Immunology & Inflammation
Animal Disease Models
Models of Rheumatoid Arthritis
Collagen-induced Arthritis (CIA) in mice shares both immunological and pathological features with human RA, and has been widely used for testing RA therapeutics. To initiate the arthritis, a systemic autoimmune response is induced in mice by intradermal injections of type II collagen emulsified in Complete Freund’s Adjuvant (CFA). The clinical score, incidence of disease, and paw swelling, are determined to assess the severity of the resulting arthritis. Pain-related assays including, the Hargreave’s assay and the Von Frey stimulation, can be applied to assess arthritic pain. The majority of prior studies have shown that among mouse strains, DBA1/LacJ mice bearing the H-2q haplotype are the most sensitive to CIA, and C57BL/6J mice carrying H-2b are resistant. By using a modified immunization procedure with an increased concentration of CFA and injecting collagen/CFA at several sites, we have successfully induced CIA in C57BL/6J mice, a strain commonly used in gene-targeting studies.
Antigen-Induced Arthritis (AIA). This model utilizes methylated bovine serum albumin (mBSA) as the trigger of arthritis. AIA can be induced in most strains of mice by intra-articular injection of mBSA. Prior systemic immunization with mBSA in CFA is required. T lymphocytes are the major, if not the only, determinant of the disease state in this model. B cell activation and anti-mBSA antibodies do not contribute to the disease pathogenesis.
Experimental Autoimmune Encephalitis (EAE)
EAE is a commonly used murine model for human multiple sclerosis (MS). EAE shares both immunological and pathological features with MS. This autoimmune disease is thought to be triggered by auto-reactive myelin-specific CD4+ T cells. The disease is characterized by perivascular infiltration of CD4+ T- and mononuclear cells, followed by primary demyelination of axonal tracks in the central nervous system (CNS), leading to progressive hind-limb paralysis. The severity of the disease correlates with the levels of cytokines in the CNS, and IL-17. These cytokines are produced by encephalogenica, TNF-gmainly, IFN- T-cells, activated CNS-resident mononuclear cells (microglia and astrocytes), and infiltrating antigen presenting cells (APCs). We offer two mouse model of EAE: the chronic EAE model that is induced in C57BL/6 female mice by active priming with a Myelin Oligodendrocyte Glycoprotein peptide (MOG35-55) and the relapsing-remitting EAE model that is induced in SJL female mice by active priming with a Proteolipid Protein peptide (PLP139-151). In both models, we monitor the clinical disease score and perform flow cytometric analysis of spinal cord samples during different clinical stages of the disease. In addition, we evaluate cytokine levels in the CNS and the serum, and antigen-specific CD4+T- and B- cell responses.
Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a chronic, relapsing and remitting inflammatory condition that can be subdivided into Crohn’s disease and Ulcerative Colitis based on the specific clinical manifestations. Experimental IBD can be initiated in mice by rectal instillation of trinitrobenzene sulfonic acid (TNBS) or by adding dextran sodium sulfate (DSS) to the drinking water. TNBS-induced Colitis, an animal model of Crohn’s disease, results from the rapid induction of an IL-12-mediated, Th1-driven response that precludes response. Emerging evidence suggestsbdevelopment of a counter-regulatory TGF- that this response is genetically controlled, most probably by genes that regulate the magnitude of the initial IL-12 induction by substances in the mucosal microflora. Oral administration of DSS in drinking water causes a reproducible, acute, colitis, followed by a slow regeneration of the colonic epithelium with a concomitant chronic inflammation showing high mucosal IFN-γ and IL-4 levels. This chronic DSS-induced colitis shares morphological and pathophysiological features with Ulcerative Colitis, and is characterized by focal epithelial regeneration and a Th1, as well as a Th2, cytokine profile. To evaluate the progression and severity of the experimentally induced colitis, the mice are monitored for the clinical signs of colitis (wasting syndrome, diarrhea, bloody stool, rectal prolapse, etc). At the end of the experiment, the colons are examined macroscopically for signs of inflammation and the colon cytokine levels are determined.
