Humanized mouse models are created by implanting human genes, cells, tissues, or organs into a mouse (Allen et al., 2019; Chen et al., 2022). They aim to recreate parts or the whole of the human immune system in a murine host, thereby providing an in vivo preclinical model to investigate human immune conditions and diseases (e.g. HIV) or to test the safety of drugs that target human-specific immunoreceptors. In addition, humanized models are widely used to study the mechanisms of and potential treatments for different types of cancer, including novel immune-modulating anticancer drugs (Chuprin et al, 2023).
Many of these models are based on reconstituting human immune cell types in immunodeficient mice, which possess genetic defects that cause impaired development of the mouse’s own immune system. This reduces the risk of graft rejection and facilitates expression of human immune function in the host.
Inflammatory Bowel disease (IBD) is a multifaceted and polygenic disorder that is characterized by persistent inflammation in the GI tract. It encompasses two primary phenotypes (Ulcerative Colitis and Crohn’s disease). Attaining a comprehensive insight into the intricate mechanisms and the pathogenesis of IBD is of paramount importance for the development of efficacious therapeutic interventions. In this context, animal models have emerged as indispensable tools, playing a major role in both the advancement of and the understanding of IBD. The establishment of both robust and reliable animal models has made significant contributions to giving us a better insight into the complexities of IBD. These models enable the simulation of the intricate interplay between genetic predisposition, environmental factors, and dysregulated immune responses, which collectively contribute to the initiation and the development of the disease process. By providing a controlled experimental environment, animal models facilitate the systematic investigation of the fundamental pathogenic mechanism underlying IBD and serve as platforms for the evaluation of potential therapeutic modalities. Among the plethora of animal models available for studying IBD, two stand out: The Dextran Sodium Sulfate (DSS) colitis model, and the 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis model.
Overview of IBD
Inflammatory bowel disease stands as a perplexing and intricate medical condition posing a significant challenge to medical professionals and researchers alike1. This chronic inflammatory disorder involves a complex interplay of genetic, environmental, and immunological factors within the gastrointestinal tract leading to sustained inflammation and tissue damage1. The immune system’s dysregulated responses further contribute to the pathophysiology of IBD, perpetuating the disease’s chronicity. Patients afflicted with IBD endure a range of distressing symptoms including persistent abdominal pain, frequent diarrhea, unintended weight loss, and fatigue, significantly impairing their quality of life. Despite significant advancements in medical knowledge and therapeutic options, current treatments for IBD remain predominantly focused on symptom management and reducing inflammation rather than achieving a cure1. Immunosuppressants, corticosteroids, and biological therapies targeting specific cytokines are among the treatments commonly employed. However, these therapeutic approaches often carry adverse effects and may not provide sustained remission or long-term relief of patients1. To address the complexity of IBD and improve patient outcomes, researchers are dedicatedly delving into the disease’s underlying mechanisms2. The role of gut microbiota, immune cell pathways, and intricate inflammatory cascades is being extensively investigated to identify potential therapeutic targets2. Advancements in pharmacology and precision medicine offer promising avenues for more tailored and effective treatments aiming to restore the disrupted immune responses and achieve sustained remission for IBD patients.
DSS Colitis Model
The DSS Colitis model is a well-established and widely used experimental approach to better study and understand the acute phase of IBD in rodents. The induction protocol involves the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide, in the drinking water of experimental animals. DSS exposure leads to colonic epithelial injury and disruption of the gut barrier function, triggering an inflammatory response3.
Researchers can modulate the severity of colitis in this model by adjusting the concentration and duration of DSS exposure. Shorter exposure durations generally induce milder inflammation, while longer exposures lead to more severe and sustained colitis. This allows for the study of various disease stages and the evaluation of therapeutic interventions at different time points during the inflammatory process. The DSS colitis model is commonly employed in mice and rats, with the C57BL/6 mouse strain being one of the most frequently used due to its well-characterized immune response. The model’s induction is relatively straightforward reproducible, making it an attractive choice for researchers investigating the early stages of IBD.
Clinically, researchers assess disease progression in DSS-treated animals by monitoring various parameters, such as body weight loss, stool consistency, and the presence of rectal bleeding. Additionally, histological examination of colonic tissue samples allows researchers to study the extent of mucosal damage, inflammatory cell infiltration, goblet cell depletion, and other key features characteristic of IBD pathology4.
TNBS Colitis Model
The TNBS colitis model is another widely used experimental approach employed to study the chronic phase of IBD. In this model trinitrobenzene sulfonic acid (TNBS) is typically dissolved in ethanol and instilled directly into the colon, leading to a T-cell-mediated immune response and inducing a delayed hypersensitivity reaction. This results in chronic inflammation and tissue injury in the colon5. Similar to the DSS model, the severity of colitis in the TNBS model can be controlled by adjusting the concentration and volume of TNBS instillation.
The model is commonly conducted in rats, such as the Wistar or Lewis strains, and mice, including the BALB/c and C57BL/6 strains. The strains have been extensively characterized in IBD research, providing valuable insights into the inflammatory responses seen in both acute and chronic phases of the disease. TNBS colitis model researchers assess disease severity by monitoring weight loss, disease activity index, and the occurrence of diarrhea. Additionally, histopathological analysis of colon sections allows for the examination of immune cell infiltration, granuloma formation, and transmural inflammation, which are key features observed in IBD patients particularly those with Crohn’s disease6.
