Host Resistance Assays.

The goal of immunotoxicity testing is to obtain data useful for immunotoxicity safety assessment. Guidance in the performance of immunotoxicity safety evaluations is provided in documents from the US EPA for chemicals and the ICH S8 document for pharmaceuticals. The ICH S8 document outlines a tiered approach that includes (1) standard toxicity studies with associated hematology, immune system organ weights, and histopathology data; (2) functional assays, such as cytotoxic T lymphocyte (CTL) assays, natural killer (NK) cell assays, respiratory burst, phagocytosis, and T-cell-dependent antibody response (TDAR) assays; and (3) host resistance assays. Host resistance assays are considered the gold standard in immunotoxicity testing and provide a critical overview of the extent to which innate, adaptive, and homeostatic regulatory immune functions are integrated to protect the host. Both comprehensive and targeted host resistance assays are available, each with distinct advantages. This chapter serves to provide a general overview of the various assays that may be used, as well as a summary of procedures.

Evaluation of Cell-Mediated Immune Function Using the Cytotoxic T-Lymphocyte Assay.

Evaluation of cell-mediated immunity (CMI) is a significant component in any assessment designed to predict the full range of potential immunotoxic risk underlying health risks. Among measures of CMI, the cytotoxic T-lymphocyte (CTL) response is recognized as perhaps the most relevant functional measure that reflects cell-mediated acquired immune defense against viral infections and cancer. The CTL response against T-dependent antigens requires the cooperation of at least three different major categories of immune cells. These include professional antigen-presenting cells (e.g., dendritic cells), CD4+ T helper lymphocytes, and CD8+ T effector lymphocytes. It is also among the few functional responses dependent on and, hence, capable of evaluating effective antigen presentation via both class I and class II molecules of the major histocompatibility complex (MHC). For this reason, the CTL assay is an excellent candidate for evaluation of potential immunotoxicity. This chapter provides an example of a mouse CTL assay against influenza virus that has been utilized for this purpose.

Metabolic Syndrome and Associated Diseases: From the Bench to the Clinic.

Metabolic Syndrome and Associated Diseases: From the Bench to the Clinic, a Society of Toxicology Contemporary Concepts in Toxicology (CCT) workshop was held on March 11, 2017. The meeting was convened to raise awareness of metabolic syndrome and its associated diseases and serve as a melting pot with scientists of multiple disciplines (eg, toxicologists, clinicians, regulators) so as to spur research and understanding of this condition. The criteria for metabolic syndrome include obesity, dyslipidemia (low high-density lipoprotein and/or elevated triglycerides), elevated blood pressure, and alterations in glucose metabolism. It can lead to a greater potential of type 2 diabetes, lipid disorders, cardiovascular disease, hepatic steatosis, and other circulatory disorders. Although there are no approved drugs specifically for this syndrome, many drugs target diseases associated with this syndrome thus potentially increasing the likelihood of drug-drug interactions. There is currently significant research focusing on understanding the key pathways that control metabolism, which would be likely targets of risk factors (eg, exposure to xenobiotics, genetics) and lifestyle factors (eg, microbiome, nutrition, and exercise) that contribute to metabolic syndrome. Understanding these pathways could also lead to the development of pharmaceutical interventions. As individuals with metabolic syndrome have signs similar to that of toxic responses (eg, oxidative stress and inflammation) and organ dysfunction, these alterations should be taken into account in drug development. With the increasing frequency of metabolic syndrome in the general population, the idea of a "normal" individual may need to be redefined. This paper reports on the substance and outcomes of this workshop.

Evaluation of 4-methylcyclohexanemethanol (MCHM) in a combined irritancy and Local Lymph Node Assay (LLNA) in mice.

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 µL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact.

Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.

Influenza vaccine response in adults exposed to perfluorooctanoate and perfluorooctanesulfonate.

