy in Animal Models first appeared on Creative Diagnostics.

What is cAMP?

It was identified in 1957 and is an information molecule that is made after the first messenger attaches itself to the target cell. It’s involved in all manner of biological processes: hormone release, glycogen-processing, smooth-muscle relaxation, heart beat, learning, memory, etc. The standard cAMP signalling system is extracellular agonists bind to G protein-coupled receptors (GPCRs) and alter their conformation. Gs protein activates ACs and activates AC to oxidise adenine triphosphate (ATP) to produce cAMP. cAMP’s intracellular rise will also activate protein kinase A (PKA) and exchange protein (Epac) directly induced by cAMP, signaling to direct downstream reactions. GPCRs, ACs and PDE actively manage cAMP signaling. Intracellular cAMP level is modulated by PDE, which turns cAMP into 5′-adenine nucleotides (5’-AMP) and stops its signal transduction. Further, AKAP keeps cellular cAMP compartmentalised through the creation of a complex, which regulates certain cellular effects. Already in preliminary work, cAMP once raised also activated PKA and Epac, which in turn induced effectors on various channels, resulting in relaxation of airway smooth muscle, inhibition of airway smooth muscle growth, and modulation of cytokine production.

Figure 1. Overview of compartmentalization of cAMP signaling.

Proteases that Regulate cAMP

PDEs

PDEs can hydrolyse cAMP and cyclic guanosine monophosphate. We now recognise 11 isoenzyme-containing PDE subtypes (PDE1-PDE11) in mammals. PDE4 is the main cAMP hydrolyser among them. There are four subtypes of PDE4: PDE4A, PDE4B, PDE4C and PDE4D. It’s abundantly present in tissues and plays roles in physiology: the modulation of inflammation, microvascular permeability and fibrosis, contraction and expansion of smooth muscles of the bloodstream and airways. The negative correlation between PDE4 and cAMP is the primary driver of several pathological and physiological conditions including airway inflammation, COPD, asthma, psoriasis and rheumatoid arthritis. PDE4 is mostly found in lung structural cells (smooth muscle cells, airway epithelial cells, inflammatory cells) that maintain and relax airway smooth muscles and engage in a broad range of cell-life processes.

ACs

In 1989, the first mammalian AC was discovered in the bovine brain and purified and cloned. Subsequently, 9 AC subtypes were discovered. Currently, it has been determined that there are 10 subtypes of mammalian AC, 9 transmembrane AC subtypes (mAC) and one soluble AC (sAC). As a soluble enzyme, sAC is different from other AC subtypes in mammals. sAC is not regulated by G protein. The stimulation of calcium and bicarbonate is the main factor affecting it. It is mainly related to sperm motility and the growth of neuronal processes. mAb consists of a nitrogen terminus (N-terminus), two transmembrane domains, which are connected by C1a and C1b regions, and connected to the carbon terminus (C-terminus) by C2a and C2b in the cell. The N-terminus and C-terminus extend into the cytoplasm. C1 and C2 are two highly homologous cytoplasmic regions, which are the catalytic domains of AC and affect the activity of AC. The sequence and length of the N-terminus are significantly different in different ACs. Most tissues and many cell types express more than one AC subtype, and the expression of each subtype is inconsistent between different species. AC1 is highly expressed in the brain, mainly affecting neuronal function, and is a suitable neuron-specific drug target for chronic pain. AC8 is mainly expressed in the brain and is believed to play a role in learning and memory; AC3 is most abundant in the olfactory epithelium and plays a role in olfaction; AC5 and AC6 are dominant in the heart and are related to cardiac contraction; AC4, AC7, and AC9 are commonly found in mammalian tissues; AC2 is more widely distributed and is expressed in the brain, lungs, skeletal muscle, and heart, and the subtypes highly expressed in the lungs are AC2, AC6, AC8, and AC9.

cAMP and COPD

Complex reaction signalling in cells is extremely controlled by cAMP. When it detects and amplifies signals, it regulates related physiological effects through cAMP-dependent enzymes or proteins. After the cells are ‘inflamed’ by the outside world, it affects the functions of AC and PDE enzymes, modulates intracellular cAMP, which affects downstream PKA, EPAC and AKAP, then nuclear factor B (NFB) and cAMP response element binding protein. (CREB), and extracellular signal-regulated kinase (ERK) activation, which leads to airway smooth muscle relaxation and diminished inflammation and airway fibrosis. Consequently, controlling the cAMP pathway is a great candidate for both prevention and cure of long-term respiratory conditions like COPD.

cAMP and Airway Smooth Muscle Relaxation

In the respiratory system of COPD patients, we see the obvious signs of airflow inhibition: wall remodeling and mucus formation. Among them, distorted growth and hypertrophy of airway smooth muscle cells (ASMCs) is one of the most important features of COPD airway remodelling. One of the most important medications for treating airflow obstruction in COPD is 2-adrenergic receptor agonists. They act as bronchodilators because intracellular cAMP levels spike. The two main effectors of cAMP, PKA and EPAc, are then mobilised and participate in smooth muscle relaxation of the airway. EPAc reverses the phenotypic conversion of airway smooth muscle with ERK, regulates contraction and relaxation of ASMCs, and skewed Ras homologous gene family member A and Ras-associated C3 botulinum toxin substrate (Rac) to Rac, to suppress myosin. light chain Phosphorylation ultimately relaxes airway smooth muscle. Another research has shown that a boost in the levels of cAMP in the smooth muscle of the airway and the bronchial epithelium can both decrease intraepithelial acetylcholine and ameliorate bronchial symptoms. Recently, it has been discovered that mutating or demethylating ATP-binding box subfamily C member 1 decreases the release of cAMP from ASMCs that is stimulated by 2 receptors, increasing intracellular cAMP, easing tension on airway smooth muscle cells and inhibiting COPD airway smooth muscle remodeling in several different ways.

cAMP and Inflammation

COPD’s root cause is chronic inflammation, whose airway inflammation is defined by increased numbers of neutrophils, alveolar macrophages and lymphocytes. The cAMP anti-inflammatory effect is varied, it regulates several biological processes, and its regulatory effect depends on the type of cell. In the COPD inflammatory process, neutrophils generate inflammatory mediators and proteases. We have found that COPD patients’ neutrophils migrate irregularly, and that they migrate to the chemoattractant leukotriene B4 (LTB4) and the chemokine CXC ligand 1 (CXCL1) with increased velocity. We also discovered that increasing the intracellular cAMP level can prevent neutrophils from chemotaxis to CXCL1 and LTB4. Bronchial macrophages, interstitial macrophages, and alveolar macrophages are the most prominent ones in the lungs; alveolar macrophages are the major players here. On alveolar macrophages, researchers have observed that macrophages spit out cytokines and chemokines after they have been activated and stimulated by CSE or other stimulants, and that cAMP induces the release of pro-inflammatory factors like tumor necrosis factor- (TNF-) and the chemokine C-C ligand 3 (CCL3), but not the anti-inflammatory cytokines like IL-10.

