A low-dose, high-resolution CT technique is detailed for longitudinal visualization and quantification of lung pathology in mouse models of respiratory fungal infections, specifically in models of aspergillosis and cryptococcosis.

Fungal infections, specifically those caused by Aspergillus fumigatus and Cryptococcus neoformans, are frequent and life-threatening in immunocompromised patients. Menadione in vitro Acute invasive pulmonary aspergillosis (IPA) and meningeal cryptococcosis are severe forms of the condition that significantly affect patients, resulting in high mortality rates, despite current therapeutic interventions. Further investigation into these fungal infections is critically needed, given the substantial unknowns that still exist. This research should extend beyond clinical observations to include controlled preclinical experiments, in order to deepen our comprehension of virulence factors, host-pathogen interactions, infection progression, and effective treatment strategies. To delve deeper into some of these needs, preclinical animal models stand as vital instruments. However, the quantification of disease severity and fungal load in mouse models of infection frequently suffers from the use of less sensitive, single-time, invasive, and variable methodologies, such as colony-forming unit determination. Bioluminescence imaging (BLI), performed in vivo, can alleviate these problems. Individual animal disease development, from the onset of infection to potential dissemination to various organs, is tracked by BLI, a noninvasive tool offering longitudinal, dynamic, visual, and quantitative data on fungal burden. From mouse infection to BLI data collection and quantification, a comprehensive experimental protocol is outlined, enabling non-invasive, longitudinal tracking of fungal burden and dissemination. This protocol can be readily used by researchers for preclinical studies into IPA and cryptococcal disease pathophysiology and treatment

In the quest to comprehend the intricacies of fungal infection pathogenesis and to develop innovative therapeutic strategies, animal models have been instrumental. Despite its uncommon occurrence, mucormycosis carries a significant risk of fatality or debilitating illness. Mucormycoses arise from diverse fungal species, each potentially entering the body through unique infection pathways, while affecting patients with varying underlying diseases and risk profiles. Subsequently, different types of immunosuppression and infection pathways are employed in clinically pertinent animal models. Moreover, it gives step-by-step instructions for intranasal administration, aimed at creating pulmonary infections. At last, the discussion turns to clinical parameters capable of informing the development of scoring systems and the determination of humane endpoints in mice.

Pneumocystis jirovecii pneumonia is a prevalent complication for immunocompromised individuals. Understanding host-pathogen interactions and drug susceptibility testing are hampered by the presence of the diverse species within Pneumocystis spp. The in vitro environment is not suitable for their viability. The absence of a continuous culture method for this organism significantly curtails the identification of potential new drug targets. The constrained nature of the system has made mouse models of Pneumocystis pneumonia incredibly valuable to researchers. Menadione in vitro This chapter presents an overview of chosen methodologies employed in murine infection models, encompassing in vivo propagation of Pneumocystis murina, transmission routes, available genetic mouse models, a P. murina life cycle-specific model, a murine model of PCP immune reconstitution inflammatory syndrome (IRIS), and the associated experimental parameters.

Dematiaceous fungal infections, particularly phaeohyphomycosis, are increasingly recognized as a global health concern, presenting diverse clinical manifestations. Phaeo-hyphomycosis, mimicking dematiaceous fungal infections in humans, finds a valuable investigative tool in the mouse model. Significant phenotypic variations were detected in a mouse model of subcutaneous phaeohyphomycosis developed in our laboratory, contrasting Card9 knockout and wild-type mice. This pattern corresponds to the heightened susceptibility seen in CARD9-deficient human cases. The mouse model of subcutaneous phaeohyphomycosis and accompanying experiments are detailed in this work. We expect this chapter to be beneficial to the study of phaeohyphomycosis, thereby prompting the development of more effective diagnostic and therapeutic methods.

In the southwestern United States, Mexico, and selected areas of Central and South America, coccidioidomycosis, a fungal disease, is a result of infection by the dimorphic pathogens Coccidioides posadasii and Coccidioides immitis. Pathology and immunology of disease studies predominantly utilize the mouse as a model organism. Due to their remarkable susceptibility to Coccidioides spp., mice pose a challenge in studying the host's adaptive immune responses that are critical for coccidioidomycosis control. This report outlines the methodology for infecting mice to produce a model of asymptomatic infection accompanied by controlled, chronic granulomas, and a slow, ultimately fatal disease progression, with kinetics akin to human disease.