Historic Overview of the DSS Colitis Model
The DSS Colitis model has a relatively recent history with its development and application dating back to the late 1980s and early 1990s. The model was initially introduced by Okayasu and colleagues in 1989 as another method to induce acute and chronic ulcerative colitis in mice3. The researchers recognized the need for a reproducible and reliable experimental model that could mimic the characteristics of human ulcerative colitis. To achieve this the utilized dextran sodium sulfate (DSS), a sulfated polysaccharide known for its pro-inflammatory effects on the colonic epithelium. Over the years the DSS colitis model has undergone refinement and optimization, becoming a widely used and well-established experimental tool in IBD research. Its versatility, reproducibility, and ability to simulate the acute phase of colitis make it valuable for studying the initial inflammatory events and assessing potential therapeutic interventions.
Histopathology Slide from UC Patient
Historic Overview of the TNBS Colitis Model
The TNBS Colitis model has a longer history, dating back to the 1960s, when researchers were exploring the immune mechanisms underlying hypersensitivity reactions. Trinitrobenzene sulfonic acid (TNBS) was initially used as a hapten to study delayed-type hypersensitivity in rodents5. In the late 1980s, researchers recognized the potential of TNBS as a colitis-inducing agent, leading to its application in the study of Inflammatory Bowel Disease. By administering TNBS into the colons of experimental animals, researchers could elicit a delayed-type hypersensitivity reaction, triggering chronic colitis and granulomatous inflammation. As the TNBS model was further investigated and characterized, researchers identified its relevance for studying fibrosis, a hallmark feature of IBD. The model offered insights into the fibrotic processes observed in Crohn’s disease, contributing to a more comprehensive understanding of IBD pathology.
DSS Colitis and TNBS Colitis V/S Other Models
The DSS colitis model and TNBS colitis model are valuable tools for studying Inflammatory Bowel Disease (IBD), but they are not the only experimental models used for this indication. Other models, such as the IL-10 knockout model and the adoptive T cell transfer model, have also been employed in IBD research. Each model offers unique advantages and focuses on different aspects of IBD pathology. As mentioned previously, the DSS colitis model and TNBS colitis model primarily represent chemically-induced models, involving the administration of DSS or TNBS to induce acute or chronic colitis, respectively. These models allow for the investigation of gut inflammation, immune cell infiltration, and tissue damage, making them well-suited for understanding the early and chronic stages of IBD. In contrast, the IL-10 knockout model mimics the genetic component of IBD, as IL-10 plays a pivotal role in maintaining intestinal homeostasis. The absence of IL-10 leads to spontaneous colitis in mice, resembling the chronic and relapsing nature of IBD in humans. This model is particularly useful for studying the role of immune dysregulation and the contribution of genetic factors to IBD development. The adoptive T cell transfer model focuses on immune cell-mediated inflammation. In this model, T cells isolated from mice with colitis are transferred into immunocompromised recipient mice, resulting in colitis development. Researchers can investigate the pathogenicity of specific T cell populations and study immune cell interactions and signaling pathways associated with IBD.
Best Practices for DSS Colitis Model
Standardization: Use standardized DSS concentration and administration protocols to ensure consistency in results across experiments. Consider factors such as animal strain, DSS concentration, duration of exposure, and water restriction carefully.
Animal Welfare: Monitor the health and welfare of experimental animals regularly. Assess and record parameters such as body weight, stool consistency, and the presence of rectal bleeding throughout the study period.
Water and Food Access: Ensure access to regular drinking water and a balanced diet for animals not receiving DSS treatment. Ad libitum access to water and food should be restored after DSS treatment to promote recovery.
Cage Conditions: Maintain optimal cage conditions, including appropriate temperature, humidity, and light cycles, to minimize stress and promote the well-being of experimental animals.
Randomization and Blinding: Randomize the assignment of animals to experimental groups and employ blinding during data collection and analysis to reduce bias and increase the reliability of results.
Sample Collection: Collect tissue samples consistently and systematically from the same regions of the colon to ensure accurate comparison and analysis between experimental groups.
Ethical Considerations: Adhere to ethical guidelines and obtain proper approval from the institutional animal ethics committee before conducting any experiments involving animals.
Best Practices for TNBS Colitis Model
TNBS Preparation: Prepare TNBS solutions freshly and with precision to ensure the stability and accuracy of the colitis induction process.
Ethanol Content: Use an appropriate amount of ethanol as a vehicle for dissolving TNBS to avoid potential non-specific effects of ethanol on the colonic tissue.
Dosage: Administer TNBS at the appropriate dosage to elicit a robust immune response and induce colitis consistently.
Animal Strain: Choose animal strains that are responsive to TNBS induction and have established literature support for colitis development.
Sterile Technique: Practice strict sterile techniques during TNBS administration and tissue sampling to minimize contamination and potential confounding factors.