The other cAMP signalling mechanism in the immune system is to turn on the downstream nuclear transcription factor CREB, bind PKA to the NF-B inhibitory protein (IB) molecules to create a complex, and the signal that breaks down IB turns on the catalytic subunit of the PKA, which phosphorylates the p65 molecule. Phosphorylated CREB could block the NF-B association with the gene in question and can block gene transcription via a binding to activated NF-B to prevent inflammation.

cAMP and Epithelial-Mesenchymal Transition

And from earlier research, evidence for EMT involvement in the COPD pathogenesis is mounting. – Smoke and pollution bombard the lungs with excess oxidants (especially reactive oxygen species) which increases oxidative stress and EMT leading to a radical degeneration of COPD. Moreover, mesenchymal transition of airway, lung epithelial cells, and bronchial epithelial cells in COPD is considerably elevated. They’ve discovered that cAMP sits in particular subcellular microdomains and regulates the activity of downstream effector proteins PKA and Epac; AKAP attaches to PKA in a small -helical loop to drive EMT. It’s now even known that CSE-induced release of transforming growth factor- in Be-as-2b cells is a trigger for EMT, for the activation of type I collagen (COLI) and for the inhibition of the epithelial cell marker E-cadherin. But increasing cAMP or disrupting the direct interaction between AKAP and PKA will significantly decrease COLI upregulation, impact epithelial-mesenchymal transition in COPD, and therefore reduce COPD symptoms.

Treatment of COPD with Drugs that Balance Camp Pathway

In practice, COPD prevention and treatment medications consist mostly of inhaled glucocorticoids, long-acting 2-adrenergic receptor agonists, and long-acting M receptor antagonists, all of which are very bad. There are still few drugs available to prevent and treat COPD, and no treatment that prevents the disease from spreading. The use of drugs that regulate cAMP pathway in COPD is currently limited to PDE4 inhibitors and AC agonists.

The post Research on Regulating cAMP Pathway to Chronic Obstructive Pulmonary Disease first appeared on Creative Diagnostics.

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterized by progressive airflow limitation. The main symptoms are cough, expectoration, and progressive worsening dyspnea. As of 2017, COPD has become the third-largest cause of death in the world and the eighth-largest cause of life loss globally in 2019. The above data suggest that COPD has attracted widespread attention as a global public health issue. Wednesday in the third week of November every year is World COPD Day. It was initiated and established by the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) in 2002. The purpose is to increase public understanding and attention to COPD as a global health issue.

Effect of COPD on the Lung

COPD is a common chronic respiratory disease. It is accompanied by an increase in the chronic inflammatory response of the airways and lungs to harmful particles or gases, resulting in obstruction of respiratory flow, difficulty breathing, and often accompanied by coughing, expectoration and other discomfort. Most of the things people often call “chronic bronchitis” and “emphysema” belong to chronic obstructive pulmonary disease. The lungs of the human body are like an inverted tree, the trachea and bronchi at all levels are like tree trunks and branches, and the alveoli are like tree crowns. The airflow flows in the trachea, bronchi at all levels, and alveoli. The farther the branch becomes, the narrower the channel of the airflow becomes. Chronic obstructive pulmonary disease can cause bronchial obstruction and alveolar destruction, which together lead to airflow restriction. As a result, patients will experience difficulty breathing, which will gradually worsen, which is also a landmark symptom of COPD.

What Factors Can Easily Lead to COPD?

Figure 1. Risk factors and symptoms of COPD.

The incidence of COPD may be the result of long-term interaction between multiple environmental factors and the body’s own factors.

(1) Smoking: Smoking is the most important causative factor for COPD. The incidence of COPD in smokers is 2-8 times higher than that in non-smokers. And the longer you smoke and the greater the amount you smoke, the higher the risk of developing COPD. Passive smoking (i.e. second-hand smoke) can also cause varying degrees of COPD depending on the smoke concentration in the environment and contact time.

(2) Inhalation of occupational dust and chemical substances: In the absence of protection, long-term contact or repeated inhalation of dust and chemical substances with excessively high concentrations, such as organic and inorganic dust, industrial waste gas, allergens, smoke, etc., can lead to the occurrence of chronic obstructive pulmonary disease.

(3) Air pollution: People who have lived in areas with severe air pollution for a long time inhale large amounts of harmful gases such as sulfur dioxide and chlorine to continuously stimulate the respiratory tract, leading to chronic airway inflammation, which is also an important cause of chronic obstructive pulmonary disease.

(4) Long-term exposure to biofuels: In some areas, a large amount of smoke generated when people use firewood, wood, charcoal, crop poles and animal manure for cooking or heating for a long time leads to indoor air pollution and may also lead to chronic obstructive pulmonary disease.

(5) Infection: Respiratory infection is another important factor in the development of COPD. Infections such as viruses, bacteria, and mycoplasma can cause airway damage and airway inflammation. Infection is also a common cause of acute exacerbation of COPD. A history of severe respiratory infections in childhood is associated with reduced lung function and respiratory symptoms in adulthood. Creative Diagnostics’ pathogen infection animal model platform provides high-quality pharmacodynamic evaluation services for the treatment of bacterial respiratory infections.

(6) Other factors: It is now believed that the relatively clear individual susceptibility factor is the lack of the α1-antitrypsin gene. In addition, airway hyperresponsiveness is also considered to be an important risk factor for the development of COPD. The incidence of COPD is related to the patient’s socioeconomic status.

Researchers speculate that the incidence of COPD is related to genetic factors, and it is currently believed that the incidence of COPD is related to the interaction of multiple genetic mutations and environmental factors. Despite extensive research, little is known about the potential mechanisms of COPD at the molecular level, so there are currently no effective treatment drugs. Animal models are an important tool for studying the pathogenesis, prevention, and treatment options of human diseases and identifying potential treatment targets and biomarkers. They provide a valuable, ethically and economically feasible experimental platform for research and play an important role in the development of medicine.

Animal Models of Chronic Obstructive Pulmonary Disease

The lung anatomy of COPD animal models should be similar to that of humans in order to better simulate the pathophysiological characteristics of human COPD. Currently, there are many types of animals for establishing COPD models, such as rats, mice, dogs, sheep, rabbits, monkeys, miniature pigs, etc. Among them, small rodents are the most commonly used COPD and AECOPD models. Rats and mice have short life cycles, many strains, are easy to raise, grow quickly, have a gentle temperament, and have low breeding and reproduction costs; their physiological and pathological characteristics are many similar to those of humans. Its advantages include clear genetic background, good operability, low breeding and maintenance costs, saving manpower and material resources, etc. Most researchers choose SPF Wistar rats, SD rats, C57BL/6 mice, BALB/C mice, Kunming mice, etc. as animal models to simulate COPD and AECOPD.

Creative Diagnostics focuses on using respiratory pathogens to infect model animals to accelerate research to clarify the mechanisms and treatments involved. These services include animal models of the bacterial respiratory tract (including the determination of MCLD, probability of survival, lung bacterial load, and histopathological analysis), assessment of vaccine immunogenicity, challenge protection testing, in vitro antibacterial testing, serum sterilization testing and plaque testing.

Respiratory bacterial infection models

Bacterial infection is considered to be the main cause of acute exacerbation of COPD. Pseudomonas aeruginosa infection is the most common among patients with COPD, so it is often used to build COPD models. Bacterial colonization induces chronic inflammation that drives progressive airway obstruction. In addition, a weakened immune response results in permanent colonization or failure to clear the pathogen.

Respiratory virus infection models

Studies have shown that patients with chronic obstructive pulmonary disease have increased susceptibility to influenza A virus (IAV) and experience excessive immune responses after infection. In acute and chronic cigarette smoke exposure models, increased lung and systemic inflammation, reduced virus clearance, and reduced response to bronchodilators were observed after IAV infection. The excessive immune response observed after cigarette smoke exposure was associated with increased expression of Toll-like receptor 3 in the lungs of mice.

