RESUMO
Immunotoxicology/immunosafety science is rapidly evolving, with novel modalities and immuno-oncology among the primary drivers of new tools and technologies. The Immunosafety Working Group of IQ/DruSafe sought to better understand some of the key challenges in immunosafety evaluation, gaps in the science, and current limitations in methods and data interpretation. A survey was developed to provide a baseline understanding of the needs and challenges faced in immunosafety assessments, the tools currently being applied across the industry, and the impact of feedback received from regulatory agencies. This survey also focused on current practices and challenges in conducting the T-cell-dependent antibody response (TDAR) and the cytokine release assay (CRA). Respondents indicated that ICH S8 guidance was insufficient for the current needs of the industry portfolio of immunomodulators and novel modalities and should be updated. Other challenges/gaps identified included translation of nonclinical immunosafety assessments to the clinic, and lack of relevant nonclinical species and models in some cases. Key areas of emerging science that will add future value to immunotoxicity assessments include development of additional in vitro and microphysiological system models, as well as application of humanized mouse models. Efforts are ongoing in individual companies and consortia to address some of these gaps and emerging science.
Assuntos
Fatores Imunológicos , Humanos , Animais , Inquéritos e Questionários , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/toxicidade , Citocinas/imunologia , Medição de Risco , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Toxicidade/métodosRESUMO
Cytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation.
Assuntos
Neoplasias , Linfócitos T Citotóxicos , Animais , Primatas , Neoplasias/terapia , Citotoxicidade ImunológicaRESUMO
Immune responses are heavily involved in the regulation and pathogenesis of human diseases, including infectious diseases, inflammatory and autoimmune conditions, cancer, neurological disorders, and cardiometabolic syndromes. The immune system is considered a double-edged sword serving as a powerful host defense mechanism against infection and cancerous cells and causing detrimental tissue damage when the immune response is exaggerated or uncontrollable. One of the challenges in studying the efficacy and toxicity of drugs that target or modulate the immune system is the lack of suitable preclinical human models that are predictive of human response. Recent advancements in human microphysiological systems (MPS) have provided a promising in vitro platform to evaluate the response of immune organs ex vivo, to investigate the interaction of immune cells with non-lymphoid tissue cells, and to reduce the reliance on animals in preclinical studies. The development, regulation, trafficking, and responses of immune cells have been extensively studied in preclinical animal models and clinically, providing a wealth of knowledge by which to evaluate new in vitro models. Therefore, the application of immunocompetent MPS in drug discovery and development should first verify that the immune response in an MPS model recapitulates the complexity of the human immune physiology. This manuscript reviews biological functions of immune organ systems and tissue-resident immune cells and discusses contexts-of-use for commonly used immunocompetent and immune organ MPS models. Current perspective and recommendations are provided to guide the continued development of immune organ and immunocompetent MPS models and their application in drug discovery and development.
RESUMO
Activation of the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1) in T cells induces stabilization of proinflammatory AU-rich element (ARE)-bearing mRNAs, by triggering the nuclear-to-cytoplasmic translocation of the mRNA-binding and -stabilizing protein HuR. However, the mechanism by which LFA-1 engagement controls HuR localization is not known. Here, we identify and characterize four key regulators of LFA-1-induced changes in HuR activity: the p38 pathway kinase MK2 and the constitutive nuclear proteins hnRNPs C, H1 and K. LFA-1 engagement results in rapid, sequential activation of p38 and MK2. Post-LFA-1 activation, MK2 inducibly associates with both hnRNPC and HuR, resulting in the dissociation of HuR from hnRNPs C, H1 and K. Freed from the three hnRNPs, HuR translocates from the nucleus to the cytoplasm, and mediates the stabilization of labile cytokine transcripts. Our results suggest that the modulation of T cell cytokine mRNA half-life is an intricate process that is negatively regulated by hnRNPs C, H1 and K and requires MK2 as a critical activator.
