Toxicologic Pathology

I was honored to be the keynote speaker at the 30th Annual Society of Toxicologic Pathology Symposium “Toxicologic Pathology and the Immune System.” I had the opportunity to reminisce about events in the 1970s that set the stage for the birth and subsequent growth of the field of immunotoxicology and to summarize my research career that has spanned the past 40 years as well. An initial focus on the immunotoxicity of pentachlorophenol led my laboratory into the aryl hydrocarbon receptor (AHR) field and the study of its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). My research career has been devoted to trying to elucidate the immunological basis of TCDD’s profound immunosuppressive activity that is mediated by activation of AHR. In recent years, my laboratory has focused on the role of CD4⁺ T cells as targets of TCDD, and we were the first to describe the induction of AHR-dependent regulatory T cells (Tregs). The ability to induce Tregs using an exogenous AHR ligand to activate the AHR-Treg pathway represents a novel approach to the prevention and/or treatment of autoimmune disease. We are currently searching for such ligands.

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.

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.

The liver is a target of many inflammatory pathologies of both infectious and noninfectious etiology. As key effectors of the innate immune system, neutrophils are critical for defense against microbial infections but are often the source of profound collateral damage to host tissues during disease states. In this article based on the authors’ presentation at the 2011 Society of Toxicologic Pathology Annual Symposium, they review the molecular mechanisms of neutrophil recruitment to the liver in response to sepsis/endotoxemia, as well as sterile inflammation, and discuss variations in the molecular choreography of neutrophil trafficking in response to these different insults. Furthermore, the authors discuss the functional contributions of neutrophils within the liver microvasculature during severe sepsis, including their contributions to both host defense and organ damage. Given that inappropriate neutrophilic inflammation contributes to the pathogenesis of many liver diseases, a thorough understanding of the molecular mechanisms that regulate the recruitment of neutrophils to the liver, and their functions therein, may reveal new avenues for therapeutic interventions to treat inflammatory liver pathologies.

In simple terms, inflammation can be defined as a beneficial, nonspecific response of tissues to injury that generally leads to restoration of normal structure and function. In this concept, resolution of the inflammatory response, once it has achieved its protective and pro-immunogenic functions, becomes a critical determinant of what might be considered the paradox of inflammation. On one hand, inflammation is essential to resolve tissue injury and maintain homeostasis. On the other, inflammation is a key participant in the great majority of human diseases. Accordingly, to achieve complete resolution of inflammation, it is necessary to both turn off inflammatory mediator production and inflammatory cell accumulation and to remove inflammatory cells and debris without initiating an autoimmune response. Much of this process involves key activities of the mononuclear phagocyte series of cells, including resident and recruited macrophages. Recognition of activated and dying acute inflammatory cells by mononuclear phagocytes has been shown to (a) enhance macropinocytic activity for removal of debris, (b) enhance uptake of the effete inflammatory cells themselves, (c) induce inflammosuppressive and immunosuppressive mediators such as TGFβ and IL-10 that can down-regulate and limit proinflammatory mediator production, and (d) induce production of growth factors for tissue cells that may play key roles in tissue repair. Defects in these highly regulated processes are associated with persistent inflammation and/or autoimmunity in overaggressive resolution mechanisms such as nonresolving fibrosis or persistent tissue destruction as in emphysema.

The complement system and toll-like receptors (TLRs) are two components of innate immunity that are critical for first-line host defense. Many pathogen-associated molecular patterns activate both complement and TLRs, and recent studies in animal models have revealed a marked synergistic interaction between the two systems. In mice deficient in a membrane complement regulator, prototypical TLR ligands such as LPS, zymosan, and polyI:C caused increased systemic complement activation, which in turn led to a profound elevation of proinflammatory cytokine biosynthesis. This phenotype required interaction between complement and TLRs because complement activation alone by cobra venom factor without TLR engagement did not lead to appreciable cytokine production. The regulatory effect of complement on TLR signaling was mediated by the anaphylatoxins C5a and C3a through a receptor-dependent mechanism and involved increased mitogen-activated protein kinase and nuclear factor κB activation. The crosstalk between complement and TLRs may also impact adaptive immunity, for example, the differentiation of T helper 17 (Th-17) cells. Given that excessive activation of either TLR or complement has been associated with inflammatory tissue injury as occurs in sepsis and autoimmune diseases, the new insight on complement and TLR crosstalk may have therapeutic implications.