Supported by several epidemiological studies and a large number of animal studies, certain polyfluorinated alkyl acids are believed to be immunotoxic, affecting particularly humoral immunity. Our aim was to investigate the relationship between the antibody response following vaccination with an inactivated trivalent influenza vaccine and circulating levels of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS). The study population consisted of 411 adults living in the mid-Ohio region of Ohio and West Virginia where public drinking water had been inadvertently contaminated with PFOA. They participated in a larger cross-sectional study in 2005/2006 and were followed up in 2010, by which time serum levels of PFOA had been substantially reduced but were still well above those found in the general population. Hemagglutination inhibition tests were conducted on serum samples collected preinfluenza vaccination and 21 ± 3 days postvaccination in 2010. Serum samples were also analyzed for PFOA and PFOS concentrations (median: 31.5 and 9.2 ng/ml, respectively). Questionnaires were conducted regarding the occurrence and frequency of recent (during the last 12 months) respiratory infections. Our findings indicated that elevated PFOA serum concentrations are associated with reduced antibody titer rise, particularly to A/H3N2 influenza virus, and an increased risk of not attaining the antibody threshold considered to offer long-term protection. Although the direct relationship between weakened antibody response and clinical risk of influenza is not clear, we did not find evidence for an association between self-reported colds or influenza and PFOA levels nor between PFOS serum concentrations and any of the endpoints examined.

Dimethylfumarate: potency prediction and clinical experience.

BACKGROUND: Dimethylfumarate (DMF) was the cause of a major outbreak of allergic contact dermatitis as a consequence of its use as an antifungal agent in leather products, particularly in furniture, with what became known as 'toxic sofa dermatitis'. OBJECTIVES: To determine whether the frequency and severity of reactions to DMF arose as a function of its intrinsic potency and/or the nature and extent of exposure. METHODS: The intrinsic potency of DMF was measured with the standard local lymph node assay (LLNA), with determination of an EC3 value, which is the threshold in the LLNA and serves as an indicator of relative skin-sensitizing potency in humans. RESULTS: The EC3 value for DMF was 0.35% when tested in dimethylformamide as a vehicle, indicating that DMF is a strong, but not an extreme, skin sensitizer in this mouse model. CONCLUSIONS: DMF appears to have a sensitizing potency in the mouse that is very similar to that of formaldehyde, which is also a strong human skin sensitizer. However, the frequency and intensity of allergic contact dermatitis reactions to DMF suggest that it was the prolonged, repeated and occlusive exposure to this chemical over large skin areas, combined with the strong sensitizing potency, that generated the 'perfect storm' conditions that caused the DMF epidemic.

Two-component polyethylene glycol surgical sealant influence on intraperitoneal infection in a refined rodent model.

OBJECTIVE: This study determined the influence of a 2-component polyethylene glycol surgical sealant (Coseal) as an adhesion prevention device on sepsis-related mortality and/or systemic bacterial translocation to the spleen. STUDY DESIGN: A bacterial inoculum and telemetry probe were implanted in 50 treated and 49 untreated rats. Telemetry probes monitored core-body temperature to determine time of death. Spleens were collected on day 3 for quantitative bacteriology of Escherichia coli and Bacteroides fragilis. RESULTS: Median survival time and mortality of treated rats (37.0 hours, 54.0%) were noninferior to untreated rats (47.5 hours, 55.1%). Median E coli titers in treated rats (2.24 log colony forming units/spleen) were significantly less than untreated rats (4.32 log colony forming units/spleen). B fragilis titers were not different. CONCLUSION: This study demonstrates intraperitoneal administration of a 2-component polyethylene glycol surgical sealant as an adhesion prevention device does not alter time to death or sepsis-related mortality and/or systemic bacterial translocation to the spleen.

Host resistance assays including bacterial challenge models.

Immunotoxicity testing is used to provide safety assessment with the major objective being the avoidance of unacceptable risk of infectious or neoplastic disease. To this end, immunotoxicity testing has employed a variety of host resistance challenge models for measuring both host resistance to disease as well as immune function. This chapter provides an overview of those viral, bacterial, fungal, and parasitic host resistance models that are most commonly used in safety assessment. It also describes in more detail the bacterial challenge models that are employed to address specific host resistance and immune function issues.

The cytotoxic T lymphocyte assay for evaluating cell-mediated immune function.

Evaluation of cell-mediated immunity (CMI) is a significant component in any assessment designed to predict the full range of potential immunotoxic risk underlying health risks. Among measures of CMI, the cytotoxic T Lymphocyte (CTL) response is recognized as perhaps the most relevant functional measure that reflects cell-mediated acquired immune defense against viral infections and cancer. The CTL response against T-dependent antigens requires the cooperation of at least three different major categories of immune cells. These include professional antigen presenting cells (e.g., dendritic cells), CD4(+) T helper lymphocytes, and CD8(+) T effector lymphocytes. It is also among the few functional responses dependent on and, hence, capable of evaluating effective antigen presentation via both class I and class II molecules of the major histocompatibility complex (MHC). For this reason the CTL assay is an excellent candidate for evaluation of potential immunotoxicity. This chapter provides an example of a mouse CTL assay against influenza virus that has been utilized for this purpose.