Experimental rodent models serve as a convenient tool for exploring the complex interplay of host and fungus during fungal illnesses. Fonsecaea sp., a causative agent of chromoblastomycosis, presents a unique challenge, as the preferred animal models typically exhibit spontaneous cures, leaving a notable absence of models capable of replicating the prolonged human chronic disease. Using a subcutaneous route, this chapter details a rat and mouse model designed for investigation of acute and chronic lesions. The study meticulously tracks lesion similarities to human conditions, including fungal burden and lymphocytic response.

The human gastrointestinal (GI) tract, a microcosm of life, is home to trillions of commensal organisms. Modifications within the host's physiology and/or the microenvironment enable some of these microbes to manifest as pathogens. One such organism is Candida albicans, which generally resides peacefully in the gastrointestinal tract as a commensal, yet has the capacity to cause severe infections. Exposure to antibiotics, neutropenia, and abdominal surgeries are associated with a heightened probability of Candida albicans infections in the gastrointestinal system. Determining the pathways by which commensal organisms evolve into harmful pathogens is a significant research priority. Mouse models of gastrointestinal fungal colonization offer a vital framework for examining the pathways that dictate the change in Candida albicans from a benign commensal to a harmful pathogen. This chapter explores a groundbreaking approach to the consistent, long-term colonization of the murine gastrointestinal system by the Candida albicans fungus.

Immunocompromised individuals are at risk for invasive fungal infections that can impact the brain and central nervous system (CNS), potentially leading to the fatal condition of meningitis. New technological capabilities have allowed for a transition in research from studying the brain's inner tissue to understanding the immune functions of the meninges, the protective lining enveloping the brain and spinal cord. Advanced microscopy has allowed researchers to visualize, for the first time, the anatomy of the meninges, along with the cellular components that drive meningeal inflammation. We present, in this chapter, the method of creating meningeal tissue mounts for confocal microscopy analysis.

For the long-term control and elimination of several fungal infections, notably those originating from Cryptococcus species, CD4 T-cells are essential in humans. To effectively address the complex issues surrounding fungal infection pathogenesis, it is imperative to delve into the mechanisms of protective T-cell immunity, providing essential mechanistic understanding. This protocol outlines a procedure for the in-vivo assessment of fungal-specific CD4 T-cell responses by utilizing the adoptive transfer of genetically engineered fungal-specific T-cell receptor (TCR) CD4 T-cells. This protocol, while utilizing a TCR transgenic model responsive to Cryptococcus neoformans peptides, holds adaptable potential for other fungal infection research settings.

Fatal meningoencephalitis, a frequent outcome of infection by the opportunistic fungal pathogen Cryptococcus neoformans, often affects patients with weakened immune responses. This intracellular microbe, a fungus, avoids the host's immune system, resulting in a latent infection (latent C. neoformans infection, or LCNI), and cryptococcal disease develops upon reactivation when the host's immunity is compromised. The intricate pathophysiology of LCNI remains elusive, hindered by the scarcity of mouse models. The established standards for the LCNI process and its reactivation are explained in this document.

Cryptococcal meningoencephalitis (CM), a condition stemming from the fungal pathogen Cryptococcus neoformans species complex, can result in high mortality or significant neurological complications in surviving patients. These complications are often associated with extreme inflammation in the central nervous system (CNS), particularly among those affected by immune reconstitution inflammatory syndrome (IRIS) or post-infectious immune response syndrome (PIIRS). Menadione in vitro Human research methods to establish causal relationships in a specific pathogenic immune pathway during central nervous system (CNS) conditions are restricted; in contrast, studies employing mouse models allow detailed analysis of possible mechanistic connections within the CNS's immunologic network. These models are especially beneficial for differentiating pathways primarily associated with immunopathology from those necessary for fungal defense. This protocol describes methods for the induction of a robust, physiologically relevant murine model of *C. neoformans* CNS infection; this model reproduces many aspects of human cryptococcal disease immunopathology, and subsequent detailed immunological analysis is performed. With the integration of gene knockout mice, antibody blockade, cell adoptive transfer, and powerful high-throughput techniques like single-cell RNA sequencing, studies employing this model will provide fresh perspectives into the cellular and molecular mechanisms underlying cryptococcal central nervous system diseases, thus encouraging the development of more efficacious therapeutic strategies.