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The immune system has been recognized for decades as a potential “target organ” of toxicity. Immune system activation can result in cytokine release resulting in severe systemic toxicity. Immunosuppression can result in impaired host defense and an increase in opportunistic infection, reemergence of latent infection, poor responses to vaccination, or increased risk of certain cancers. Several regulatory documents have addressed various aspects of immunotoxicity assessments. Nonhuman primates (NHPs) and in particular macaques are often the only relevant species for biotechnology-derived investigational new drugs based on cross-reactivity with human and NHP targets. This article reviews the challenges and opportunities associated with monitoring immune function in NHPs in the context of regulatory expectations. The article emphasizes how a comprehensive assessment of immunotoxicity remains a challenge due to interanimal variability associated with certain parameters (e.g., T-dependent antibody response)and it identifies gaps, such as the stage of development of certain assays (e.g., cytotoxic T-cell function). Despite these challenges, a thorough assessment of target biology-driven theoretical risks, in combination with proper integration of all information from the standard toxicology studies, and the refinement of certain assays should enable proper risk assessment. To this effect, emphasis should be placed on leveraging predictive in vitro assays using human cells. [ABSTRACT FROM AUTHOR]

The immune system functions primarily as a defense mechanism to provide protective immunity against microbial pathogens and cancer. The resulting protective responses occur through the complex interaction of tissues, cells, proteins, and molecular pathways that act in concert with other systems (e.g., nervous and endocrine) to provide the host with immunologic responses that cause pathologic processes seen primarily as inflammatory reactions. The pathologic responses can be attributed to either normal responses to infectious organisms and cancer cells, misdirected responses as in the case of hypersensitivity or autoimmune diseases, or deficient responses attributable to deficiencies or defects in components of the immune system. Pathologists need to have a basic understanding of the immune system to not only interpret findings as to their likely pathogenesis, but also to predict when the immune system may be a potential target. This review will be limited to a general overview of the basic immunologic responses and primary components involved. [ABSTRACT FROM PUBLISHER]

Characteristic susceptibility to environmental and pharmaceutical exposure may occur during periods in life of marked histophysiological changes of the immune system. Perinatal development is such a period; pregnancy followed by lactation is potentially another one. Here, we explored the influence of pregnancy and lactation on the model immunotoxic compound di-n-octyltin dichloride (DOTC) in rats using clinical and histopathological parameters. Female rats were exposed to 0, 3, 10, or 30 mg DOTC/kg feed during pregnancy and up to 20 (at weaning) or 56 days after delivery. Age-matched nonmated females were exposed during the same time periods. DOTC at the level of 10 and 30 mg/kg decreased thymus weight and affected thymus morphology in the lactating rats. In addition, DOTC decreased the numbers of neutrophils in the lactating rats. These effects were no longer apparent at day 56 despite continuous exposure to DOTC. This explorative study indicates that the innate and adaptive immune system may be especially sensitive to immunotoxicants during pregnancy and lactation. [ABSTRACT FROM PUBLISHER]

Perfluorinated compounds (PFCs) are environmentally widespread, persistent, and bioaccumulative chemicals with multiple toxicities reported in experimental models and wildlife, including immunomodulation. The two most commonly detected compounds, which also generally occur in the highest concentrations in environmentally exposed organisms, are perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). PFOA and PFOS have been reported to alter inflammatory responses, production of cytokines, and adaptive and innate immune responses in rodent models, avian models, reptilian models, and mammalian and nonmammalian wildlife. Mounting evidence suggests that immune effects in laboratory animal models occur at serum concentrations below, within the reported range, or just above those reported for highly exposed humans and wildlife. Thus, the risk of immune effects for humans and wildlife exposed to PFCs cannot be discounted, especially when bioaccumulation and exposure to multiple PFCs are considered. This review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA, PFOS, and other PFCs in rodent models, alternative laboratory models, and wildlife. [ABSTRACT FROM PUBLISHER]

