Impact of genotype on clinical course in sickle cell disease and the utility of neutrophil-lymphocyte ratio: a ten-year single-institution experience.

BACKGROUND: Sickle cell disease (SCD) is a diverse group of blood disorders with significant global disease burden. Contemporary interest in the underlying inflammatory paradigm of SCD has emphasized the role of the neutrophil-lymphocyte ratio (NLR) as a prognostic inflammatory marker. METHODS: We retrospectively reviewed 268 hospitalized patients with SCDs of different genotypes (HbSS, HbSß0 thalassemia, HbSß+ thalassemia, and HbSC), totaling 3329 hospital admissions over a 10-year period. Patients were stratified into SS/Sß0 and Sß+/SC groups for statistical analysis of parameters collected at steady state and at hospital admission. RESULTS: At steady state, per unit increase of hemoglobin values was associated with reduced odds of ≥ 2 hospital admissions per year in SS/Sß0 and Sß+/SC groups; per unit increase in platelet count and white blood cell count was associated with increased odds only in the SS/Sß0 group. The NLR had no association in either group. During admission, a cutoff of NLR = 3.5 discerned infection with a sensitivity of 60% and specificity of 57%. Performance improved when excluding patients on outpatient hydroxyurea therapy (cutoff of NLR = 3.5; sensitivity of 68% and specificity of 64%). CONCLUSION: This study supports the utility of NLR as an accessible adjunctive clinical tool in SCD prognostication.

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.

An inter-species protein-protein interaction network across vast evolutionary distance.

In cellular systems, biophysical interactions between macromolecules underlie a complex web of functional interactions. How biophysical and functional networks are coordinated, whether all biophysical interactions correspond to functional interactions, and how such biophysical-versus-functional network coordination is shaped by evolutionary forces are all largely unanswered questions. Here, we investigate these questions using an "inter-interactome" approach. We systematically probed the yeast and human proteomes for interactions between proteins from these two species and functionally characterized the resulting inter-interactome network. After a billion years of evolutionary divergence, the yeast and human proteomes are still capable of forming a biophysical network with properties that resemble those of intra-species networks. Although substantially reduced relative to intra-species networks, the levels of functional overlap in the yeast-human inter-interactome network uncover significant remnants of co-functionality widely preserved in the two proteomes beyond human-yeast homologs. Our data support evolutionary selection against biophysical interactions between proteins with little or no co-functionality. Such non-functional interactions, however, represent a reservoir from which nascent functional interactions may arise.

Exploring the evolution of a trade-off between vigilance and foraging in group-living organisms.

Even though grouping behaviour has been actively studied for over a century, the relative importance of the numerous proposed fitness benefits of grouping remain unclear. We use a digital model of evolving prey under simulated predation to directly explore the evolution of gregarious foraging behaviour according to one such benefit, the 'many eyes' hypothesis. According to this hypothesis, collective vigilance allows prey in large groups to detect predators more efficiently by making alarm signals or behavioural cues to each other, thereby allowing individuals within the group to spend more time foraging. Here, we find that collective vigilance is sufficient to select for gregarious foraging behaviour as long there is not a direct cost for grouping (e.g. competition for limited food resources), even when controlling for confounding factors such as the dilution effect. Furthermore, we explore the role of the genetic relatedness and reproductive strategy of the prey and find that highly related groups of prey with a semelparous reproductive strategy are the most likely to evolve gregarious foraging behaviour mediated by the benefit of vigilance. These findings, combined with earlier studies with evolving digital organisms, further sharpen our understanding of the factors favouring grouping behaviour.

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.

Preclinical evaluation of local JAK1 and JAK2 inhibition in cutaneous inflammation.

JAKs are required for signaling initiated by several cytokines (e.g., IL-4, IL-12, IL-23, thymic stromal lymphopoietin (TSLP), and IFNγ) implicated in the pathogenesis of inflammatory skin diseases such as psoriasis and atopic dermatitis (AD). Direct antagonism of cytokines, such as IL-12 and IL-23 using ustekinumab, has proven effective in randomized studies in psoriasis patients. We hypothesized that local inhibition of cytokine signaling using topical administration of INCB018424, a small molecule inhibitor of JAK1 and JAK2, would provide benefit similar to systemic cytokine neutralization. In cellular assays, INCB018424 inhibits cytokine-induced JAK/signal transducers and activators of transcription (STAT) signaling and the resultant production of inflammatory proteins (e.g., IL-17, monocyte chemotactic protein-1, and IL-22) in lymphocytes and monocytes, with half-maximal inhibitory concentration values <100 nM. In vivo, topical application of INCB018424 resulted in suppression of STAT3 phosphorylation, edema, lymphocyte infiltration, and keratinocyte proliferation in a murine contact hypersensitivity model and inhibited tissue inflammation induced by either intradermal IL-23 or TSLP. Topical INCB018424 was also well tolerated in a 28-day safety study in Gottingen minipigs. These results suggest that localized JAK1/JAK2 inhibition may be therapeutic in a range of inflammatory skin disorders such as psoriasis and AD. Clinical evaluation of topical INCB018424 is ongoing.

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.

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms.

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC(50)] = 281nM), and proliferation of JAK2V617F(+) Ba/F3 cells (IC(50) = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F(+) polycythemia vera patients (IC(50) = 67nM) versus healthy donors (IC(50) > 400nM). In a mouse model of JAK2V617F(+) MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs.

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.

Combined inhibition of Janus kinase 1/2 for the treatment of JAK2V617F-driven neoplasms: selective effects on mutant cells and improvements in measures of disease severity.

PURPOSE: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome-negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy. EXPERIMENTAL DESIGN: A novel inhibitor of JAK1/2 was characterized using kinase assays. Cellular effects of this compound were measured in cell lines bearing the JAK2V617F or JAK1V658F mutation, and its antiproliferative activity against primary polycythemiavera patient cells was determined using clonogenic assays. Antineoplastic activity in vivo was determined using a JAK2V617F-driven xenograft model, and effects of the compound on survival, organomegaly, body weight, and disease-associated inflammatory markers were measured. RESULTS: INCB16562 potently inhibited proliferation of cell lines and primary cells from PV patients carrying the JAK2V617F or JAK1V658F mutation by blocking JAK-STAT signaling and inducing apoptosis. In vivo, INCB16562 reduced malignant cell burden, reversed splenomegaly and normalized splenic architecture, improved body weight gains, and extended survival in a model of JAK2V617F-driven hematologic malignancy. Moreover, these mice suffered from markedly elevated levels of inflammatory cytokines, similar to advanced myeloproliferative disease patients, which was reversed upon treatment. CONCLUSIONS: These data showed that administration of the dual JAK1/2 inhibitor INCB16562 reduces malignant cell burden, normalizes spleen size and architecture, suppresses inflammatory cytokines, improves weight gain, and extends survival in a rodent model of JAK2V617F-driven hematologic malignancy. Thus, selective inhibitors of JAK1 and JAK2 represent a novel therapy for the patients with myeloproliferative diseases and other neoplasms associated with JAK dysregulation.

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.