Control Groups: Include suitable control groups, such as vehicle-treated or untreated animals, to provide a baseline comparison for TNBS-induced colitis.
Monitoring: Monitor animals closely for signs of colitis, such as body weight loss, stool consistency, and the presence of diarrhea or rectal bleeding, throughout the study.
Endpoint Consideration: Determine the appropriate endpoint for the study based on the research question, as TNBS colitis can manifest in various degrees of severity and duration.
FAQ
What are the key differences between the DSS colitis model and the TNBS colitis model in studying IBD?
The DSS Colitis model is induced by administering DSS orally in the drinking water, causing human ulcerative colitis-like pathologies due to its damaging effect on the colonic epithelial barrier. The inflammation resulting from DSS administration is more evenly distributed throughout the colon, primarily affecting the mucosal layer. This model activates innate immune responses, leading to a neutrophil-dominated inflammation.
On the other hand, the TNBS-induced colitis model involves a single intrarectal instillation of the haptenizing molecule TNBS. This model is commonly utilized and shares significant properties with human Crohn’s disease. In TNBS colitis, the inflammation tends to be more localized and is often concentrated in specific segments of the colon. The inflammation affects multiple layers of the colon wall, including the mucosa, submucosa, and muscularis. Additionally, this model triggers both innate and adaptive immune responses, involving T cells, cytokines, and antibodies.
Researchers often choose between these two models based on their research objectives and the aspects of IBD they wish to study. DSS Colitis is suitable for simulating ulcerative colitis-like pathologies and studying the effects of inflammation in a more widespread manner. TNBS-induced colitis, on the other hand, is preferred when investigating localized and more chronic inflammatory responses akin to Crohn’s disease. Combining both models can provide a more comprehensive understanding of IBD and contribute to a better characterization of potential therapeutic interventions.
Can the DSS colitis and TNBS colitis models be used to study specific subtypes of IBD, such as Crohn’s disease or ulcerative colitis?
While the DSS colitis model and TNBS colitis model primarily induce colitis, they have distinct pathological characteristics that resemble specific subtypes of IBD. The DSS colitis model is more commonly associated with ulcerative colitis due to its primarily colonic involvement and superficial inflammation. Conversely, the TNBS colitis model is frequently used to study aspects of Crohn’s disease, given its capacity to induce granulomatous inflammation and transmural colonic lesions. However, it is essential to recognize that these models represent simplifications of the complex spectrum of IBD, and results should be interpreted with consideration of the specific research question.
How can researchers address inter-individual variability observed in the DSS colitis and TNBS colitis models?
Inter-individual variability is inherent in animal models, including the DSS colitis and TNBS colitis models. To minimize its impact, researchers should adopt rigorous experimental design with appropriate sample sizes, randomization, and blinding. Ensuring that animals within each experimental group are from the same source and age and standardizing housing conditions can also help mitigate variability. Moreover, the inclusion of relevant control groups allows researchers to distinguish treatment effects from natural variations in disease severity.
What are the limitations of using the DSS colitis and TNBS colitis models in IBD research?
Both models have limitations that researchers should be aware of. The DSS colitis model does not fully replicate the chronic and transmural features seen in Crohn’s disease. The TNBS colitis model may induce severe inflammation, leading to animal morbidity and ethical concerns. Additionally, species differences should be considered when translating results to human patients. The models may not capture the complex interplay between genetic susceptibility, environmental factors, and the gut microbiota that contribute to IBD pathogenesis in humans.
Can the DSS colitis and TNBS colitis models be used to study the efficacy of novel biologics and immunomodulatory agents?
Yes, both models are highly relevant for studying the effects of novel biologics and immunomodulatory agents in IBD research. These models allow researchers to investigate the impact of targeted therapies on immune cell populations, cytokine profiles, and gut barrier function during acute and chronic colitis. By evaluating the modulation of inflammatory responses and tissue damage, researchers can identify potential candidates for further preclinical and clinical development.
Can the DSS colitis and TNBS colitis models be used to investigate the long-term effects of potential treatments in IBD?
Yes, both models can be adapted for long-term studies to assess the sustained effects of potential treatments in IBD. Researchers can administer therapeutic agents during the induction phase and continue treatment throughout the course of colitis development. By monitoring disease progression over an extended period, researchers can evaluate the durability and efficacy of the interventions in mitigating inflammation and promoting tissue repair.
What are some key considerations for data analysis when using the DSS colitis and TNBS colitis models?
Proper data analysis is crucial when working with the DSS colitis and TNBS colitis models. Researchers should employ appropriate statistical tests to determine the significance of differences between experimental groups. Careful consideration should be given to selecting relevant endpoints, such as histopathological scoring, cytokine levels, and molecular markers, and to choosing the appropriate timepoints for data collection. Inflammation and recovery may follow different patterns over time, so capturing data at multiple timepoints can provide a more comprehensive view of the disease process. Additionally, incorporating adequate sample size calculations and assessing for outliers will enhance the robustness and reliability of the study results.
Can the DSS colitis and TNBS colitis models be used to study the role of the gut-brain axis in IBD pathogenesis?
