From bats to pangolins: new insights into species differences in the structure and function of the immune system.

Species differences in the structure and function of the immune system of laboratory animals are known to exist and have been reviewed extensively. However, the number and diversity of wild and exotic species, along with their associated viruses, that come into contact with humans has increased worldwide sometimes with lethal consequences. Far less is known about the immunobiology of these exotic and wild species. Data suggest that species differences of the mechanisms of inflammation, innate immunity and adaptive immunity are all involved in the establishment and maintenance of viral infections across reservoir hosts. The current review attempts to collect relevant data concerning the basics of innate and adaptive immune functions of exotic and wild species followed by identification of those differences that may play a role in the maintenance of viral infections in reservoir hosts.

The lymphoid system: a review of species differences.

While an understanding of the structure and function of a generically described immune system is essential in contemporary biomedicine, it is clear that a one-size-fits-all approach applied across multiple species is fraught with contradictions and inconsistencies. Nevertheless, the breakthroughs achieved in immunology following the application of observations in murine systems to that of man have been pivotal in the advancement of biology and human medicine. However, as additional species have been used to further address biologic and safety assessment questions relative to the structure and function of the immune system, it has become clear that there are differences across species, gender, age and strain that must be considered. The meaningfulness of these differences must be determined on a case-by-case basis. This review article attempts to collect, consolidate and discuss some of these species differences thereby aiding in the accurate placement of new observations in a proper immunobiological and immunopathological perspective.

Evaluation of the utility of popliteal lymph node examination in a cyclophosphamide model of immunotoxicity in the rat.

The objective of this study was to characterize the variability of rat lymphoid organ weights and morphology following treatment with a known immunotoxicant, with a focus on the usefulness of evaluating popliteal lymph node weight and histology. Cyclophosphamide was administered to male Sprague-Dawley rats by oral gavage at doses of 2, 7 or 12 mg/kg/day for 10 consecutive days. Left and right popliteal lymph nodes (PLN), spleen and thymus were collected at necropsy, weighed, fixed and processed for histopathology. Femoral bone marrow was also collected, fixed and processed for histology. Organ weight variability was greater for PLN than for either spleen or thymus in control animals. There was a significant but weak correlation between paired left and right PLN weights (p < 0.005; r(2) = 0.2774). Significant treatment-related decreases in lymphoid organ weights were observed in spleen and thymus at ≥ 7 mg/kg/day (p < 0.01), whereas in PLN a significant decrease (p < 0.05) was noted only at 12 mg/kg/day. The inclusion of PLN did not enhance the sensitivity of detection of systemic treatment-related changes in lymphoid organs in a rat cyclophosphamide model.

Small molecule immunomodulatory drugs: challenges and approaches for balancing efficacy with toxicity.

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.

Selective inhibition of JAK1 and JAK2 is efficacious in rodent models of arthritis: preclinical characterization of INCB028050.

Inhibiting signal transduction induced by inflammatory cytokines offers a new approach for the treatment of autoimmune diseases such as rheumatoid arthritis. Kinase inhibitors have shown promising oral disease-modifying antirheumatic drug potential with efficacy similar to anti-TNF biologics. Direct and indirect inhibition of the JAKs, with small molecule inhibitors like CP-690,550 and INCB018424 or neutralizing Abs, such as the anti-IL6 receptor Ab tocilizumab, have demonstrated rapid and sustained improvement in clinical measures of disease, consistent with their respective preclinical experiments. Therefore, it is of interest to identify optimized JAK inhibitors with unique profiles to maximize therapeutic opportunities. INCB028050 is a selective orally bioavailable JAK1/JAK2 inhibitor with nanomolar potency against JAK1 (5.9 nM) and JAK2 (5.7 nM). INCB028050 inhibits intracellular signaling of multiple proinflammatory cytokines including IL-6 and IL-23 at concentrations <50 nM. Significant efficacy, as assessed by improvements in clinical, histologic and radiographic signs of disease, was achieved in the rat adjuvant arthritis model with doses of INCB028050 providing partial and/or periodic inhibition of JAK1/JAK2 and no inhibition of JAK3. Diminution of inflammatory Th1 and Th17 associated cytokine mRNA levels was observed in the draining lymph nodes of treated rats. INCB028050 was also effective in multiple murine models of arthritis, with no evidence of suppression of humoral immunity or adverse hematologic effects. These data suggest that fractional inhibition of JAK1 and JAK2 is sufficient for significant activity in autoimmune disease models. Clinical evaluation of INCB028050 in RA is ongoing.

Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors.