COPD can cause changes in patients’ lung function and form lung pathological changes, which seriously affects patients’ quality of life. Exploring its pathogenesis and related molecular markers is of great significance for clinical diagnosis and treatment. Establishing an animal model of human diseases, and through comparative research with human diseases, we can deeply explore the pathogenesis of the disease, find therapeutic targets and pathways, and develop therapeutic drugs, which is of great significance to disease research. However, the physiological and pathological differences between animals and humans, the unclear mechanism of induction methods, and the lack of unified standards for animal model evaluation have all had a certain impact on the study of animal models of COPD. Creative Diagnostics provides customers with good COPD animal models: they have similar anatomical, physiological, and pathological characteristics to humans, and the modeling methods have certain specifications to facilitate repeated reproduction, and researchers can select corresponding model animals and modeling methods according to different experimental purposes. At the same time, we will continue to improve animal models of chronic obstructive pulmonary disease to ultimately further understand, define and treat COPD.

References

1.Czarnecka-Chrebelska, Karolina H., et al. Biological and genetic mechanisms of COPD, its diagnosis, treatment, and relationship with lung cancer. Biomedicines. 11.2 (2023): 448.

2.Berenson, Charles S., et al. Impaired alveolar macrophage response to Haemophilus antigens in chronic obstructive lung disease. American journal of respiratory and critical care medicine. 174.1 (2006): 31-40.

3.Chen-Yu Hsu, Alan, et al. Targeting PI3K-p110α suppresses influenza virus infection in chronic obstructive pulmonary disease. American journal of respiratory and critical care medicine. 191.9 (2015): 1012-1023.

4.Kimura, Genki, et al. Toll-like receptor 3 stimulation causes corticosteroid-refractory airway neutrophilia and hyperresponsiveness in mice. Chest. 144.1 (2013): 99-105.

The post How Does COPD Affect the Lungs?-Animal Models Reveal the Truth first appeared on Creative Diagnostics.

Figure 1. Treatment of febrile autoimmune hemolytic anemia.

Primary AIHA

Genetic Level

Copper/zinc superoxide dismutase genes (SOD1 genes): Superoxide dismutase (SOD) is an active substance derived from living organisms that can eliminate harmful substances produced by organisms during metabolism. Continuous supplementation of SOD in the human body has a special anti-aging effect. Studies have shown that SOD is associated with a variety of autoimmune diseases, such as autoimmune cerebrospinal meningitis and rheumatoid arthritis. SOD1 is a subtype of SOD dimer containing Cu and Zn atoms. To date, most studies on the SOD1 gene have focused on changes in the relevance of amyotrophic lateral sclerosis. However, recent studies have found that oxidative stress deficiency of SOD1 can also lead to anemia and autoimmune reactions. NZB mice are mice with autoimmune diseases that will spontaneously develop AIHA after 4 to 6 months. The study found that as the course of the disease in NZB mice prolonged, the levels of methemoglobin and lipid peroxidation products in the body continued to increase, and consistent with this, the level of reactive oxygen species (ROS) in erythrocytes also continued to increase, and was positively correlated with the degree of anemia and the incidence of AIHA. However, the level of ROS in erythrocytes in hSOD1-Tg/NZB mice carrying the human SOD1 gene decreased significantly. The SOD1 gene reduces the incidence of AIHA by inhibiting the level of ROS in erythrocytes.

Protein Level

Human erythrocyte anion exchanger 1 (AE1): Human erythrocyte anion exchanger 1 is located in the third band in erythrocyte membrane protein electrophoresis, also known as band 3 protein. It is encoded by the SLC4A1 gene and is one of the most important proteins on the erythrocyte membrane. Band 3 protein is not only the main attachment point of the membrane skeleton, but also participates in the transmembrane transmission and functional regulation of various substances and information. Any changes in its structure or content may affect the integrity of the erythrocyte membrane structure and the response deformation ability of the entire cell. Membrane protein band 3 regulates the interaction with other cytoskeleton proteins and key enzymes of metabolic pathways, and mediates the phosphorylation of Lyn kinase and Src family kinase, thereby jointly maintaining the overall balance of erythrocytes. For example, streptolysin can inhibit the activity of band 3 protein, allowing chloride ions to quickly enter erythrocytes, destroying the stability of the cell membrane, and causing hemolysis. In addition, a series of serological and immunohistochemical tests were performed on 120 AIHA patients, and it was found that warm anti-self IgG antibodies can specifically act on band 3 protein, and it was also proved that it is an important target of warm antibodies in AIHA patients. Therefore, band 3 protein plays a vital role in the destruction of red blood cells and antibodies. Regulating the function of band 3 protein may reduce the hemolysis of AIHA and thus alleviate the disease process.

Stromal interaction molecule (STIM): store-operated calcium ion channel (SOCE) is one of the important channels that mediate the entry of extracellular calcium ions into cells. Its core protein is composed of the stromal interaction molecule (STIM) located on the endoplasmic reticulum and the calcium release-activated calcium channel modulator (ORAI) located on the cell membrane. At present, studies have found that STIM protein is divided into two subtypes, STIM1 and STIM2, and their main functions are slightly different. In the process of the body fighting against pathogens, STIM protein can change due to the increase and decrease of calcium ions in the cell, accumulate and translocate to the vicinity of the plasma membrane. The accumulated STIM protein will then activate the Orai1 protein, prompting the calcium release activated calcium channel (CRAC) to open. The influx of calcium ions leads to cell activation, thereby activating immune cells. STIM actually has the dual functions of regulating the opening and selectivity of CRAC channels. If the STIM1 and STIM2 proteins of T lymphocytes are deficient, autoimmune diseases of the exocrine glands, such as Sjögren’s syndrome, will occur. Researchers have found that STIM2 is closely related to cell migration and cytokine changes downstream of G protein-coupled receptors and TLR4 activation in macrophages. Like STIM1, it can cause the release of stored calcium and phagocytosis. In addition, in the AIHA model, STIM1 also activates C5aR-mediated FCγR, thereby causing the occurrence and development of the disease. Therefore, STIM1 and STIM2 act jointly and independently on macrophages in the fatal hemolysis process. After blocking the STIM protein-related functions, the mortality rate of AIHA model mice and LPS-induced septic mice was significantly reduced, further indicating that the inhibition of STIM protein may help alleviate inflammatory and autoimmune diseases.

Secondary AIHA

Gene Level

Immunoglobulin variable heavy chain region (IGVH) gene: The immunoglobulin variable heavy chain region is currently an important molecular indicator for predicting the condition and prognosis of chronic lymphocytic leukemia. It is generally believed that patients with somatic hypermutation of the IGHV gene sequence have a better prognosis and a longer overall survival than those without mutations. In a retrospective study of 585 Chronic lymphocytic leukemia (CLL) patients, it was found that the non-mutation state of IGHV was an independent influencing factor for the secondary occurrence of AIHA in CLL patients, and the median survival of AIHA disease development in non-mutated patients was significantly shortened. It is speculated that it may play an important role in specific B cell receptor subsets, but the specific molecular mechanism of action is still unclear.