Yes, these models provide opportunities to explore the bidirectional communication between the gut and the brain, known as the gut-brain axis, in IBD research. Researchers can investigate the effects of stress, anxiety, and neural signaling on gut inflammation and immune responses in the DSS colitis and TNBS colitis models. By assessing neural pathways and neurotransmitter systems, researchers can gain insights into the neuroimmune interactions involved in IBD progression and the potential for neuroimmune-targeted therapies.
References
Neurath MF. 2018. New targets for mucosal healing and therapy in inflammatory bowel diseases. Mucosal Immunology, 11(3), 5-17.
Pastorelli L, et al. 2017. New Insights into IBD Pathogenesis: Intestinal Microbiota, IL-17, and Gut Macrophages. Inflammatory Bowel Diseases, 23(8), 1494-1503.
Okayasu I, et al. 1990. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology, 98(3), 694-702.
Chassaing B, et al. 2014. Dextran sulfate sodium (DSS)-induced colitis in mice. Current Protocols in Immunology, 104, 15.25.1-15.25.14.
Morris GP, et al. 1989. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology, 96(3), 795-803.
Morrissey PJ, et al. 1993. Murine TNF receptors: differential intracellular fate for p55 and p75. Journal of Immunology, 151(1), 427-434.
Scientists have been investigating cancer for well over 100 years, with the first animal model of cancer being a squamous cell carcinoma induced on the ears of rabbits using coal tar.1,2 Since that time, many thousands of animal models of cancer have been developed, but they largely fall into one of two baskets: syngeneic models and xenograft models. Both types of models have been used by researchers for over 50 years.3,4 Understanding the differences between these models, the variations within each type of model, and how to select the most appropriate model is key to designing a successful study – one which will most accurately predict clinical relevance.
Asthma is a chronic respiratory disease that is estimated to affect 262 million people worldwide and kill approximately 455,000 annually. It is characterized by airway inflammation, bronchoconstriction and airway hyperresponsiveness. These features can significantly impact the quality of life of affected individuals, and although symptom management is feasible, there is no known cure for this condition. The complexity of asthma and the factors contributing to its onset make its treatment and management challenging even for the most experienced healthcare professionals and pharmacologists. One commonly used animal model of asthma is the ovalbumin-induced asthma model. This model involves sensitizing animals to ovalbumin, which is a common allergen, using an adjuvant such as aluminum hydroxide. The sensitization process primes the immune system to recognize ovalbumin as a foreign substance and mount an immune response against it. After sensitization, the animals are challenged with aerosolized albumin, which is delivered directly into the lungs via a nebulizer or inhalation chamber. The challenge with ovalbumin leads to an inflammatory response in the lungs, which includes airway hyperresponsiveness, mucus production, and eosinophil infiltration. In the OVA induced asthma model, this can be measured by assessing changes in lung function, such as airway resistance or lung compliance. Mucus production refers to the increased secretion of mucus in the airways, which can obstruct airflow. In the OVA-induced asthma models, this can be assessed by staining lung tissue with a dye that highlights mucus producing cells. Eosinophilic infiltration is a characteristic feature of inflammatory airway reaction occurring because of asthma. In addition to eosinophils, other immune cells, such as neutrophils, lymphocytes, and macrophages, can also be involved in the inflammatory process of asthma. The levels of these cells in the bronchoalveolar lavage fluid (BALF) can provide insights into the specific inflammatory status and immune responses associated with different asthma phenotypes or exacerbations.
With an estimated total annual cost of $81.9 billion in the United States only (factoring in medical care, absenteeism, and mortality), the burden of asthma on the healthcare system needs to be dealt with swiftly.
Although the exact cause of asthma remains unknown, OVA-induced asthma models have been instrumental in uncovering the underlying mechanisms of the disease.
Overview of the OVA-Induced Asthma Model:
Recent studies have continued to use OVA-induced asthma models to investigate the pathophysiology of asthma and potential therapeutic interventions. For instance, a 2021 study by Chelladurai et al. demonstrated that blocking the interaction between OX40L and its receptor OX40 using an antibody reduced airway hyperresponsiveness and inflammation in an OVA-induced asthma model in mice (Chelladurai et al., 2021). Another study by Liu et al. (2020) used an OVA-induced asthma model to investigate the potential therapeutic effects of a novel molecule, FXYD6, which is involved in regulating ion transport in airway epithelial cells. The study found that FXYD6 overexpression attenuated airway inflammation, hyperresponsiveness, and remodeling in the OVA-induced asthma model in mice (Liu et al., 2020).
H&E Stain of a Lung Section
Role of IgE in the Model:
In the OVA-induced asthma model, IgE can play a significant role in allergic reactions by sensitizing mast cells and basophils to ovalbumin (OVA). However, it is important tot note that in order to effectively replicate the model, multiple mucosal OVA challenges are required to drive the IgE-mediated response. Upon re-exposure to OVA, the allergen binds to the IgE antibodies on sensitized mast cells and basophils, leading to the release of various inflammatory mediators that contribute to the development of asthma symptoms. These mediators, such as histamine, leukotrienes, and cytokines, can induce airway hyperresponsiveness, mucus production, and airway inflammation, which are considered important aspects of asthma pathogenesis.
