Rheumatoid arthritis (RA) is a chronic disease involving inflammation of the joints. While the etiology of RA remains unknown, evidence exists for a significant contribution of the major histocompatibility complex (MHC). Environmental factors are also indicated as playing an etiological role. Since it is impossible to define the mechanisms contributing to disease onset and progression in humans, mouse models have been used widely. While many animal models have been generated by immunization with self-proteins to induce arthritis, type II collagen (CII)-induced arthritis is one of the most commonly used models utilized to understand the immunopathology of RA. CII constitutes 80% to 90% of total collagen content of hyaline cartilage found in joints and is a genetically conserved sequestered protein. Immunization with heterologous CII with an adjuvant in mice leads to cellular and humoral responses to heterologous and autoreactive CII-specific responses and collagen-induced arthritis (CIA). Mice immunized with CII develop inflammatory arthritis that shares many similarities in clinical, serological, and radiological features with RA in humans. However, selecting an antigen for inducing arthritis and the mouse strain are important, as not all strains are susceptible to CIA. A critical difference between RA and CIA is that in mice that lack the expression of human MHC II, the development of CIA is linked to the H2A locus, which is the homologue of HLA-DQ, while most human studies have linked RA susceptibility with HLA-DR alleles. Mice expressing HLA-DQ and HLA-DR molecules have been used to understand the role of MHC genes in susceptibility to RA. The protocols for inducing CIA in the HLA expressing transgenic mice described in this article can be used to understand how the different HLA molecules confer susceptibility to RA. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Use of mouse strains and humanized mice for modeling rheumatoid arthritis Basic Protocol 2: Use of humanized mice for therapeutic protocols.
An estimated 40 million people are infected with human immunodeficiency virus type 1 (HIV-1) and there is no effective cure. Humanized mice are a useful model to study transmission, pathogenesis, prevention, and treatment of HIV-1, which only infects human cells. Here we provide detailed protocols to humanize immunodeficient mice, e.g., NSG or MISTRG6 strains, using human hematopoietic stem cells isolated from cord blood and a flow cytometry panel of antibodies to assess human immune cell reconstitution. Furthermore, we describe how to produce, concentrate, and titer HIV-1 in vitro, which can be used to challenge humanized mice either systemically or mucosally. Finally, we adapted a sensitive quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay with internal control to detect HIV-1 RNA in longitudinal plasma samples to measure viremia over time in the mice. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of CD34+ cells from human cord blood Basic Protocol 2: Injection of human CD34+ cells in immunodeficient mice Basic Protocol 3: Flow cytometry of peripheral blood mononuclear cells from mouse blood Basic Protocol 4: Production and titration of HIV-1 Basic Protocol 5: Systemic HIV-1 challenge of humanized mice Alternate Protocol: Mucosal HIV-1 challenge of humanized mice Basic Protocol 6: Single-copy HIV-1 RNA detection in mouse plasma Support Protocol 1: Retrovirus internal control for plasma virus isolation and RT-qPCR Support Protocol 2: Production of HIV-1 standard for RT-qPCR.