Testing human biologicals in animal host resistance models.

The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment. Host resistance assays are the best measure of a toxicant's effect on the overall ability to mount an effective immune response and protect the host from infectious disease. An outline is presented for immunotoxicological evaluation using host resistance assays. The influenza virus host resistance model is useful to evaluate the overall health of the immune system and is one of the most thoroughly characterized host resistance models. Viral clearance requires all aspects of the immune system to work together and is the ultimate measure of the health of the immune system in this model. Mechanistic immune functions may be included while measuring viral clearance and include: cytokines, macrophage activity, natural killer (NK) cell activity, cytotoxic T-lymphocyte (CTL) activity, and influenza-specific IgM and IgG. Measurement of these immunological functions provides an evaluation of innate immunity (macrophage or NK activity), an evaluation of cell-mediated immunity (CMI) (CTL activity), and an evaluation of humoral-mediated immunity (HMI) (influenza-specific IgM or IgG). Measurement of influenza-specific IgM or IgG also provides a measurement of T-dependent antibody response (TDAR) since influenza is a T-dependent antigen. There are several targeted host resistance models that may be used to answer specific questions. Should a defect in neutrophil and/or macrophage function be suspected, Streptococcus pneumoniae, Pseudomonas aeruginosa, or Listeria monocytogenes host resistance models are useful. Anti-inflammatory pharmaceuticals or therapeutics for rheumatoid arthritis or Crohn's disease that target TNFalpha may also be evaluated for immunotoxicity using the S. pneumoniae intranasal host resistance assay. Marginal zone B (MZB) cells are required for production of antibody to T-independent antigens such as the polysaccharide capsule of the encapsulated bacteria that are so prominent in causing blood-borne infections and pneumonia. Intravenous infection with Streptococcus pneumoniae, an encapsulated bacterium, results in a blood-borne infection that requires MZB cells for clearance. The systemic S. pneumoniae host resistance assay evaluates whether a therapeutic test article exerts immunotoxicity on MZB cells and measures the T-independent antibody response (TIAR). Suppression of CMI or in some cases HMI may result in reactivation of latent virus that may result in a fatal disease such as progressive multifocal leukoencephalopathy (PML). The murine cytomegalovirus (MCMV) reactivation model may be used to evaluate a pharmaceutical agent to determine if suppression of CMI or HMI results in reactivation of latent virus. Candida albicans is another host resistance model to test potential immunotoxicity. Host resistance assays have been the ultimate measure of immunotoxicity testing for environmental chemicals and pharmaceutical small molecules. Human biologicals are now an important component of the drug development armamentarium for biotech and pharmaceutical companies. Many human biologicals are fusions of IgG, and/or target immune mediators, immunological receptors, adhesion molecules, and/or are indicated for diseases that have immune components. It is therefore necessary to thoroughly evaluate human biological therapeutics for immunotoxicity. Numerous biologicals that are pharmacologically active in rodents can be evaluated using well-characterized rodent host resistance assays. However, biologicals not active in rodents may use surrogate biologicals for testing in rodent host resistance assays, or may use host resistance assays in genetically engineered mice that mimic the effect of the human biological pharmacological agent.

Influenza virus host resistance model.

Host resistance (HR) models are used to evaluate the effect of a test article on clearance of an infectious microorganism in order to assess total immunocompetence. HR models serve as biomarkers of net immunological health or immunological well-being. Immunotoxicity can result either in an impaired clearance of an infectious agent, increased susceptibility to an opportunistic microorganism, prevention of immunization, or exacerbation of latent viral infections. The purpose of immunotoxicity testing is to obtain data that is meaningful for safety assessment, and for immunosuppression the major objective is to determine the significance with respect to increased susceptibility to infectious disease. Host resistance models provide the only sure method of examining the influence of test articles on the functional integrity of the immune system and its ability to eliminate pathogenic microorganisms and tumor cells. They provide the means to directly assess the functional reserve of the immune system. Clearance of influenza virus requires an intact and functional immune system that incorporates a cascade of immune responses. Mechanistic studies can be included in the influenza virus host resistance model by measuring the effect of a test article on innate immunity (cytokine and interferon production, macrophage function, and natural killer (NK) cell function) and acquired or adaptive immunity (cytotoxic T lymphocyte (CTL) activity as well as influenza-specific IgM and/or IgG antibody).