MicroRNA: MicroRNA is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes, which participates in post-transcriptional gene expression regulation in animals and plants. It has been found that microRNA is differentially expressed in many immune diseases. Therefore, drugs that act on abnormal microRNAs may be a new research direction to achieve therapeutic effects, such as systemic lupus erythematosus, chronic idiopathic urticaria, etc. In the process of studying the physiological and pathological mechanisms of AIHA secondary to CLL, it was found that a total of 9 miRNAs (miR-19a, miR-20a, miR-29c, miR-146b-5p, mir-186, miR-223, mir-324-3p, mir-484miR-660) were downregulated in expression. Two of the miRNAs (miR-20a and miR-146b-5p) are involved in autoimmune phenomena, especially miR-146b-5p, which is involved in both autoimmune diseases and chronic lymphocytic leukemia. Further research on this microRNA found that miR-146b-5p is closely related to regulating CD80, which is a molecule closely related to the synapse between B lymphocytes and T lymphocytes and the restoration of cell antigen presentation ability. Therefore, research on microRNA may become a new hotspot for the treatment of related immune diseases in the future.

Protein Level

B cell adsorption factor 1 (BCA-1): B cell adsorption factor 1, also known as B cell chemoattractant (BLC) or CXCL13 (chemokineCXCligand13), is a member of the CXC chemokine family and is secreted by follicular dendritic cells and macrophages in secondary lymphoid organs. This chemokine selectively attracts B cells, including two subsets, B-1 and B-2, and exerts its effect by interacting with the chemokine receptor CXCR5. The CXCR5 receptor is currently the only known ligand, which is expressed in mature B cells, follicular helper T cells, Th17 cells, and regulatory T cells. Many studies have shown that abnormal expression of CXCL13 is associated with the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus. Since secondary AIHA is mostly secondary to autoimmune diseases, especially systemic lupus erythematosus. Through the case analysis of clinical AIHA patients, the researchers traced the relationship between it and systemic lupus erythematosus and found that CXCL13 and hemoglobin values ​​were negatively correlated, while another chemokine CCl4 (chemokine (C-Cmotif) ligand4) and reticulum erythematosus values ​​were positively correlated. They can be used as sensitive markers for the growth and decline of AIHA disease. The plasma CXCL13 level can reflect the severity of the disease, and CCl4 can be used as an indicator for determining the bone marrow hyperplasia of AIHA patients. At the same time, higher plasma soluble tumor necrosis factor receptor II levels may have a strong guiding significance for distinguishing whether it is AIHA secondary to SLE. Therefore, the use of CXCL13 antagonists may have a certain degree of delaying effect on the development of AIHA.

In short, the pathogenesis of AIHA involves the interaction of multiple factors such as genes and proteins. As the research continues to deepen, these possible influencing factors will continue to be discovered, which can provide possible drug targets for clinical treatment and provide further in-depth directions for the study of its pathogenesis.

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Avian influenza virus (AIV) is a common type A influenza virus, belonging to the genus influenza virus of the family Orthomyxoviridae, and is classified as a Class A epidemic by the World Organization for Animal Health (OIE). It is usually transmitted among poultry and birds, especially when accompanied by migratory birds, which will further increase its spread and cause a large-scale epidemic across regions and countries. There are two main protein protrusions on the envelope of influenza viruses, namely hemagglutinin (HA) and neuraminidase (NA), which can be used to divide influenza viruses into four types: type A (IAV), type B (IBV), type C (ICV) and type D (IDV). Among them, type A influenza virus uses a variety of birds including waterfowl and poultry as hosts, and can also infect other species such as pigs, dogs, cats, and horses. Type B influenza virus is mainly transmitted by infecting humans, and the host of type C infection is also humans. Type D influenza virus mainly uses livestock as hosts, such as pigs, cattle and sheep. There are also obvious differences in the epidemics caused by the above four influenza viruses.

Figure 1. Avian influenza transmission flow from the natural reservoir (aquatic birds) to poultry, humans, and other animal species.

Influenza A virus and type B influenza virus can both cause widespread seasonal epidemics. On this basis, influenza A virus can further cause zoonotic pandemics, while influenza B virus has not been found to be associated with pandemics. Seasonal influenza caused by influenza A virus and influenza B virus can reach 5 million cases and 500,000 deaths each year, of which infections caused by influenza B virus account for about 25%. On this basis, according to the different pathogenicity of different avian influenza viruses, they can be further divided into low pathogenic avian influenza virus (LPAIV) and highly pathogenic avian influenza virus (HPAIV). Usually, if the HA protein in the avian influenza virus has a multi-base cleavage site, it is a highly pathogenic avian influenza virus. Most avian influenza viruses are low pathogenic avian influenza viruses, and common highly pathogenic influenza viruses include two subtypes: H5N1 and H7N9. The two subtypes of H5N1 and H7N9 can infect mammals including humans, often causing severe lower respiratory tract diseases, while low pathogenic avian influenza viruses mainly infect the upper respiratory tract, usually with complications of conjunctivitis.

Structure of Avian Influenza Virus

The genetic material of avian influenza virus of the family Orthomyxoviridae is composed of 8 single-stranded negative-strand RNA (vRNA), namely, PB2, PB1, PA, HA, NA, M and NS. It is also composed of a variety of essential and non-essential proteins. The essential proteins include hemagglutinin, neuraminidase, polymerase protein (PB1, PB2, PA), nucleoprotein (NP), non-structural protein (NS) and matrix protein (M). The diameter of the avian influenza virus particles composed of the above three is 80-120nm in spherical state. The avian influenza virus isolated from clinical samples will expand from filaments to spheres after chicken embryo culture. The filamentous virus particles are 200-300nm high and 20nm in diameter at the bottom.

Pathogenicity of H5N1 Avian Influenza Virus to Animals

Avian influenza viruses are generally classified as either low pathogenic or highly pathogenic according to their pathogenicity to poultry. H5N1 avian influenza virus belongs to the latter, which is the result of the accumulation of multiple basic amino acids in the HA cleavage site. Unlike low pathogenic avian influenza, highly pathogenic avian influenza develops rapidly and has a high mortality rate. After the outbreak of H5N1 avian influenza in 1996, H5N1 avian influenza occurred in various countries around the world to varying degrees, characterized by large-scale sudden deaths of poultry, waterfowl and wild birds, which confirmed the high pathogenicity of H5N1 to poultry. H5N1 avian influenza virus can also infect mammals, but this is determined by many factors. PA-X is a fusion protein of influenza A virus (IAV). Studies have shown that the absence of PA-X protein enhances the pathogenicity and replication of highly pathogenic H5N1 avian influenza virus to mice. At the same time, analysis of viruses isolated from Vietnam in 2012 and 2013 found that two amino acid changes in the PA protein in the polymerase complex affected the activity and virulence of the viral polymerase complex in mice. In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that recombination events could lead to a highly pathogenic influenza pandemic. Studies have shown that obtaining the NS fragment from H1N1pdm09 enhances the virulence of H5N1 in mice.

Detection of Highly Pathogenic H5N1 Avian Influenza Virus

According to the regulations of the World Organization for Animal Health (OIE), chicken embryo virus isolation and culture are often used for the detection of avian influenza. In recent years, many studies have used biosensors to detect viruses, such as silver nanocluster fluorescent molecular beacons based on DNA templates. As long as the corresponding recognition sequence is embedded, the well-designed molecular beacons can be conveniently used to detect a variety of virulence genes, including the genes of H5N1 virus. Aptamer sensors can also be used for detection. Aptamers represent an alternative to antibodies as recognition agents in diagnosis and detection analysis and have advantages. In 2016, scholars proposed an analytical platform for fluorescence detection of H5N1 virus gene sequences combined with near-infrared light and 2-photon excitation. At the same time, a digital microfluidic technology immunoassay method based on surface-enhanced Raman scattering has been proposed, and a sandwich immunoassay has been designed to sensitively detect H5N1 virus.