Airway Hyperresponsiveness in the OVA-induced Asthma Model:
Several mechanisms have been implicated in the development of AHR in the OVA model. OVA-induced inflammation in the airways can lead to increased airway smooth muscle contraction, which contributes to airway narrowing. This inflammation can be driven by various immune cells such as Th2 cells, eosinophils, mast cells, and basophils. The activation of these immune cells leads to the release of cytokines and chemokines that recruit more immune cells, promote inflammation, and contribute to AHR. In addition, the recruitment of inflammatory cells, such as eosinophils and mast cells, can release various mediators that can further promote airway hyperresponsiveness, such as histamine, leukotrienes, and prostaglandins. These mediators can increase bronchial smooth muscle tone, enhance microvascular permeability, and promote mucus secretion, all of which contribute to AHR.
History of the OVA-Induced Asthma Model:
The ovalbumin-induced asthma model was first described in the 1970s and has since become one of the most widely used models for studying asthma (1). The model was initially used to investigate the role of immunoglobulin E (IgE) in the development of asthma. Since then, the model has been used to study various aspects of the disease, including the role of cytokines, chemokines, and other immune system molecules in the pathogenesis of asthma.
Comparison of OVA-Induced Asthma vs. Other Asthma Models:
Several animal models have been developed to study asthma, such as the house dust mite model, the cockroach antigen model, and others. While each model has its advantages and limitations, the ovalbumin-induced asthma model is still considered one of the most reliable and widely used models for studying asthma (2). The model is cost-effective and there are genetically engineered transgenic mice available for investigating OVA-specific responses, which is essential for studying the mechanisms of the disease, and for evaluating new biologics. However, it is important to note that no single animal model can fully replicate the complexity and heterogeneity of human asthma.
Best Practices When Using the Model:
Using high-quality ovalbumin to minimize batch-to-batch variability, standardizing the animal vendors, and the route and dose of ovalbumin exposure to ensure consistency. Additionally, using a suitable control group to account for any nonspecific effects of ovalbumin and monitoring the animals for signs of distress or illness while ensuring appropriate care are crucial aspects of conducting the study.
FAQ:
Q: How does the ovalbumin-induced asthma model compare to human asthma?
A: While animal models cannot fully replicate human disease, the ovalbumin-induced asthma model can provide valuable insights into the underlying mechanisms of asthma. However, caution should be exercised when extrapolating findings from animal models to human disease. For example, there may be differences in the immune response between mice and humans, and ovalbumin may not be a relevant allergen for all patients with asthma. The OVA model is best used to model patients with a TH2 asthma phenotype.
Q: What are the advantages of the ovalbumin-induced asthma model compared to other animal models?
A: The ovalbumin-induced asthma model is one of the most widely used and reliable models for studying asthma. One of the main advantages of this model is the ability to mimics key inflammatory features of asthma, including airway hyperresponsiveness, mucus production, eosinophilic infiltration, and cytokine release. These features closely resemble the characteristics seen in human asthma, making the model relevant for studying underlying mechanisms and evaluating anti-inflammatory interventions.
Q: What are the limitations of the ovalbumin-induced asthma model?
A: Like all animal models, the ovalbumin-induced asthma model has its limitations. One limitation is that the immune response in mice may not accurately reflect the immune response in humans. For example, the relative dominance of Th2 immune responses in the ovalbumin model may not fully reflect the immune profiles seen in all types of human asthma, which can involve various immune cell types and cytokines. Additionally, the ovalbumin-induced asthma model is an acute model of asthma that does not fully capture the chronic and progressive nature of the disease. Furthermore, ovalbumin is not a common human allergen, and the model does not account for the contribution of non-allergic triggers to the development of the disease. The HDM model is more mixed TH1/2/17 phenotype model is also offered by PL,
Q: Can ovalbumin-induced asthma models be used to study the effects of therapeutic interventions?
A: Yes, ovalbumin-induced asthma models have been used to study the effects of various therapeutic interventions, including anti-inflammatory agents and bronchodilators. By using these models, researchers can test the efficacy and safety of potential asthma treatments before moving to human clinical trials.
Q: What are some best practices for using ovalbumin-induced asthma models?
A: To ensure reliable and reproducible results, it is essential to follow best practices when using ovalbumin-induced asthma models. These practices include using high-quality ovalbumin to minimize batch-to-batch variability, standardizing the animal vendors, and the route and dose of ovalbumin exposure to ensure consistency. Additionally, using a suitable control group to account for any nonspecific effects of ovalbumin and monitoring the animals for signs of distress or illness while ensuring appropriate care are crucial aspects of conducting the study. Furthermore, researchers should consider using multiple models of asthma to validate their findings and to account for the limitations of each model, such as the HDM Asthma Model.
Q: Are OVA-induced asthma models only in rodents? What would an analogous asthma model be in non-human primates?
A: Ovalbumin (OVA)-induced asthma models have been primarily used in rodents, such as mice and rats, to study the pathophysiology of asthma and to evaluate potential therapeutic interventions. However, there have been some studies that have also used OVA-induced asthma models in other species, such as guinea pigs and rabbits.