Session 3 of the Toxicologic Pathology and the Immune System Symposium, presented as part of the 30th Annual Symposium of the Society of Toxicologic Pathology in 2011, focused on the biological advances in control of selected cellular and secretory components of acquired immunity. Acquired immunity goes beyond innate immunity to provide controlled recognition and memory for specific antigenic challenges. Predominately involving activation of T and B lymphocytes, the resulting cellular- and secretory-mediated activity provides immediate and long-term host defenses to antigenic challenge. This session highlighted the biological advances in function and dysfunction of acquired immunity through regulatory T cells, the pathophysiology of effector cells and secretory molecules in immunosuppression, allergic inflammatory disease, and dysregulation that leads to loss of tolerance and autoimmune disease. A brief overview of major concepts in acquired immunity and summaries of the above themes are covered herein, and discussions of these themes are covered in greater detail in this issue of Toxicologic Pathology.

Regulatory T-cells (T_REG) are diverse populations of lymphocytes that regulate the adaptive immune response in higher vertebrates. T_REG delete autoreactive T-cells, induce tolerance, and dampen inflammation. T_REG cell deficiency in humans (i.e., IPEX [Immunodysregulation, Polyendocrinopathy and Enteropathy, X-linked syndrome]) and animal models (e.g., “Scurfy” mouse) is associated with multisystemic autoimmune disease. T_REG in humans and laboratory animal species are similar in type and regulatory function. A molecular marker of and the cell lineage specification factor for T_REG is FOXP3, a forkhead box transcription factor. CD4⁺ T_REG are either natural (nT_REG), which are thymus-derived CD4⁺CD25⁺FOXP3⁺ T-cells, or inducible (i.e., Tr1 cells that secrete IL-10, Th3 cells that secrete TGF-β and IL-10, and Foxp3⁺ Treg). The proinflammatory Th17 subset has been a major focus of research. T_H17 CD4⁺ effector T-cells secrete IL-17, IL-21, and IL-22 in autoimmune and inflammatory disease, and are dynamically balanced with T_REG cell development. Other lymphocyte subsets with regulatory function include: inducible CD8⁺ T_REG, CD3⁺CD4⁻CD8⁻ T_REG (double-negative), CD4⁺Vα14⁺ (NKT_REG), and γδ T-cells. T_REG have four regulatory modes of action: secretion of inhibitory cytokines (e.g., IL-10 and TGF-β), granzyme-perforin-induced apoptosis of effector lymphocytes, depriving effector T-cells of cytokines leading to apoptosis, or inhibition of dendritic cell function. The role of T_REG in mucosal sites, inflammation/infection, pregnancy, and cancer as well as a review of T_REG as a modulatory target in drug development will be covered.

Cytokines are critical in allergic intercellular communication networks, and they contribute to disease pathology through the recruitment and activation of pro-inflammatory leukocytes and in chronic disease to pro-fibrotic/remodeling events. Th2 cytokines predominate primarily in mild to moderate allergic asthma, although clinical trials with inhibitors of IL-4 and IL-5 have not provided the robust efficacy observed in animal models of allergy. These results not only highlight the complexity of allergic disease, but they also point to the importance of other cytokine networks in driving pathology. The heterogeneous nature of the disease is emphasized by the fact that the Th2/Th1/Th17 cytokine balance can be influenced by the initiating allergic trigger. For example, the house dust mite allergen Der p 2 mimics the activity of MD-2 by presenting lipopolysaccharide to Toll-like receptor-4 for the activation of inflammatory genes including innate-type cytokines. Here we discuss the functions of the novel cytokine players, thymic stromal lymphopoetin (TSLP), IL-33, IL-25, and IL-9 and delineate nonredundant roles for IL-4 and IL-13 in allergic disease. Persistent efforts in the characterization of these and other cytokine networks will be essential for understanding the complex pathogenic mechanisms that underpin allergic disease and for guiding targeted therapeutic interventions.