Vaccine for Highly Pathogenic H5N1 Avian Influenza Virus

Inactivated Vaccine

Using reverse genetics technology, an inactivated vaccine strain of H5NI with extremely weak pathogenicity and well adapted to growing in chicken embryos was artificially created, and the inactivated vaccine for avian influenza was made using this strain.

Recombinant Viral Vector Vaccine

Compared with inactivated vaccines, recombinant viral vector vaccines can induce more helper T cells to produce immune responses. A protective foreign antigen is injected into the viral gene to obtain the recombinant virus, and the immune system produces the corresponding target protein, thereby inducing an immune response. The recombinant viral vector vaccines currently developed for H5N1 avian influenza mainly include fowl pox virus vector vaccine, Newcastle disease virus vector vaccine, turkey herpes virus vector vaccine, duck enteritis virus vector vaccine and infectious laryngotracheitis virus vector vaccine.

DNA Vaccine

Compared with other vaccines, DNA vaccines have more advantages. Studies have reported that optiHA was inserted into the pCAGGS vector to construct a DNA vaccine, which caused a strong immune response when injected into chickens intramuscularly. In order to improve the efficacy of DNA vaccines against H5N1 influenza virus, studies have inserted three repeated KappaB (κB) motifs upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal, which were separated by a 5 bp nucleotide spacer. Variants with κB sites in chickens stimulated stronger humoral responses against the target antigen.

Inactivated IAV H5N1 Native Antigens

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Animal Models for Vaccine Development

Using animal models to test the effectiveness of vaccines is one of the key steps in vaccine development and is often a key point in the long process of vaccine development and registration. Hundreds of different animal models can be used to evaluate multiple aspects of the immune response. Although many countries are promoting scientific experiments with alternative animals as much as possible and minimizing the number of animals used in scientific research, since there are currently no other feasible methods to evaluate or test the immune response after vaccination, the use of animals in experimental research in vaccine development remains the key. Choosing the most suitable animal model for this purpose is the key to the success of the experiment and to reducing the use of animals and research funding. Most vaccines have been evaluated in some way on small rodents, mostly mice. The advantages of using mice are low cost, easy manipulation, and specific genetic background and immune functions. In addition, a large number of immune components in mice can be used to explain the specific immune response caused by the vaccine. Because other species have much less immune components, this limits the level of detail in immune response analysis. However, large animal species such as pigs, cattle and sheep also have advantages, as they are closer to humans in terms of physiological functions and immune characteristics, and they are often hosts of the same or closely related pathogens. In addition, large animal species mainly reproduce outbreeding, and the normal distribution of vaccine responders and non-responders can be fully understood, which is very important for vaccine development. Animal models may be described in a number of ways: induced (experimental), spontaneous (natural), genetically modified, negative, orphan, and surrogate. These models vary widely in scope of application, cost, and their requirements for special infrastructure. The following are the advantages and disadvantages of commonly used animal models in vaccine development (Table 1):

Table 1. The advantages and disadvantages of conventional animal models in development of vaccine.

Screening of Animal Models

During the development of vaccines, not only should appropriate animal models be selected based on the delivery route of the vaccine being developed, but also the ethical issues when using animal models should be considered. Therefore, before using animal models, various factors need to be comprehensively considered to select the most suitable animal model to solve specific problems during the experimental process. For the development of a vaccine, there may be more than one animal model suitable for it. The following table summarizes the advantages and disadvantages of common vaccine delivery routes and the corresponding animal models (Table 2).

Table 2. The vaccine delivery routes and related animal models.

All in all, animal models are crucial and indispensable for vaccine development. It is essential when determining the quality and quantity of the immune response of a vaccinator, evaluating the safety and toxicity of the vaccine formulation, determining the effectiveness of the vaccine in providing protection against attacking infections, and evaluating the likelihood that the vaccine will prevent the transmission of disease in a specific population. At the same time, the selection of the most suitable animal model should be based on the special needs of the research project, and should not be influenced by factors such as price and difficulty of handling. In addition, in the long run, selecting the right animal model when developing new vaccines can save a lot of money, time and valuable resources.

Creative Diagnostics provides a large number of infectious animal models and a large library of viral pathogens for the development of new vaccines and antiviral drugs. These models include:

In addition, we provide well-designed analytical services that allow researchers to obtain well-researched animal models of viral infection. These models provide a multi-faceted perspective on infection dynamics, covering a range of parameters such as clinical symptoms, virus proliferation rates, clinical pathological characteristics, and cellular and humoral immune responses. Working with us ensures that a single scientific study for each project is supported by carefully designed methods, thereby promoting breakthrough discoveries in the pathogenesis and treatment of the virus.

Reference

Kiros, Tadele G., et al. The importance of animal models in the development of vaccines. Innovation in Vaccinology: from design, through to delivery and testing. 2012: 251-264.

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Autoimmune diseases can involve any organ and affect people of any age. More than 100 autoimmune diseases have been discovered. Common autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, etc. Patients with autoimmune diseases have autoantibodies against their own tissues, organs, cells and intracellular components, which are important signs in disease diagnosis. Each autoimmune disease is accompanied by a characteristic autoantibody spectrum.

Common Autoimmune Diseases

Autoimmune diseases caused by type II hypersensitivity: autoimmune hemolytic anemia, immune thrombocytopenic purpura, myasthenia gravis, toxic diffuse goiter, anti-glomerular basement membrane nephritis, anti-tubular basement membrane Nephritis etc.

Autoimmune diseases caused by autoantibodies-immune complexes: systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, polymyositis, dermatomyositis, scleroderma, etc.

Autoimmune diseases caused by T cell responses to self-antigens: type I diabetes, multiple sclerosis, etc.

Autoantibody Testing

Antinuclear Antibodies

Antinuclear antibodies (ANA) are a general term for a group of autoantibodies that use various nuclear components of one’s own eukaryotic cells as target antigens. The nature of ANA is mainly IgG, but also includes IgM, IgA and IgD. So far, more than twenty kinds of antinuclear antibodies against different components of the nucleus have been discovered. ANA mainly exists in serum, but can also exist in pleural effusion, joint synovial fluid and urine. Currently, indirect immunofluorescence (IIF) is most commonly used as the ANA screening test.

Anti-ENA Antibody Profile

ENA is the general term for extractable nuclear antigens. ENA antigens can be extracted from the nucleus with saline or phosphate buffer. Different autoimmune diseases produce different anti-ENA antibodies. Depending on the molecular weight and antigenic characteristics of the ENA antigen, different immune methods can be used to detect these autoantibodies. Currently, the most commonly used methods are immunoblotting technology (IBT) and dot enzyme immunoassay technology (dot-ELISA). The anti-ENA antibody profile is a confirmatory test for laboratory diagnosis of autoimmune diseases.

Antineutrophil Cytoplasmic Antibodies

Antineutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies that target human neutrophil cytoplasmic components and are closely related to a variety of clinical small vessel inflammatory diseases. This group of antibodies can be expressed as IgG, IgM or IgA. It has been confirmed that this antibody is the serum signature antibody of systemic vasculitis and is of great significance for the diagnosis, classification and prognosis of vasculitis. Detection can be divided into two types: medium ANCA and specific ANCA detection. The IIF method is usually used to detect total ANCA, and the ELISA method is most commonly used to detect specific ANCA.