Sourcing Your Asthma Study:
Our OVA-induced asthma model faithfully reproduces pulmonary inflammation (eosinophil infiltration), airway hyperresponsiveness, and the elevated IgE levels found in asthma. Coupled with our many years of experience in pharmacology, PharmaLegacy has the expertise and resources to provide comprehensive and reliable preclinical services for respiratory drug development. Choose right when looking for a CRO. Choose PharmaLegacy.
References:
Alcorn, J. F., Crowe, C. R., Kolls, J. K. (2011). TH17 cells in asthma and COPD. Annual Review of Physiology, 73, 495-516.
Carroll, O. R., Pillar, A. L., Brown, A. C., Feng, M., Chen, H., & Donovan, C. (2023). Advances in respiratory physiology in mouse models of experimental asthma. Frontiers in Physiology, 14. https://doi.org/10.3389/fphys.2023.1099719
Chelladurai, P., Sehgal, I. S., Dhooria, S., Agarwal, R. (2021). Targeting the OX40-OX40L pathway in allergic asthma: a preclinical study. Immunotherapy, 13(1), 15-27.
Das, S., Miller, M., Broide, D. H. (2019). Chromogranin A: a novel mediator of asthma pathogenesis and therapeutic target. Journal of Allergy and Clinical Immunology, 143(4), 1372-1379.
Fallon, P. G. (2019). The high and lows of type 2 asthma and mouse models. Journal of Allergy and Clinical Immunology, 105(2). https://doi.org/10.1016/s0091-6749(00)70138-2
Fujisawa, T., Joshi, B. H., Puri, R. K. (2019). IL-13 regulates cancer invasion and metastasis through IL-13Rα2 via ERK/AP-1 pathway in mouse model of human ovarian cancer. Molecular Cancer Therapeutics, 18(5), 903-914.
Liu, Z., Wang, Q., Wang, J., Yao, X., Huang, J., Yang, Z., … & Lu, X. (2020). Overexpression of FXYD6 attenuates airway inflammation, hyperresponsiveness and remodeling in a murine model of OVA-induced asthma. Molecular Immunology, 117, 1-10.
Reber, L. L., Marichal, T., Galli, S. J. (2019). New models for analyzing mast cell functions in vivo. Trends in Immunology, 40(4), 361-373.
Sathish, V., Thompson, M. A., Bailey, J. P., Pabelick, C. M., Prakash, Y. S., Sieck, G. C. (2020). Electrostatic atomization-based pulmonary administration of ovalbumin in mice: development of a novel in vivo asthma model. Journal of Aerosol Medicine and Pulmonary Drug Delivery, 33(2), 98-110.
Yan, X., Zhang, Y., Wang, L., Chen, H., Wang, J., Gao, J., & Zou, X. (2020). Plumbagin alleviates airway inflammation in ovalbumin-induced asthma mice through suppressing the activation of nuclear factor kappa B and mitogen-activated protein kinases. Journal of Immunology Research, 2020, 1-13.
Zhang, Y., Tang, H., Cai, L., Zhao, J., Liu, X., Yao, D., … & Zhang, H. (2021). Nanoparticle-based targeted delivery of ovalbumin to dendritic cells for enhanced cellular immune response and asthma therapy. Journal of Materials Chemistry B, 9(19), 4072-4081.
Drug metabolism and pharmacokinetics (DMPK) are essential to understanding important characteristics of drug candidates, including absorption, distribution, metabolism, excretion, toxicity (ADMET), and pharmacokinetic (PK) properties. DMPK studies are conducted at the preclinical and clinical stages as well as post-approval. Preclinical DMPK studies involve both in vitro studies to evaluate drug candidate characteristics such as protein binding affinities and interactions with transporters and enzymes as well as in vivo analyses to assess toxicity and pharmacokinetics such as half-life, bioavailability, excretion, absorption, and distribution in preclinical animal models. Drug characteristics are further evaluated during clinical trials, including assessment of safety, dosage, side effects, efficacy, and drug-drug interactions (DDIs) in human participants. Post-approval studies involve preparing labeling restrictions for the market as well as collecting safety and effectiveness data in larger populations once available.
Nearly 90% of drug candidates fail after entering clinical trials. This high failure rate at late stages in the drug development pipelines results in wasted time and resources that could have been utilized for more promising candidates. In a 2016 estimation from the Tufts Center for the Study of Drug Development, each approved drug has an estimated price tag of $2.87 Billion ($3.65 Billion in 2022 dollars). The main driving factor for this high cost is the failure rate for other products. Analyses have revealed that the top reasons for drug candidate failures between 2010 and 2017 from the largest pharmaceutical companies were:
Lack of clinical efficacy (40-50%)
Unmanageable toxicity (30%)
Poor drug properties (10-15%)
Lack of market/commercial strategy (10%)
Proper preclinical DMPK studies can address the second and third most frequent causes of attrition: unmanageable toxicity and poor drug properties. Combined, these account for 40-45% of all drug failures. Identifying these characteristics earlier in the drug development pipeline could cut costs for pharmaceutical companies and shorten the time to effective therapeutics for patients. The risk is even higher for smaller companies. Smaller biotech companies tend to pursue riskier drug candidates, which are critical for innovative therapies, but when combined with limited resources and experience for conducting proper DMPK studies, contribute to a high rate of attrition.