Autoimmune disease (AIDx) results from failure to sustain tolerance to self molecules. Dozens of AIDx involving one or multiple organ systems afflict 3% or more of people worldwide (>75% women). Predisposing factors for AIDx include genetic background, hormonal status, pathogens, and xenobiotic exposures. The incidence of AIDx is higher in individuals living in developed nations, including recent immigrants. Patients may have several AIDx simultaneously. Certain AIDx can prevent other AIDx. A history of AIDx raises the risk for developing hematopoietic neoplasia. Some common mechanisms for losing self-tolerance include reduced deletion or enhanced activation of autoreactive CD4⁺ T-helper (Th) lymphocytes, defective immunomodulation by CD4⁺ regulatory (Treg) and CD8⁺ suppressor (Ts) T-lymphocytes, dysregulated signaling (leading to a relative increase in pro-inflammatory cytokines), comparable structure between self-antigens and foreign molecules, or expression of new epitopes on previously hidden or xenobiotic-modified self proteins. Organ-specific AIDx is generally a cell-mediated (Th1 or Th17) process, while multi-organ AIDx also incorporates a robust autoantibody (Th2) component. Cytokine signatures of different AIDx overlap incompletely; for a given AIDx, different patients have divergent cytokine profiles. Newer anti-AIDx agents are based on our increasing knowledge of AIDx pathogenesis and usually attempt to reverse lymphocyte dysfunction, quell pro-inflammatory signaling, or restore self-tolerance.

Developmental immunotoxicity (DIT) occurs when exposure to environmental risk factors prior to adulthood, including chemical, biological, physical, or physiological factors, alters immune system development. DIT may elicit suppression, hyperactivation, or misregulation of immune responses and therefore may present clinically as decreased resistance to pathogens, allergic and autoimmune diseases, and inflammatory diseases. When evaluating DIT in an animal model, specific endpoints are assessed, which can reveal the potential for a risk factor to alter immune system development. However, linking DIT evaluation in an animal model with clinical realities observed in human populations requires that DIT testing regimens evaluate critical windows in immune system development. In addition, pathways leading to DIT may not be apparent without the stressors that induce aberrant and detectable responses. This review contains brief descriptions of recently published work that addresses disease patterns associated with DIT and solutions for altering such patterns of disease. We also comment on gaps between DIT testing in animal models and the clinical manifestation of immune-based diseases in children that can be filled by a better understanding of critical windows in immune system development and DIT testing that includes multiple functional assays.

The liver is the primary hematopoietic organ of the mammalian body during the fetal stage. The postnatal liver retains immunologically important functions and contains a substantial population of immunologically active cells, including T and B lymphocytes, Kupffer cells, liver-adapted natural killer (NK) cells (pit cells), natural killer cells expressing T cell receptor (NKT cells), stellate cells, and dendritic cells. The liver is the major site of production of the acute phase proteins that are associated with acute inflammatory reactions. Kupffer cells have an important role in the nonspecific phagocytosis that comprises a major component of the barrier to invasion of pathogenic organisms from the intestine. Hepatic NK and NKT cells are important in the nonspecific cell killing that is important in resistance to tumor cell invasion. The liver has a major role in deletion of activated T cells and induction of tolerance to ingested and self-antigens. Disposal of waste molecules generated through inflammatory, immunologic, or general homeostatic processes is accomplished via the action of specific endocytic receptors on sinusoidal endothelial cells of the liver. Age-related changes in sinusoids (pseudocapillarization), autophagy, and functions of various hepatic cell populations result in substantial alterations in many of these immunologically important functions.

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.

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.

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.

The first effective immunosuppressive drug (ISD) was azathioprine, approved in 1968. Early experience with this drug suggested that patients might have an excess risk of tumors including lymphoma and skin tumors. Comparison among various registries has shown that the cumulative risk of tumors increases over time. The risk is additionally increased by the more intense immunosuppressive regimens needed for lung or heart-lung transplants. The link between immunosuppression and tumorigenesis was further reinforced by the high concordance of tumor types between transplant and HIV patients. The role of the immune system in tumor defense includes both direct tumor surveillance and immunity against oncogenic viruses. In transplant patients, at least two-thirds of the lymphomas are Epstein-Barr virus (EBV)–positive. Existing methods of testing for carcinogenicity are not considered adequate to identify the hazard of tumorigenesis due to these drugs. Research is ongoing in Food and Drug Administration laboratories and at collaborators’ laboratories to evaluate experimental systems that may have the ability to adequately identify this class of hazard. Initial work is on various model systems similar to EBV. These include the MHV-68 mouse model, lymphocryptovirus (LCV-1) in the cynomolgus monkey, and preliminary work with mice with humanized immune systems using EBV directly.