Developmental immunotoxicity (DIT) testing is centered around the concern that exposure to immunotoxicants early in development may result in enhanced susceptibility of, or unique or more persistent effects on, the immune system, in comparison to adult exposure. Developmental immunotoxicity has been the focus of numerous workshops and reviews for at least fifteen years. Most of these earlier activities have focused on both environmental chemicals and pharmaceuticals and have concluded that the best approach to DIT is to address the possible impacts of exposure during all of the critical windows of development. This article will emphasize the critical role played by exposure during the juvenile stage of development. This article will also highlight several key issues that distinguish DIT testing of pharmaceuticals. Representatives from the pharmaceutical, biotechnology, academic, and regulatory sectors (both FDA and EMA) were brought together during a two-day workshop in May 2010 to consider the current state of the science of DIT as it pertains to the testing of pharmaceuticals. It is important to emphasize at the onset that there are currently no regulatory guidelines for either drugs or nondrug chemicals specifically focused on assessment of DIT, although some general guidelines are included in both developmental and reproductive toxicity and general immunotoxicology guidance documents. [ABSTRACT FROM PUBLISHER]

Current immunotoxicity testing guidance for drugs, high production–volume chemicals, and pesticides specifies the use of animal models to assess potential biomarkers of immune system effects (e.g., lymphoid organ and bone marrow indices, histopathology) or actual measures of immune function (e.g., responses to challenge with antigens or pathogens). These assays are resource intensive and often require special training or experience to ensure reliable results. Alternative in vitro assays to detect immunosuppression and allergic hypersensitivity have the potential to reduce animal use and testing costs and increase immunotoxicity screening and prioritization efforts. Alternative models to detect immunosuppression tend to address broad modes of action because suppression may be caused by a wide variety of events; current in vitro models access the supply of innate and adaptive immune system cells as well as cellular markers associated with function, including gene expression, protein synthesis, and proliferation. Events leading to the induction of allergic hypersensitivity, particularly contact hypersensitivity, are more restricted, and alternative methods currently exploit chemical properties and activation of defined cell populations to detect and estimate the potency of skin sensitizers. [ABSTRACT FROM PUBLISHER]

Enhanced histopathology (EH) of the immune system is a tool that the pathologist can use to assist in the detection of lymphoid organ lesions when evaluating a suspected immunomodulatory test article within a subchronic study or as a component of a more comprehensive, tiered approach to immunotoxicity testing. There are three primary points to consider when performing EH: (1) each lymphoid organ has separate compartments that support specific immune functions; (2) these compartments should be evaluated individually; and (3) semiquantitative descriptive rather than interpretive terminology should be used to characterize any changes. Enhanced histopathology is a screening tool that should be used in conjunction with study data including clinical signs, gross changes, body weight, spleen and thymus weights, other organ or tissue changes, and clinical pathology. Points to consider include appropriate tissue collection, sectioning, and staining; lesion grading; and diligent comparison with concurrent controls. The value of EH of lymphoid organs is to aid in the identification of target cell type, changes in cell production and cell death, changes in cellular trafficking and recirculation, and determination of mechanism of action. [ABSTRACT FROM PUBLISHER]

As the molecular pathobiology of immunologically based diseases, such as rheumatoid arthritis, has become clearer, pharmaceutical researchers have responded with highly efficacious and selective biological compounds. In contrast to older, nonspecific small-molecule therapeutics, the exquisite species sensitivity of monoclonal antibodies has introduced new challenges to preclinical safety studies. Repeated exposure of animals to biopharmaceutical compounds tends to be restricted in the species in which these compounds have pharmacological action, and it tends to stimulate antidrug immune responses with acceleration of clearance, thereby limiting the duration of repeat-dose studies and potentially resulting in hypersensitivity reactions. Thus, the safety testing of biopharmaceutical compounds has necessitated the use of relatively short-term studies in rodents, whereas nonhuman primates have become the primary tool for large-animal, repeat-dose studies. However, as the number of highly targeted and efficacious small-molecule immunomodulators rapidly increases, these molecules will be developed in a manner similar to that of other small molecules with regard to safety assessment. Because such approaches inherently push drug levels to achieve maximally tolerated doses, the pharmacologic specificity of these new small-molecule drugs may be lost as they affect additional receptors and pathways. Therefore, toxicologic pathologists must refamiliarize themselves with the consequences of profound immunosuppression in species other than nonhuman primates. The interrelationships of cytokine signaling and receptor biology are complex, highly integrated, and at times paradoxical, and the loss of specificity at high doses may result in unforeseen consequences caused by the impact on complex down-stream pathways that culminate in exaggerated and adverse responses. The species specificity of such responses may not be inherently familiar or anticipated. [ABSTRACT FROM PUBLISHER]