Allergen-induced Pulmonary Inflammation
In vivo experimental models of airway response to allergen sensitization and challenge have contributed significantly to the understanding of the pathogenesis of human asthma. In these models, sensitized mice that are challenged acutely with inhaled ovalbumin (OVA) develop allergic airway inflammation, which is characterized by OVA-specific IgE production, airway eosinophilia, increased pulmonary B and T lymphocyte populations, and airway hyper-reactivity. Th2 type cells play a central role in this model by initiating and perpetuating the inflammatory response. The Th2 cells secrete several cytokines. One of them, interleukin 4 (IL-4), is essential for OVA-specific antibody isotype switching to IgE and for the maintenance of the immune response. Another Th2 cytokine, IL-5, plays a key role in the activation and recruitment of eosinophils to the airways. In this model, the animals receive repeated, systemic, sensitization with ovalbumin (OVA) followed by subsequent airway challenge with the same antigen. Cytokines and anti-OVA IgE levels in the lungs and/or broncho-alveolar lavage fluids are determined. Inflammatory cell influx to the lungs is also assessed.
Contact Hypersensitivity Model
Contact hypersensitivity is an in vivo assay for evaluating the antigen-presenting cell (APC) component of the cell-mediated immune response. Repetitive exposure of the skin to exogenous haptens, such as 2,4-dinitro-1-fluorobenzene (DNFB), results in a delayed type hypersensitivity (DTH) reaction that consists of the initial sensitization phase, and the elicitation phase. In this model, the Langerhans cells are the critical antigen-presenting cells. These cells present the antigen to CD4+ T lymphocytes, which results in the expansion of the antigen-specific CD4+ T-cell population. Repeated exposure to the antigen triggers the elicitation phase of the reaction. During the elicitation phase, the antigen-specific CD4+T-cells secrete lymphokines and recruit eosinophils and other inflammatory cells to the site of the antigen application. This results in local skin inflammation that, among other features, is characterized by increased thickness of the skin. In our protocol, the sensitization phase is initiated by the exposure of the shaved abdominal skin of the experimental mice to DNBF. Five days later (the time required for expansion of the antigen-specific CD4+ T-cells) the baseline ear thickness is measured and the elicitation phase is initiated by epicutaneous application of the hapten solution to the ears of the sensitized mice. Ear thickness is measured again, 24 hrs after the application of hapten, and the ear swelling is determined. In addition, histological evaluation of the inflamed skin may be performed.
Lipopolysaccharide (LPS)-Induced Lethal Endotoxemia (Sepsis)
Endotoxic shock (sepsis) is a potentially lethal clinical condition that develops as a result of a dysregulated host response to bacterial infection. In contrast to a properly regulated immune response to LPS that is beneficial for an organism and is critical for elimination of infection, in the endotoxic shock the inflammatory response contributes to pathological processes resulting in cell damage, tissue necrosis, and vascular disruption. At the whole organism level, unabated progression of the condition invariably results in respiratory and renal failure and an ever increasing oxygen debt, which eventually culminates in multiple organ failure and often death. This assay is designed to assess the effects of genetic modifications or potential pharmacological compounds on mouse resistance to endotoxic shock. Endotoxic shock is induced in mice by a lethal dose of LPS and the survival of the mice is monitored.
Lipopolysaccharide (LPS)-Induced Pulmonary Inflammation
Non-allergic pulmonary inflammation is induced by introduction of LPS into the airways of the mouse. LPS recruits neutrophils and monocytes to the airway lumen followed by their activation and release of various mediators of inflammation including IL-1, IL-6, IL-8, TNFa and platelet-activating factor. The extended activation of the cells and the release of inflammatory mediators can lead to degradation of the alveolar matrix. Alveolar matrix degradation is associated with a variety of pulmonary diseases, including emphysema, idiopathic pulmonary fibrosis, adult respiratory distress syndrome, chronic bronchitis and cystic fibrosis. A reduction in neutrophil and monocyte recruitment to the lungs in these patients might have therapeutic value. To induce pulmonary inflammation in this model, the mice are administered LPS via intra-tracheal instillation or by inhalation. At the end of the experiment, broncho-alveolar lavage is performed and the cellular composition of the lavage fluids and lung infiltrates, IL-8,ais determined by FACS analysis. The levels of cytokines such as TNF and IL-17 in the lungs and peripheral blood may be also determined.bIL-1
Other in vivo Bioassays
Lipopolysaccharide (LPS)-Induced Non-lethal Endotoxemia
Lipopolysaccharides (LPS), or endotoxins, are expressed on the outer membrane of the cell wall of Gram-negative bacteria. LPS-induced non-lethal endotoxemia is an experimental model of systemic inflammation that is mediated by variety of cellular inflammatory responses, including the up-regulation of expression of different pro-inflammatory cytokines, such as interleukins IL-1b, ), and other pro-inflammatory proteins,a (TNF-aIL-6 and tumor necrosis factor- such as the enzyme cyclooxygenase-2 (COX-2) and the inducible nitric oxide synthase (iNOS). To initiate the inflammatory reaction, each mouse is administered a non-lethal dose of LPS by intraperitoneal injection. The blood from the experimental mice is collected at several time points after the LPS administration. Serum levels of pro-inflammatory and anti-inflammatory cytokines/ and chemokines are determined.