Malignant tumors arise, in part, because the immune system does not adequately recognize and destroy them. Expression of indoleamine-2,3-dioxygenase (IDO; IDO1), a rate-limiting enzyme in the catabolism of tryptophan into kynurenine, contributes to this immune evasion. Here we describe the effects of systemic IDO inhibition using orally active hydroxyamidine small molecule inhibitors. A single dose of INCB023843 or INCB024360 results in efficient and durable suppression of Ido1 activity in the plasma of treated mice and dogs, the former to levels seen in Ido1-deficient mice. Hydroxyamidines potently suppress tryptophan metabolism in vitro in CT26 colon carcinoma and PAN02 pancreatic carcinoma cells and in vivo in tumors and their draining lymph nodes. Repeated administration of these IDO1 inhibitors impedes tumor growth in a dose- and lymphocyte-dependent fashion and is well tolerated in efficacy and preclinical toxicology studies. Substantiating the fundamental role of tumor cell-derived IDO expression, hydroxyamidines control the growth of IDO-expressing tumors in Ido1-deficient mice. These activities can be attributed, at least partially, to the increased immunoreactivity of lymphocytes found in tumors and their draining lymph nodes and to the reduction in tumor-associated regulatory T cells. INCB024360, a potent IDO1 inhibitor with desirable pharmaceutical properties, is poised to start clinical trials in cancer patients.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

A data-based assessment of alternative strategies for identification of potential human cancer hazards.

The two-year cancer bioassay in rodents remains the primary testing strategy for in-life screening of compounds that might pose a potential cancer hazard. Yet experimental evidence shows that cancer is often secondary to a biological precursor effect, the mode of action is sometimes not relevant to humans, and key events leading to cancer in rodents from nongenotoxic agents usually occur well before tumorigenesis and at the same or lower doses than those producing tumors. The International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) hypothesized that the signals of importance for human cancer hazard identification can be detected in shorter-term studies. Using the National Toxicology Program (NTP) database, a retrospective analysis was conducted on sixteen chemicals with liver, lung, or kidney tumors in two-year rodent cancer bioassays, and for which short-term data were also available. For nongenotoxic compounds, results showed that cellular changes indicative of a tumorigenic endpoint can be identified for many, but not all, of the chemicals producing tumors in two-year studies after thirteen weeks utilizing conventional endpoints. Additional endpoints are needed to identify some signals not detected with routine evaluation. This effort defined critical questions that should be explored to improve the predictivity of human carcinogenic risk.

The role of histopathology in the identification of immunotoxicity.

Recently finalized regulatory guidance documents concerned with the identification of immunotoxicity (CPMP: Note for Guidance on Repeated Dose Toxicity; FDA: Guidance for Industry, Immunotoxicology Evaluation of Investigational New Drugs; ICH S8) state that immunotoxicity testing should be performed on all new investigational drugs or medicinal products. In addition, all documents clearly identify gross and microscopic examination of lymphoid tissues as necessary and pivotal first steps in the assessment of new xenobiotics for immunotoxic potential. However, as is true for the evaluation of other organs systems, there are numerous approaches to the histopathologic examination of lymphoid tissues. To assist in a more uniform and consistent histopathologic assessment of the immune system, the Society of Toxicologic Pathology (STP), has recently prepared "best practice" recommendations concerning the collection, interpretation and reporting of organ weights, gross and microscopic observations, and other pathology data relevant to the immune system. The STP recommendations are intended to provide a scientifically sound and well-considered guidance document for routine pathology evaluation of the immune system. This presentation will consider the implications of this "best practice" document and place these recommendations in the context of normal animal tissue variability.

Species differences in the structure and function of the immune system.

With the recent publication of regulatory guidelines from both the FDA and the CPMP addressing the investigation of immunotoxicity of new chemical entities has come the requisite increased application of immunotoxicology protocols. Importantly, the fulfillment of these protocols may require the use of different species, and while in many cases information concerning the structure and function of the immune system can be readily translated across species, there are numerous and significant species differences that need to be considered. In some cases, the generation of meaningful immunotoxicology data can be adversely affected by the choice of a species that does not adequately share the immune function of concern with man. Likewise immunotoxicology testing in one species may produce negative data in one species but positive data in another. Knowing the mechanistic basis through an understanding of species differences in the structure and function of the immune system is pivotal to success. This becomes especially true as pharmaceutical companies design and develop highly specific immunomodulatory molecules that demonstrate species-specific pharmacology. This review is an exploration of various species differences in the structure and function of the immune system and an attempt to identify those differences that may be important in the conduct of immunotoxicity tests.