A number of therapeutic immunomodulatory biologics, including antibodies, fusion proteins, and recombinant proteins, have been causally linked with serious adverse effects in humans. In nearly all cases, these serious adverse effects have been directly associated with the immunomodulatory biologic’s intended pharmacologic activity or exaggerated pharmacology. Examples of immunomodulatory biologics known to cause serious adverse effects in the clinic ranging from immunostimulation and cytokine release syndrome (e.g., TGN1412) to immunosuppression with increased risk of opportunistic infections (e.g., TNF-α antagonists, anti-integrins) are presented. Specific examples of the nonclinical testing strategy used for the clinical risk assessment of these immunomodulatory biologics are discussed, with an emphasis on the clinical relevance and predictivity of the models. Infectious challenge animal models, in particular, were critically evaluated for their utility in evaluating clinical risk assessment versus understanding mechanism of action. The nonclinical safety testing strategy for an immunomodulatory biologic should be custom tailored to interrogate the biology of the immunologic target in order to best assess potential clinical risk. This nonclinical strategy should include mechanistic and efficacy models of pharmacologic activity and immunologic signaling pathways, in vitro immunologic assays such as cytokine release, and immunophenotypic assessment by flow cytometry, immunohistochemistry, and/or immunofluorescence, as appropriate.

The ICH S8 immunotoxicity testing guideline for human pharmaceuticals was published in 2006 and was intended to provide guidance for assessing the immunotoxicity potential of low-molecular-weight drugs that are not intended to alter the immune system. For drugs intended to modulate the immune system, immunotoxicity testing strategies are generally developed on a case-by-case approach since the targets, intended patient population, and mechanisms of action of the test compound will determine the type of testing needed. Some of the general principles of ICH S8, however, may be applied to immunotoxicity testing strategies for immunomodulatory drugs. A weight-of-evidence approach using factors discussed in ICH S8 in concert with an assessment of the potential value of additional immunotoxicity testing should be considered. For most situations, immunotoxicity studies with immunomodulatory compounds evaluate off-target effects on the immune system and exaggerated pharmacology. The potential use of data from these studies and considerations such as translatability to humans are discussed.

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.

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.

Air pollution has long been associated with health risks such as increased susceptibility to respiratory infections and potentiation of asthmatic-type responses. Experimental evidence in rodents indicates that air pollutants including diesel exhaust particles (DEPs), gases, and metals cause lung injury, inflammation, reduce aspects of host defense, and may potentiate allergic airway responses. Here we present evidence that diesel exhaust particles delivered by inhalation or aspiration can exacerbate allergic lung disease depending on the material’s chemical properties. Genomic analysis of mouse lungs following instillation or inhalation of DEPs shows an alteration spectrum of pathways associated with immune signaling, cell metabolism, and oxidative stress. Diesel exposure also may worsen respiratory infections through depression of protective immune responses. Here we show that mice exposed to diesel and co-infected with influenza had increased influenza virus titers as well as higher levels of lung injury and inflammation in association with increased Th2 cytokines, and a concomitant decrease in Th1 polarization. A simplified model explains how the potentiation of the Th2 arm of immunity by diesel exhaust results in increased allergic sensitization, whereas cell-mediated (protective) immunity against viral infections is simultaneously reduced.

The Deepwater Horizon (DWH) oil spill was the largest environmental disaster and response effort in U.S. history, with nearly 800 million liters of crude oil spilled. Vast areas of the Gulf of Mexico were contaminated with oil, including deep-ocean communities and over 1,600 kilometers of shoreline. Multiple species of pelagic, tidal, and estuarine organisms; sea turtles; marine mammals; and birds were affected, and over 20 million hectares of the Gulf of Mexico were closed to fishing. Several large-scale field efforts were performed, including assessments of shoreline and wildlife oiling and of coastal waters and sediments. The assessment of injuries, damages, and restoration options for the DWH spill is ongoing. Although petroleum and the polycyclic aromatic hydrocarbon component of oils are known to affect the immune systems of aquatic organisms and wildlife, immunotoxicity is not typically assessed during oil spills and has not been a focus of the DHW assessment. The effects of oil spill contaminants on immune responses are variable and often exposure dependent, but immunotoxic effects seem likely from the DHW spill based on the reported effects of a variety of oils on both aquatic and wildlife species.