Antiphospholipid Antibodies

Antiphospholipid antibodies (APLA) are autoantibodies directed against a group of antigenic substances containing phospholipid structures. These antibodies include anticardiolipin antibodies, antiphosphatidic acid antibodies, and antiphosphatidylserine antibodies. This group of antibodies can be divided into IgG, IgM and IgA types, with the IgG type being the most common. Among them, anticardiolipin antibodies are the most representative, because they have the strongest specificity and are the most studied in relation to various diseases. For the detection of anticardiolipin antibodies, ELISA is the most commonly used method.

Rheumatoid Factor

Rheumatoid factor (RF) is an antibody against the antigenic determinant of the Fc fragment of human or animal IgG molecules. It is an autoantibody with denatured IgG as the target antigen. Common RF types include IgM, IgG, IgA and IgE, with IgG being the main type and the type measured by conventional methods. Detection methods include latex particle agglutination test, rate scattering nephelometric method, and ELISA method.

Anti-Keratin Antibodies

Anti-keratin antibodies (AKA), also known as anti-keratin antibodies (ASCA), are mainly common in patients with rheumatoid arthritis. Indirect immunofluorescence analysis is commonly used for detection.

Autoantibodies also include anti-cyclic citrullinated peptide antibodies, anti-smooth muscle antibodies, anti-mitochondrial antibodies, anti-acetylcholine receptors, anti-skeletal muscle antibodies, etc. Autoantibody detection has become an important laboratory indicator in clinical immune testing.

Although autoantibodies are valuable biomarkers for diagnosis and classification, there is often no clear link between the specificity of autoantibodies and the resulting immunopathology, especially when the autoantigen is widely expressed intracellularly. In organ-specific autoimmune diseases, the association between autoantibodies and histopathology is often more pronounced than in systemic autoimmune diseases. Although these associations are strong, the reasons for the association between certain autoantibodies and clinical manifestations are unclear.

Autoantigen Recognition

The diverse clinical manifestations of autoimmune diseases require exploration of autoreactivity, especially for conditions that are not yet understood. In these studies, the first step is to search for autoantigens to provide a framework to consider the role of B and/or T cell autoreactivity in disease pathogenesis.

The recognition of autoantibodies and autoantigens is closely intertwined, as the analysis of autoantibodies relies on the recognition of the target antigen. Currently, screening of autoantigens is accomplished via large protein arrays, peptide arrays, or phage display libraries for target identification of antibodies in patient serum, using tissue extracts as the antigen source. These techniques allow screening thousands of proteins or peptide fragments for autoantigenicity and can complement traditional immunochemical techniques.

Autoantibodies with the same specificity can be expressed in different diseases. For example, antibodies against the protein glutamate decarboxylase (GAD) can occur in stiff-person syndrome as well as type 1 diabetes (insulin-dependent diabetes mellitus or IDDM). In this case, differences in the specificity of the autoantibodies for the antigen or their quantity may lead to different clinical manifestations.

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Epidemiology of Respiratory Syncytial Virus Infection

Respiratory syncytial virus is the most important viral pathogen causing acute lower respiratory tract infections in children under 5 years of age worldwide. Respiratory syncytial virus is the leading cause of severe (and even fatal) lower respiratory tract infections in early postnatal life. It is the main cause of bronchiolitis in infancy and pneumonia in early childhood. Respiratory syncytial virus infection is the leading cause of hospitalization for viral respiratory infections in infants and young children, seriously endangering children’s health, especially premature infants, infants and young children with congenital heart disease or primary immune deficiency.

Pathogenesis of Respiratory Syncytial Virus Infection

The pathogenesis of respiratory syncytial virus infection is complex and involves the combined effects of pathogenic factors, airway epithelial cell-related factors, immune system response, nervous system response, host factors and environmental factors. Respiratory syncytial virus infection is most likely to affect the respiratory system, and its main mechanisms are airway obstruction, bronchial smooth muscle spasm, and subsequent airway hyperresponsiveness.

Respiratory syncytial virus infection can cause airway cilia and airway epithelial cells to shed. The shed airway epithelial cells, neutrophils, cellulose, and lymphocytes accumulate in the airway, causing airway obstruction. At the same time, excessive secretion of mucus and airway obstruction Edema worsens airway obstruction.

The tracheal and bronchial epithelium can be damaged and shed due to inflammatory reactions, resulting in the exposure of sensory nerve endings and the release of active substances, causing bronchial smooth muscle spasm; the exposure of nerve endings causes airway hyperresponsiveness. These mechanisms are mutually responsible and cause a series of symptoms.

Respiratory Syncytial Virus Detection Method

Currently, technologies such as pathogen isolation and culture, tissue culture, enzyme-linked immunosorbent assay (ELISA), molecular biology, gene chips, and rapid colloidal gold are mainly used to detect Respiratory syncytial virus in clinical practice.

Figure 1. Rapid Fluorescent Immunochromatographic Test to Detect Respiratory Syncytial Virus.

In the early days, culture was a common method for isolating pathogens. This method selects appropriate tissues and cells for inoculation based on the bacterial tropism of the pathogen. Subsequently, immunological and hemoadsorption methods are used to detect protein markers on the surface of infected cells, thereby confirming the detection and identification of pathogen proliferation. Although the virus isolation and culture method can objectively isolate the type of infectious virus, this method is complex to operate, takes a long time to detect, is costly, is prone to false negative results, and the test results are affected by many factors. Therefore, its clinical application rate is not high.

Molecular-level detection has high sensitivity and specificity, but its application in primary medical institutions is limited due to high requirements on laboratory conditions.

Since RSV-specific antibodies are produced slowly in the body and have low titers, the detection sensitivity is not high. Therefore, diagnosing RSV infection by measuring patients’ serum antibodies cannot provide clinicians with a timely basis for diagnosis and treatment. Moreover, the immunology of this method of detecting antibodies the method reflects indirect indicators of infection and cannot replace direct pathogenic testing. Therefore, detecting RSV antigen has become an important way to diagnose the disease in the early stage. However, because the virus titer is low in secretions, it is difficult to diagnose RSV infection by serological methods or antigen examination. The purpose of rapid cell culture centrifugation is to speed up the entry of virus particles into cells, the virus isolation time is much shorter than conventional virus isolation. Compared with direct detection of specimen smears using immunohistochemistry, it increases the proliferation of sensitive cells, which can improve the sensitivity and specificity of detection. This method is especially suitable for viruses with lower titers, but requires the presence of viable virus particles. Early diagnosis of RSV infection should detect both live and dead viruses, that is, cell culture and non-cell culture methods for detecting RSV antigens complement each other. Non-cell culture can detect dead viruses, and cell culture can detect low-titer live viruses.

Related Products

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Figure 1. Specific mediators, metabolic pathways, and organs responsible for procalcitonin production in response to bacterial infections or inflammation compared to normal physiological conditions.

The diagnosis of infection has always been a major problem faced by clinicians. Although there are many monitoring methods, there is still a lack of sensitive and specific dynamic monitoring indicators. Since it was first discovered in 1993 that the concentration of procalcitonin in the blood of patients with sepsis was significantly elevated, procalcitonin has become an important marker for the diagnosis of bacterial infection. Procalcitonin has higher accuracy and specificity than traditional biological markers. At the same time, PCT concentration is related to the severity of the disease and can be used to guide antibacterial drug treatment.