A variety of DMPK techniques can be used to assess the preclinical toxicology of drug candidates. Toxicity is temporal, with the potential for both acute and chronic effects. Proper in vivo preclinical toxicology studies can evaluate the acute effects of multiple dosing strategies as well as long-term effects such as carcinogenicity or impacts on reproductivity. Toxicology studies may evaluate any metabolites that might form or transcriptional changes that result from administration and may indicate toxicity based on previously characterized compounds. By using multiple species for toxicity studies, investigators can choose species that have similar metabolic profiles to humans, allowing for more relevant comparisons.
Poor drug properties such as absorption, distribution, metabolism, excretion (ADME), or pharmacokinetics account for 10-15% of drug attrition. Fifty years ago, these poor drug properties accounted for nearly 40% of candidate attrition. The progress of DMPK scientists in assessing these characteristics has come a long way in that time. These measurements can be obtained via in vitro assays with human-derived cells or tissues as well as in vivo animal studies. Two of the approaches currently used to predict human PK include allometric scaling and invitro-in vivo extrapolation. Allometric scaling is used to predict human PK by relating in vivo-derived parameters collected in animal models. In vitro-in vivo extrapolation is an estimate of metabolic clearance by measuring the clearance in human cells or tissue in vitro and then scaling up.
Twenty years ago, the focus of many DMPK studies for pharmaceuticals was primarily to characterize the kinetic properties of drug candidates for clinical trial design and regulatory registration. Since then, the field has shifted dramatically with new analyses including pharmacogenetics, pharmacogenomics, drug-drug interaction (DDI) predictions, and measurements of asymmetric organ or tissue exposure based on the organ-specific expression of drug transporters and metabolizing enzymes.
In 2020, the FDA released updated guidance regarding DDI evaluation for drug candidates. These guidelines outlined in vitro experimental conditions as well as details for model-based DDI prediction strategies. Preclinical DDI studies are used for the design of trials to inform participant recruitment as well as for generating label restrictions and guidance post-approval.
Pharmacogenomics is an emerging field of DMPK that evaluates the accumulative effect of genetic variants on drug responses and efficacy. Genetic polymorphisms in genes that encode metabolizing enzymes or transports can vastly affect the pharmacokinetic properties of a given drug as well as how patients respond to therapy. These biomarkers can affect dosing, and risks associated with the therapy, both important for labeling guidance and restrictions. They can also impact trial design and endpoints. The FDA pharmacogenetic biomarkers in drug labeling list contains FDA-approved drugs with known pharmacogenomic biomarkers that affect the therapeutic response.
Proper design and execution of DMPK studies can reduce drug candidate attrition, cut costs, and conserve resources for the most promising drug candidates. Smaller biotech companies often lack the resources to conduct robust DMPK studies, making it even more important to outsource these studies to a CRO with extensive experience and validated models. Because no individual model is a perfect comparison to humans, in vivo DMPK studies can be conducted in multiple models or species to compare ADMET properties across species. PharmaLegacy has an extensive set of preclinical animal models (> 600 validated models) to conduct DMPK studies from multiple species, including non-human primate models, with the capacity to run more than 200 concurrent animal studies. Scientists at PharmaLegacy also have years of experience performing DMPK/ADMET studies to help speeding up your drug discovery effort, minimizing toxicity liabilities, and ultimately reducing attrition rate in the clinic. PharmaLegacy can meet your company’s DMPK needs while remaining compliant with all applicable regulatory agencies.
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If you’ve been keeping up with the latest advancements in cancer research, you might come across the usage of humanized mouse models as a promising tool in cancer management and treatment.
This trend is driven by advances in the development of new rodent models which enabled scientists to further investigate and harness the power of immunotherapies in fighting tumors by replacing the mouse’s immune system with a functioning human one, hence the term “humanized”.
But first, what is a humanized mouse model?
A humanized mouse model is established by modifying specific genes that render it immunodeficient, lacking T, B, and NK cells, and replacing them with human functioning immune cells. This model serves as a preclinical tool in biomedical research by receiving transplantation of human tumor tissue known as “xenograft” derived from the patient’s cell line to explore cancer’s pathogenesis and evaluate different therapeutic effects.
Here, we highlight three different types of humanized mice models that are used in immuno-oncology studies:
Humanized (hu) CD34+ Mouse Models
Humanized (hu) PBMC Mouse Models
Knock-in Humanized Mouse Models
CD34+ humanized mouse models are preferably used in long-term oncology studies.
Due to their ability to stably ingraft with huCD34+ hematopoietic stem cells, and their capacity in producing multi-lineage human immune cells that are viable up to nine months post-production, severely immune-deficient mice are exposed to whole-body irradiation followed by the injection of human CD34+ cells, this process makes the models ready for tumor implantation.
PBMC humanized mouse models are cost-effective in short-term oncology studies.