The 2011 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was held in Denver, Colorado in advance of the Society of Toxicologic Pathology’s 30th Annual Meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers’ presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include: proliferative lesions from various fish species including ameloblastoma, gas gland hyperplasia, nodular regenerative hepatocellular hyperplasia, and malignant granulosa cell tumor; spontaneous cystic hyperplasia in the stomach of CD1 mice and histiocytic aggregates in the duodenal villous tips of treated mice; an olfactory neuroblastoma in a cynomolgus monkey; various rodent skin lesions, including follicular parakeratotic hyperkeratosis, adnexal degeneration, and epithelial intracytoplasmic accumulations; oligodendroglioma and microgliomas in rats; a diagnostically challenging microcytic, hypochromic, responsive anemia in rats; a review of microcytes and microcytosis; nasal lesions associated with green tea extract and Ginkgo biloba in rats; corneal dystrophy in Dutch belted rabbits; valvulopathy in rats; and lymphoproliferative disease in a cynomolgus monkey.

Expression of antigens in cells and tissues can be readily studied immunohistochemically with the use of antibodies. A panel of antibodies to cell-specific markers can be used to diagnose lesions, including tumors, in the hematopoietic and lymphoid systems. This review discusses the use of readily available antibodies and procedures to identify antigens expressed in normal tissues and in proliferative and inflammatory lesions in formalin-fixed, paraffin-embedded (FFPE) murine specimens.

The authors performed a retrospective study to determine the incidences and range of spontaneous pathology findings in the lymphoid and haemopoietic systems of control Charles River CD-1 mice (Crl: CD-1(ICR) BR). Data was collected from 2,560 mice from control dose groups (104-week and 80-week carcinogenicity studies; 13-week studies), from regulatory studies evaluated at the authors’ laboratory between 2005 and 2010. Lesions of the lymphoid and hematopoietic systems were uncommon in 13-week studies but were of high incidence in the carcinogenicity studies (80- or 104-week duration). The most common finding overall was lymphoid hyperplasia within the spleen, thymus, and lymph nodes. The finding of benign lymphoid hyperplasia of the thymus is unusual in other mouse strains. The most common cause of death in the carcinogenicity studies was lymphoma. It is hoped that the results presented here will provide a useful database of incidental pathology findings in CD-1 mice on carcinogenicity studies.

Conventional light microscopic evaluation of a semen ejaculate does not fully utilize potential indicators of functional impairment in sperm organelles. The technique described here facilitates critical evaluation of morphological features of spermatozoal organelles at an ultrastructural level and helps identify vulnerable targets. Compared with a battery of sperm function assays employed in andrology clinics, this relatively less expensive technique efficiently uses semen as biopsy material and thus serves as a comprehensive means to assess the impact of toxicants on the male reproductive system.

The first electron microscopic images of biological specimens were made in the 1940s, and the next 30 years comprised an era of descriptive ultrastructure during which transmission electron microscopy (TEM) was integral to an explosion in cellular and molecular biology. However, when questions could no longer be answered by ultrastructural information alone, the use of TEM in biological research declined. Innovative molecular techniques and newer imaging technologies such as confocal fluorescence microscopy filled the gap, providing faster answers with less rigorous training as a prerequisite to data collection. The use of TEM in toxicologic pathology has paralleled the rise and fall of its popularity in other disciplines. However, TEM remains an essential resource that provides direct and unequivocal data to explain and address safety concerns in preclinical toxicity studies. There is still an important place for TEM in preclinical safety evaluation and mechanistic studies, particularly when visualization of subcellular structures provides a link to other endpoints. This review reinforces the value of TEM in preclinical safety testing and model development and encourages best practices for ultrastructural evaluation.

This article presents an overview of microscopy and its ability to assist in understanding what happens in cells and tissues. From the 1960s to 1980s, electron microscopy was the best way to understand cell processes, but the advent in the mid-1980s of light microscopy and the ability to do fluorescence imaging displaced electron microscopy in this area. However, the 21st century has seen several improvements in electron microscopy that, along with the need for more detailed ultrastructural information, make it again very attractive in the study of cells, tissues, and organs, and electron microscopy has resumed its place as the preeminent method in understanding cell processes.