The oncogenic Epstein Barr virus (EBV) is an exclusively human pathogen with related lymphocryptoviruses (γ1-herpesviruses) only present in monkeys. Therefore, experimentation with EBV infection in a small animal model requires reconstitution or adoptive transfer of human lymphocyte populations, primarily EBV's main host cell, the human B cell. In this protocol we describe human immune system reconstitution after neonatal transfer of CD34+ hematopoietic progenitor cells in lymphodeplete immune compromised mouse strains, using NOD-scid γc -/- (NSG) mice as a commonly used example. Such reconstituted humanized mice allow intraperitoneal and intranasal infection with EBV and we describe injection of 105 infectious particles of the prototypic EBV strain B95-8 that can be produced from a recombinant bacmid (p2089) in HEK293 cells. Infection with this dose mimics symptomatic primary EBV infection, infectious mononucleosis (IM), with high viral loads plateauing 4 weeks after infection, with CD8+ T-cell lymphocytosis at week 5 and 6 after infection. This IM-like primary EBV infection in humanized mice leads to clonal EBV-induced B-cell lymphoproliferations that resemble large B-cell lymphomas with the latency III program of EBV infection. We describe Basic Protocols to monitor viral loads, immunohistochemistry of infected tissues and spectral flow cytometry to characterize protective T-cell expansion. The described mouse model has been used by us and others to characterize mutant EBV infections, cell-mediated immune control of EBV, modulation of EBV pathogenesis by co-infections with human immunodeficiency virus (HIV) and Kaposi sarcoma associated herpesvirus (KSHV), as well as passive transfer of vaccine elicited antibodies to test their protection against EBV infection. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CD34+ human hematopoietic progenitor cell isolation and characterization Basic Protocol 2: Human immune system reconstitution and characterization Basic Protocol 3: Recombinant EBV production and humanized mouse infection Basic Protocol 4: Viral load quantification, lymphoma assessment, and immunohistochemistry after EBV infection of humanized mice Basic Protocol 5: Human T-cell response analysis after EBV infection of humanized mice.
Saliva plays a central role in maintaining oral homeostasis by supporting tooth integrity, providing lubrication, and functioning as an antimicrobial wash. It also serves as a transport medium, carrying byproducts and signaling metabolites across oral niches and through the gastrointestinal tract. Because of its biological relevance and ease of collection, saliva is increasingly used as a noninvasive biospecimen for measuring cortisol, cytokines, and metabolites. However, the validity and reliability of saliva as an indicator of local and systemic biomarkers remain under investigation across diverse populations and research applications. Specific patient populations (e.g., individuals with alcohol use disorder) are particularly vulnerable to oral health problems, periodontal disease, and high rates of nicotine use. In addition to behavioral factors (e.g., food, drink, toothbrushing, and mouthwash), patient-specific variables can introduce contaminants such as nicotine and blood into saliva, potentially compromising the accurate measurement of analytes of interest. Protocols that account for possible contaminants are essential to ensure rigorous and reproducible biomarker research. Assessing factors such as pH, flow rate, and visible discoloration helps reduce limitations in analysis and improves interpretation in studies that include heterogeneous populations and health behaviors. Yet, the literature provides limited guidance on standardized methods for saliva collection, processing, and measurement of patient-specific confounders alongside analytes of interest. This protocol addresses these gaps by presenting detailed methodologies for saliva collection and processing, assessment of quantitative and qualitative salivary properties, and quantification of patient-specific modifiers. These approaches support reproducible diagnostics and have applications in populations with high rates of smoking, periodontal disease, and alcohol use. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Saliva collection by the passive drool method Basic Protocol 2: Processing, storage, and characterization of saliva (visual assessment scale, pH, and flow rate) Basic Protocol 3: Quantification of cotinine in salivary supernatant Basic Protocol 4: Quantification of transferrin in salivary supernatant.
Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by tense blisters and itchy erythema, predominantly affecting elderly individuals. The pathogenic autoantibodies mainly target collagen XVII (COL17), leading to subepidermal blister formation and infiltrations of immune cells, including eosinophils and neutrophils. Although systemic oral corticosteroids remain the mainstay of treatment, their use in elderly patients is often limited by serious complications and adverse effects, highlighting the unmet need for novel therapeutic targets. Several mouse models for BP have been reported; however, the inconsistency of disease induction has hindered therapeutic investigations. The active BP mouse model has emerged as a reliable system that recapitulates key disease features, making it valuable for both preclinical therapeutic studies and elucidation of BP pathophysiology. Furthermore, autoantigen-humanized mouse models, including the active BP model and the neonatal passive IgG transfer model, provide significant advantages for the development of antigen-specific therapies. Here, we describe detailed materials and methods for mouse models for BP using the COL17-humanized mouse, including the active BP mouse model and the neonatal passive IgG transfer model. Protocol modifications may be necessary when using different donor mice and antibodies. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Active BP mouse model Basic Protocol 2: Passive IgG transfer mouse model using neonatal mice.