Production and Release of Procalcitonin

Procalcitonin is the propeptide of calcitonin and consists of 116 amino acids. Normally procalcitonin is encoded by the Calc-1 gene located on the short arm of chromosome 11 in thyroid C cells. In thyroid C cells, the Calc-1 gene is translated into a PCT precursor peptide containing 141 amino acids, and then enters the endoplasmic reticulum to form PCT under the action of glycosylase and specific enzymes. Subsequently, PCT is processed by specific endopeptidase, cleaved into N-PCT, calcitonin and calstatin.

Under normal circumstances, PCT is almost not secreted from cells, and the blood content is <0.1ng/ml. Once bacteria invade the body, the PCT concentration in the blood can quickly increase to 5,000 times. The reason for this is that some studies believe that bacterial infection stimulates specific transcription factors in tissues to activate the Cala-1 regulatory gene, thereby activating PCT transcription. Others believe that under normal circumstances, PCT transcription is inhibited by specific transcription factors. After bacterial infection, this inhibitory factor is cleaved, resulting in PCT transcription.

Unlike C-reactive protein, almost all peripheral tissues are involved in the production of PCT during bacterial infection. Studies on thyroidectomy patients have found that blood PCT concentrations remain high during bacterial infection. Researchers injected endotoxin into baboons and found that PCT-mRNA was expressed in almost all tissues. The liver and kidney were the main tissues producing PCT 6 hours after the injection of endotoxin. About 24 hours after the injection, the lungs, stomach, the heart produces PCT at its peak. The half-life of PCT in the circulation is approximately 22 to 26 hours and does not depend on renal excretion.

It is controversial whether peripheral blood cells, in addition to parenchymal cells, participate in the production of PCT during bacterial infection. Scientists studied the peripheral blood of 17 patients with moderate to severe infections and found that PCT could be detected in monocytes and lymphocytes. By intracellular antibody staining, the researchers confirmed the expression of PCT in monocytes and neutrophils. However, the researcher believes that peripheral blood mononuclear cells cannot produce PCT even when stimulated by endotoxins and cytokines. Other investigators found that PCT-mRNA was not detected in endotoxin-stimulated macrophages or viable isolated peripheral blood mononuclear cells. Therefore, whether peripheral blood cells are involved in the release of PCT during bacterial infection still requires further study.

Current research suggests that there are two pathways for bacteria to induce the release of PCT, namely the direct and indirect pathways. In the direct pathway, bacterial tissue structures (DNA, fimbriae, peptidoglycan, etc.) directly induce intracellular signaling to release PCT. In the indirect pathway, pathogens stimulate the body to produce intermediaries (such as pro-inflammatory cytokines) that then act on target cells to produce PCT. Some researchers believe that during bacterial infection, the pathogen directly acts on the specific microorganism-related receptor at the No. 5 start site of the Calc-1 gene, causing a large amount of PCT to be released. On the contrary, another researcher found that during bacterial infection, bacterial endotoxin stimulates the body to produce a series of pro-inflammatory cytokines (such as TNFa, IL-1β, IL-8, IL-6) that stimulate tissues other than the thyroid gland (intestine, lung , immune cells) release PCT into the blood. Both clinical and animal studies have confirmed that direct injection of endotoxin results in the release of PCT in the blood. Some researchers add endotoxin to cells cultured in vitro can cause intracellular production of PCT. Intravenous injection of TNFa and IL-2 into cancer patients causes rapid and large release of PCT. Interestingly, infusion of anti-TNFa monoclonal antibodies into human leukocytes inhibited bacterial-induced stimulation of PCT release. Another study found that IFN-r inhibits the release of PCT.

Application of PCT in Infectious Diseases

Differentiate bacterial infections from non-bacterial infectious diseases

The Infectious Diseases Society of America and the American Society of Critical Care Medicine jointly recommend PCT as an auxiliary diagnostic marker to distinguish sepsis from non-infectious systemic inflammatory responses. When bacterial infection causes a systemic inflammatory reaction, PCT concentration will increase significantly. However, when viral infection, cancer fever, transplant-host rejection and other inflammatory reactions occur, the blood PCT concentration will not increase or only increase slightly.

Evaluate the degree of infection and prognosis

Blood PCT concentration is not only a specific indicator of infectious diseases, but also can monitor the severity of host infection and judge prognosis by continuously monitoring PCT levels. The concentration of PCT is related to the severity of bacterial infection and bacterial load.

Application of PCT in Infections in Special Populations

Neonatal bacterial infection is one of the most important health problems, and early diagnosis and treatment are very important due to the non-specific symptoms and signs and high mortality rate after neonatal bacterial infection. Studies found that neonates with sepsis, urinary tract infection, and meningitis had significantly higher blood PCT concentrations. Current research has confirmed the value of PCT in neonatal infections. Based on these research results, the sensitivity and specificity of PCT They were 76.9% and 100% respectively, and the positive prediction rate and negative prediction rate were 100% and 78% respectively.

Related Products

PCT

Cat.	Product name	Specificity	Applications (Pairing Table: Capture – Detection)
DMAB1357MH	Anti-PCT monoclonal antibody, clone 14C10	Epitope: Calcitonin domain (aa 72 – 81) No cross-reaction with: Calcitonin, Katacalcin, CGRP1 and CGRP2.	Best sensitivity pair for human PCT detection in LFIA and CLIA. DMAB1357MH – DMABT-H1296MH	Inquiry
DMABT-H1296MH	Anti-PCT monoclonal antibody, clone 34C23	Epitope: N-term PCT (aa 11-25) No cross-reaction with: Calcitonin, Katacalcin, CGRP1 and CGRP2.	Best sensitivity pair for human PCT detection in LFIA and CLIA. DMAB1357MH – DMABT-H1296MH	Inquiry
DMAB1352MH	Anti-PCT monoclonal antibody, clone 39G12	Epitope: N-term PCT (aa 21-40)	Recommended pair for human PCT detection in ELISA. DMAB1352MH – CABT-L6524Z	Inquiry
CABT-L6524Z	Anti-PCT monoclonal antibody, clone 33B22	Epitope: Katacalcin domain (aa 96-105)	Recommended pair for human PCT detection in ELISA. DMAB1352MH – CABT-L6524Z	Inquiry
DCAB-TJ170	Magic Anti-PCT monoclonal antibody, clone 29C8	Epitope: Katacalcin domain (aa 102-111)	Recommended pair for human PCT detection in ELISA. DCAB-TJ170 – CABT-WN1111	Inquiry
CABT-WN1111	Anti-Calcitonin monoclonal antibody, clone 25B3	Epitope: Calcitonin domain (aa 72 – 81)	Recommended pair for human PCT detection in ELISA. DCAB-TJ170 – CABT-WN1111	Inquiry
DMABP-L38	Mouse Anti-PCT monoclonal antibody, clone IN089	Epitope: N-term PCT	Recommended pair for human PCT detection in LFIA and CLIA. DMABP-L38 – DMABP-L39 DMABP-L38 – CABT-L6523Z (Sensitivity: 0.2ng/ml)	Inquiry
DMABP-L39	Mouse Anti-PCT monoclonal antibody, clone IN330	Epitope: Katacalcin domain (C-term PCT)	Recommended pair for human PCT detection in LFIA. DMABP-L38 – DMABP-L39 (Sensitivity: 0.2ng/ml)	Inquiry
CABT-L6523Z	Mouse Anti-PCT monoclonal antibody, clone IN192	Epitope: Calcitonin domain (Middle region PCT)	Recommended pair for human PCT detection in LFIA and CLIA. DMABP-L38 – CABT-L6523Z (Sensitivity: 0.2ng/ml)	Inquiry
DCABH-001H	Mouse Anti-PCT monoclonal antibody	Epitope: Katacalcin domain (aa 96-116) Specificity: React with human PCT No cross-reaction with: Canine PCT	Recommended pair for human PCT detection in LFIA and CLIA. DCABH-001H – DCABH-002H	Inquiry
DCABH-002H	Mouse Anti-PCT monoclonal antibody	Epitope: Calcitonin domain (aa 78-81) Specificity: React with human PCT	Recommended pair for human PCT detection in LFIA and CLIA. DCABH-001H – DCABH-002H	Inquiry
DMAB1342MH	Mouse Anti-PCT monoclonal antibody	Epitope: Katacalcin domain (aa 90-113) Specificity: React with human PCT		Inquiry
DMAB1337MH	Mouse Anti-PCT monoclonal antibody	Epitope: N-term PCT (aa 26-60) Specificity: React with human PCT		Inquiry