Immunotoxicology has developed into an integral regulatory requirement of the toxicological assessment of xenobiotics. Histopathological assessment of lymphoid tissues can provide genuine insight into perturbations of lymphoid cell populations. To facilitate retrospective examination of lymphoid organs should concerns over immunotoxicity be raised, we have endeavored to develop a panel of immunohistochemical techniques to demonstrate T-cells and T-cell subsets in formalin-fixed, paraffin-embedded rat lymphoid tissues. We were successful in developing methods for CD3 and CD8 but failed to arrive at a satisfactory technique for the direct demonstration of CD4 in these tissues. Taking the assumption that the majority of mature T-cells are either CD4+ or CD8+, we have combined our methods for CD3 and CD8 in a novel dual-labeling IHC method to simultaneously demonstrate CD3, CD8, and, by implication, CD4 in rat spleen, thymus, lymph node, and Peyer's patch. [ABSTRACT FROM AUTHOR]

The responses to insults including chemical toxins, irradiation and infectious agents involve morphologic, biochemical and molecular changes in the immune system. The changes in specific tissues and cells often can be detected by histopathology and its associated field of immunohistochemisty (IHC). Cells normally express specific proteins (antigens) that can be detected by IHC. When responses to xenobiotics occur, cells often up or down regulate proteins. The art of IHC requires specialized procedures for detection of antigens. Fixation, tissue processing, immunoreactions and antigen retrieval methods are important elements of IHC. We review the antibodies, their sources, use of frozen or fixed paraffin-embedded tissues and specific IHC methods including antigen retrieval and illustrate how they can be effectively used to characterize the immunotoxicologic effects of agents. [ABSTRACT FROM AUTHOR]

Lymph nodes function as filters of tissues and tissue fluids and are sites of origin and production of lymphocytes for normal physiological functions. As part of this normal function, they react to both endogenous and exogenous substances with a variety of specific morphological and functional responses. Lesions can be both proliferative and nonproliferative, and can be treatment-related or not. The histological evaluation of lymph nodes is necessary in order to understand the immunotoxic effects of chemicals with the resulting data providing an important component of human risk assessment. It is the challenge of the toxicologic pathologist to interpret the pathology data within the complete clinical evaluation of the entire animal. Daily insults, ageing and toxins can alter the normal histology and primary function of lymph nodes. Therefore it is important to distinguish and differentiate lesions that occur naturally during normal development and ageing from those that are induced by xenobiotics. To achieve this goal, comparison with strain- age- and sex-matched controls is crucial. [ABSTRACT FROM AUTHOR]

The thymus is a primary lymphoid organ that manifests dynamic physiological changes as animals age in addition to being exquisitely sensitive to stress and toxic insult. It is typically the first lymphoid tissue to respond to immunotoxic xenobiotics, with the first change being loss of cortical lymphocytes by apoptosis. This is followed by removal of the apoptotic cellular debris and, in the absence of recovery, may lead to loss of the cortico-medullary demarcation and organ atrophy. Nonneoplastic proliferative changes include focal lymphoid hyperplasia and proliferation of medullary epithelial cells, often with formation of ribbons, cords, or tubules. Thymomas are relatively rare tumors that exhibit a wide spectrum of morphologic types but do not metastasize. Thymic lymphomas are common in some mouse strains and can become leukemic with hematogenous spread throughout the body. [ABSTRACT FROM AUTHOR]