Methylated Bovine Serum Albumin (mBSA)-Induced Delayed Type Hypersensitivity
This assay is another way to assess cell-mediated immune response. It is a Th-1 type model that is employed for evaluating T-cell component of the cell-mediated immune response. The sensitization phase is initiated by immunization of experimental mice with a specific protein antigen (mBSA), which is delivered intradermally as emulsion with complete Freund’s adjuvant (CFA). The elicitation phase of the reaction, which is triggered by a second intradermal injection of mBSA into the hind paws or ears of the immunized animals (recall), results in the a local inflammation that is measured 24 hrs after the recall and expressed as footpad or ear swelling.
In Vivo B-cell Response by Antibody Production
B cells produce antibodies, which form a crucial defense mechanism against antigens in the extracellular space. Antibodies can eliminate pathogens by several mechanisms including complement activation and direct lysis of bacteria, neutralization of viruses and toxins to prevent their entry into host cells, and opsonization.
To assess the function of B cells in mice, we offer the in vivo antibody production assay in response to immunization with an antigen. The mice are immunized with either chicken egg ovalbumin (OVA) or Keyhole Limpet Hemocyanin (KLH). Then the levels of IgM, IgG1, IgG2a, IgG2b and IgE antibodies are detected by ELISA in the serum of these mice. The antibody levels may be assayed after primary and/or secondary stimulation with the antigens.
Thioglycollate-Induced Peritonitis
Recruitment of leukocyte from the circulation, and their subsequent influx into the sites of inflammation is critical for host defense and wound healing. This is a multistep process, which is regulated, in part, by the selectin family of adhesion molecules and chemokines that are upregulated during inflammation. Thioglycollate-Induced Peritonitis is designed to mimic an acute inflammatory response in the peritoneum. An intra-peritoneal injection of thioglycollate generates local inflammation and initiates the migration of inflammatory cells, particularly neutrophils and monocytes, to the site of inflammation. The thioglycollate-induced peritoneal inflammation can be used to assess a potential effect of a genetic manipulation or a compound on the ability of neutrophils and monocytes to migrate to the site of inflammation.
Ex vivo Bioassays
Immunophenotyping by Multi-color FACS Analysis
FACS, Fluorescence-based flow cytometry analysis of lymphocytes, is used to ascertain subpopulation frequencies and relative amounts of cellular antigenic determinants or receptor complexes in the cells of the immune system. The immune system consists of various unique cell types with diverse roles in immunity. However, in certain disease states only one or a few of these cell types may be affected and.FACS analysis is the method of choice for identification of those affected cell types. In our program, we use FACS to evaluate the cellular composition of various compartments of the immune system, such as thymus, Payer’s patches, spleen, lymph nodes and peripheral blood in genetically modified animals and/or in response to pharmacological treatment. In addition, inflammatory infiltrates to various tissues in multiple disease models can be evaluated.
Assessment of Proliferative Responses of T and B cells (in vitro)
Proliferation of T and B cells is an important element of immune system response against bacterial and viral infections, tumors and other foreign antigens. These lymphocytes play a crucial role in the elimination or containment of microbial and viral infections, malignant transformation, parasites, etc. We use anti-CD3/ anti-CD28 and LPS stimulation for the assessment of T and B cell proliferation in vitro, respectively. The proliferation is assessed by measuring incorporation of 5-bromo-2-deoxyuridine (BrdU, a thymidine analog) into the DNA of different populations of T and B cells using Flow Cytometry.
Serum and Tissue Cytokine Profiling
Cytokines, a diverse group of polypeptides that are generally associated with inflammation, immune activation, and cell differentiation or death, include Interleukins (IL), Interferons (IFN), tumor necrosis factors (TNF), Chemokines and growth factors. In the immune system, cytokines are pivotal in regulating the magnitude of the immune response. In addition, such cytokines, as IL-12 and IL-4, determine whether the immune response is predominantly cell mediated or humorally mediated. In our platform various cytokine levels are measured in response to a physiological challenge in the serum or tissue homogenates of genetically modified and/or pharmacologically treated animals.
To assess an effect of genetic modification on T Cell Receptor (TCR) signal transduction and cytokine production, a primary T cell culture is stimulated with anti-CD3 and anti-CD28 antibodies, and the levels of IL-2, INF-g and other cytokines are detected by ELISA.
* In development