PCT Related Kits, Antigens

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GLP-2 and Glucagon-Derived Peptides

For mammals, GLP-2 uses the proglucagon gene (PG) as a template and is transcribed in pancreatic A cells, intestinal L cells, hypothalamus, brainstem and other central neurons, and is expressed and translated. It is processed into a polypeptide containing 33 amino acids with a molecular weight of 3900. The proglucagon gene includes 6 exons and 5 introns, and its mRNA is translated into a single-chain precursor protein containing 160 amino acids. It is tissue-specific and is activated by prohormone convertase ( PCs) undergo post-translational processing to generate a series of proglucagon-derived peptides (PGDP) with different biological activities. PGDP includes glucagon (glucagon), proglucagon fragment (MPGF), and glucagon-related pancreatic polypeptide (GRPP) secreted by pancreatic A cells, as well as enteroglucagon and oxyntomodulin in the intestine and brain, glucagon-like peptide-1 (GLP-1), glucagon-like peptide 2 (GLP-2), intermediate peptide 1 (IP-1) and intermediate peptide 2 (IP-2), etc. The main form of GLP-2 in the blood circulation is complete GLP-2, and there is also partial GLP-2 formed after the first two amino acid residues at the amino terminus are hydrolyzed by diacyl peptidase IV (DPPIV).This form of GLP-2 is inactive.

Figure 1.Main biological actions of GLP-1 and GLP-2.

Physiological Functions and Pharmacological Effects of GLP-2

Different from other polypeptide growth factors, GLP2 is an intestinal epithelial-specific growth factor with intestinal specificity and stronger growth-promoting effect. Researchers used subcutaneous injection to compare the growth-promoting effects of GLP-2 EGF (epidemal growth factor), IGF-1 (in-sulin-growth factor-1) and GH (growth homone) on normal intestinal mucosa in mice., found that GLP2 has the most obvious effect. In the study on the effect of intestinal adaptation in short-intestine rats, it was found that the GLP-2 group had a significantly higher effect on increasing the weight of the jejunal intestine and the height of villi than the GH group and the KGF (keratinocyte growth factor) group.In addition, research data shows that GLP-2 mainly promotes the proliferation of intestinal epithelial cells and has no effect on the proliferation of other important organ tissues such as heart, liver, and kidney cells. No pathological changes have been seen in these tissues after long-term application of GLP-2.

GLP-2 Promotes Normal Small Intestinal Growth

GLP-2 can promote the increase in the weight and length of the stomach and small intestine of newborn rats during the lactation period. GLP2RmRNA is expressed at high levels in the stomach and small intestine of rat embryos and neonatal stages. The concentration of GLP2 in plasma increases before the birth of fetal pigs. It is significantly increased and maintained at a high level throughout the entire period of breast milk nutrition, which suggests that GLP2 may play an important role in the development and maturation of the gastrointestinal tract.

GLP2 Promotes Recovery of Damaged Intestinal Mucosa

A phase I clinical trial using teduglutide (a GLP-2 analogue resistant to DPPIV degradation) to treat patients with ulcerative colitis showed that the remission rate of ulcerative colitis in the treatment group was higher than that of the placebo group, and the therapeutic effect was dose-dependent.

GLP-2 Affects Gastric Acid Secretion and Gastrointestinal Motility

GLP2 inhibits human gastric acid secretion caused by sham feeding. Sham feeding causes gastric acid secretion to increase approximately 5 times. This increase is reduced by 65% in the GLP-2 perfusion group compared with the normal saline perfusion group, indicating that GLP-2 is a powerful inhibitory factor for human gastric acid secretion. As an inhibitory factor, animal experiments have also proven that GLP-2 can inhibit gastric emptying in pigs.

GLP-2 Increases Intestinal Blood Supply

In newborn piglets fed total parenteral nutrition, administration of GLP-2 can increase portal blood flow by 25%, and this reaction is dependent on nitric oxide. Interesting, GLP-2 increases portal blood flow only in the small intestine, because it was found that the increase in hemoglobin content and the expression of GLP-2 receptors were mainly concentrated in the small intestine.

GLP2 and Feeding

Studies have found that the plasma level of GLP-2 in healthy people increases significantly after eating, but physiological levels of GLP2 have no significant effect on appetite and energy intake.

GLP-2 Inhibits Bone Resorption

Porosis may be the result of bone loss due to an imbalance in bone remodeling, which causes bone resorption to exceed bone formation. Common treatments inhibit bone resorption by reducing osteoclast number, activity, and longevity, but subsequently bone formation is also inhibited. The study found that exogenous GLP-2 caused a sharp and sustained reduction in bone resorption but had no effect on immediate bone formation. It can be seen that GLP-2 can affect the balance of bone remodeling, and the tilt of this balance toward osteogenesis may eventually lead to an increase in bone mass and bone strength, thus providing a new model for the treatment of osteoporosis. GLP-2 treatment reduces bone resorption but does not affect bone formation, and may have a more positive impact on bone health than therapies that reduce bone resorption and bone formation at the same time.

Post-receptor Signal Transduction Mechanism of GLP-2

A large number of studies have shown that GLP2 regulates the proliferation of intestinal epithelial cells and inhibits their apoptosis by acting on the GLP2 receptor (GLP-2R), thereby protecting intestinal cells. The human GLP2R gene is located on chromosome 17p13.3. Cloning of human and rat cDNA-encoded GLP2R shows that it is a member of the G protein-coupled receptor superfamily, with 7 transmembrane domains, similar to GLP-1 glucagon and GIP (Glucose-dependent insulinotropic polypeptide) receptors have a high degree of homology. A series of studies using immunohistochemistry, in situ hybridization, confocal laser microscopy, RT-PCR and Westem blot analysis separately and/or in combination showed that GLP-2R is expressed in intestinal epithelial cells, gastric epithelial cells, intestinal intrinsic neurons. It is expressed in enteroendocrine cells, intestinal, submucosal myofibroblasts, vagal afferent fibers, cerebral cortex, cerebellum, hypothalamus, amygdala, hippocampus, dentate gyrus and lungs. The distribution density of GLP2R in the gastrointestinal tract is in the jejunum, duodenum, ileum, colon, and stomach. However, the signal transduction mechanism of GLP2R is still unclear, mainly due to the lack of cell models for studying GLP-2R.

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