The thymus is a primary or central lymphoid organ in which T lymphocytes undergo diffentiation and maturation autonomously within the cortex, without the need for antigenic stimulation, and it is essential for the normal development and function of the immune system. The thymus has been shown to be a sensitive target organ following exposure to immunotoxicants and endogenous corticosteroids, and a decrease in size or weight is often one of the first noted measures of compound-induced effects with cortical lymphocytes (thymocytes) being especially susceptible. Therefore, changes in thymus histopathology and architecture are considered to be of particular relevance for immunotoxicity screening. The separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments (Haley et al., 2005). Therefore, enhanced histopathological evaluation of the thymus involves the determination of the size and cellularity of the cortex and medulla, which should be noted separately. Other changes to evaluate include, but are not limited to, increased lymphocyte apoptosis, lymphocyte necrosis, cortex:medulla ratio and an increase or decrease in the epithelial component of the thymus. [ABSTRACT FROM AUTHOR]

Routine histopathology of lymphoid organs is the cornerstone in the identification of immunotoxic and immunomodulatory compounds. Enhanced histopathology is a systematic approach that can be used to further characterize, both qualitatively and semi-quantitatively, the immunomodulatory effects that may occur within both primary and secondary lymphoid organs. The lymph nodes are the major route of entry for antigens and pathogens, via the afferent lymph flow, and they can be sensitive indicators of compounds with regional or systemic immunomodulatory/toxic effects and should therefore be included in the battery of lymphoid organs to evaluate for enhanced histopathology. As with all lymphoid organs, the separate compartments should be evaluated independently and descriptive rather than interpretive terminology should be used to characterize changes within those compartments. This data, in conjunction with gross findings, clinical pathology and changes in organ weight (i.e., thymus), will enable the pathologist to determine if a significant effect on the immune system is present. Moreover, this data may enable the pathologist to determine the critical site or compartment in the targeted tissue, provide some indication of target cell population (B or T cell) and characterize a dose-response relationship. [ABSTRACT FROM AUTHOR]

Exposure to natural environmental products, biopharmaceuticals, or investigational adjuvants has the potential to negatively impact the immune system, resulting in either up- or downregulation of immune function (immunomodulation). Many current protocols for primate toxicologic testing call for the evaluation of changes in immune cell number (peripheral blood or tissue), alterations in the weights of immune system organs (lymph nodes, spleen, thymus), and/or increases in the overall incidence of infections or neoplasms; these data are relied upon to suggest altered immune function. However, these are informative only when clear differences in frequency and/or severity of effects can be distinguished across control and dosed groups. In the absence of such distinct morphologic or clinical pathologic changes, the identification of potential immunomodulatory effects can present a much greater challenge. Additional evaluations may be needed to detect altered immune system integrity; these are based on in vivo assessments in primates of cellular or humoral responsiveness. Immunomodulatory effects can be characterized by in vitro or in vivo immune function tests; these tests require prestudy planning to integrate assessments into ongoing toxicology programs. These methods also involve specialized training and equipment, particularly if the intent is to evaluate parameters in a GLP laboratory setting. In primate toxicology, the added costs required to perform a complete functional analysis of the immune system can be substantial, but may be warranted depending on the clinical development plans. Two analytical methods that are easily incorporated into the standard toxicology profile in primates are flow cytometry and immunohistochemistry. Flow cytometry (FC) is used to assess... [ABSTRACT FROM AUTHOR]

The article presents a systematic approach to Enhanced Histopathology which involves the subjective and semi-quantitative histological evaluation of various lymphoid tissues to identify specific cellular and architectural changes. Enhanced histopathology can assist pathologists to identify immunomodulatory drugs and chemicals. The article finally concludes that standard operating procedures in the laboratories should be established and appropriate guidelines should be followed.