Because of their rapid engraftment with human immune cells, humanized PBMC mouse models are used in studies that aim to evaluate compounds for T cell immune modulation. This is done post the intravenous engraftment of human peripheral blood monocyte cells (PBMCs) in severely immune-deficient mice before or following the implantation of the xenograft. This allows for quick evaluation of a novel immuno-therapeutic with human, specifically with a focus on T cell immunology. From study planning to start, PBMC models can be accomplished is just a few weeks. This makes the model well suited for rapid results. However, PBMC models are limited in duration by GVHD onset within 4 to 8 weeks. For longer duration models, CD34+ humanized models are a better choice.
Knock-in humanized mouse models offer a distinctive anti-tumor response in research.
By expressing chimeric proteins made of a humanized extracellular domain, knock-in mouse models with fully functioning immune systems are primarily used to evaluate the anti-tumor response of the immune checkpoint inhibitors related to human targets. First generation humanized mice broadly supported human T cell engraftment. Recent advances permitted the addition of human cytokines such as GM-CSF, IL-3 and/or IL-15 creating 2nd generation humanized mice models. Second, generation mice support an even more completed human immune system with increase in the engraftment of granulocytes, macrophages, NK and dendritic cells.
Regardless of 1st or 2nd generation model type, humanized mice models have the capacity of bearing both the human immune system and human tumors, but their successful engraftment of human immune cells depends strongly on the immunodeficiency of the recipient mice.
While ectomy procedures, radiotherapy, and chemotherapy remain the most effective methods for treating tumors, the five-year survival rate post-operation remains insufficient. The significant progress in onco-immunology enabled immunotherapy to attack a tumor by potentiating functioning lymphocytes instead of killing it directly which marked the forward leap in cancer treatment.
In addition, recent studies demonstrated that humanized mouse models opened new perspectives in immunotherapy by bearing both human immune cells and human tumors. This is translated through the model’s ability to be engrafted with tumors either in a form of cell-line-derived xenograft (CDX) or patient-derived xenograft (PDX), with the later method being extensively used in cancer research today. Even though CDXs consume less time, studies showed that in vitro culture, a step before engraftment may cause a substantial loss of features in primary tumors. PDXs on the other hand, are fragments of fresh human tumors engrafted directly onto the recipient mouse model which makes them challenging to establish and may lead to potential loss of their associated human stroma over time.
Using humanized mouse model as a preclinical application for cancer immunotherapies requires the reproduction of the tumor microenvironment (TME) in the hu-PDX mice model. TME comprises varying elements such as blood vessels, lymph vessels, stromal cells, immunocytes, fibroblasts, and the extracellular matrix (ECM). The establishment of this complex milieu is a dominant factor in oncobiology studies as it not only offers the environment for tumor development and metastasis but also aids in its diagnosis, prevention, and prognosis. Due to its major role in tumorigenesis and cancer development alongside the interplay between immunocytes and tumor cells post mice humanization and PDX implantation, TME can provide new strategies for future therapy.
Pairing humanized mice with an accomplished flow cytometry core is essential to understand the TME. Pharmalegacy (PL) is fortunately to have an excellent cytometry core with multiple cytometers supporting up to 16 color staining. If you need to study T cell exhaustion markers, or a specialized subset of DC1 cells, PL can design a FACS panel to track all your cell populations of interest.
Additionally, the usage of humanized mice model to engineer genetic-modified T cells was reported to be of beneficial value in cancer patients receiving adoptive cellular therapy (ACT) which denotes expanding immunocompetent cells in vitro followed by reinjecting them back to the patients. The procedure of ACT infusion is based on T cells engineered to express transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs), which aids in improving the affinity with tumor-associated antigens (TAAs).
Also, a humanized mouse model was helpful in immune checkpoint blockade therapy. Several signaling pathways and inhibitory receptors grouped under immune checkpoints like programmed cell death protein-1 (PD-1), cytotoxic T-lymphocytes-associated protein-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and T cell immunoglobulin-3 (TIM-3), all suppress excessively activated T cells preventing a self-tissue attack, hence eliminating the occurrence of autoimmune effects. The blockage of some immune checkpoints was marked as a unique setup in cancer treatment when PD-1 and CTLA-4 inhibitors won the Nobel Prize in physiology and medicine in 2018. The remarkable superiority of the humanized mice model in studying immune checkpoint inhibitors was established to test the effectiveness of individual clinical consultations for cancer patients.
First generation humanized mice have been highly predictive of human clinical success for check point therapies. Next generation of immune-oncology therapies will target processes beyond check point blockade. Future therapies will require a more complete TME that includes additional immune cell types commonly found in ‘cold’ tumors that do not respond to check point treatment. Second generation humanized mice promise to be the models of the future. Specifically, therapies that target NK cells, MDSC, and TAM can all benefit from 2nd generation humanized mice.
Current studies are undergoing tremendous efforts to ensure the reliable usage of humanized mouse models as a representative tool in preclinical immune-oncology studies by tackling its major setbacks. At PharmaLegacy, we understand that the need for humanized models is of utmost importance; This is why our team of experts are always hard at work to provide you with reliable and cost-effective models that fit your studies. Additionally, we manufacture our own humanized mice. This allows for customization to meet our client’s needs. We can transfect lentivirus expressing cytokines, screen donor CD34 stems cells, or additionally customize the model systems based on our customer’s needs.