Assuntos
Proteína Semelhante a ELAV 1/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade de RNA/fisiologia , Linfócitos T/metabolismo , Animais , Técnicas de Cultura de Células , Citoplasma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Jurkat , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Proteoma , RNA Mensageiro/metabolismo , Transdução de Sinais , Linfócitos T/citologiaRESUMO
Tofacitinib, an oral Janus kinase (JAK) inhibitor for treatment of rheumatoid arthritis, targets JAK1, JAK3, and to a lesser extent JAK2 and TYK2. JAK1/3 inhibition impairs gamma common chain cytokine receptor signaling, important in lymphocyte development, homeostasis and function. Adult and juvenile cynomolgus monkey and rat studies were conducted and the impact of tofacitinib on immune parameters (lymphoid tissues and lymphocyte subsets) and function (T-dependent antibody response (TDAR), mitogen-induced T cell proliferation) assessed. Tofacitinib administration decreased circulating T cells and NK cells in juvenile and adult animals of both species. B cell decreases were observed only in rats. These changes and decreased lymphoid tissue cellularity are consistent with the expected pharmacology of tofacitinib. No differences were observed between juvenile and adult animals, either in terms of doses at which effects were observed or differential effects on immune endpoints. Lymphomas were observed in three adult monkeys. Tofacitinib impaired the primary TDAR in juvenile monkeys, although a recall response was generated. Complete or partial reversal of the effects on the immune system was observed.
Assuntos
Envelhecimento/imunologia , Inibidores de Janus Quinases/toxicidade , Piperidinas/toxicidade , Pirimidinas/toxicidade , Pirróis/toxicidade , Administração Oral , Animais , Antígenos/imunologia , Contagem de Eritrócitos , Feminino , Hematócrito , Hemocianinas/imunologia , Hemoglobinas/análise , Inibidores de Janus Quinases/farmacocinética , Contagem de Leucócitos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Linfoma de Células B/induzido quimicamente , Macaca fascicularis , Masculino , Tamanho do Órgão/efeitos dos fármacos , Piperidinas/farmacocinética , Pirimidinas/farmacocinética , Pirróis/farmacocinética , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Baço/patologia , Timo/efeitos dos fármacos , Timo/patologia , Testes de Toxicidade CrônicaRESUMO
Aberrant blood vessel formation contributes to a wide variety of pathologies, and factors that regulate angiogenesis are attractive therapeutic targets. Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is a neuropilin-related transmembrane protein expressed in ECs; however, its potential effect on VEGF responses remains undefined. Here, we generated global and EC-specific Esdn knockout mice and demonstrated that ESDN promotes VEGF-induced human and murine EC proliferation and migration. Deletion of Esdn in the mouse interfered with adult and developmental angiogenesis, and knockdown of the Esdn homolog (dcbld2) in zebrafish impaired normal vascular development. Loss of ESDN in ECs blunted VEGF responses in vivo and attenuated VEGF-induced VEGFR-2 signaling without altering VEGF receptor or neuropilin expression. Finally, we found that ESDN associates with VEGFR-2 and regulates its complex formation with negative regulators of VEGF signaling, protein tyrosine phosphatases PTP1B and TC-PTP, and VE-cadherin. These findings establish ESDN as a regulator of VEGF responses in ECs that acts through a mechanism distinct from neuropilins. As such, ESDN may serve as a therapeutic target for angiogenesis regulation.
Assuntos
Endotélio Vascular/fisiologia , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Antígenos CD/fisiologia , Vasos Sanguíneos/embriologia , Caderinas/fisiologia , Células Cultivadas , Orelha Externa/irrigação sanguínea , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Isquemia/fisiopatologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropilinas/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/fisiologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Vasos Retinianos/crescimento & desenvolvimento , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
The development and regulatory approval of immunomodulatory pharmaceuticals to treat many human diseases has increased significantly over the last two decades. As discussed by FDA and ICH guidelines, all human pharmaceuticals in development should be evaluated for potential adverse effects on the immune system. Developmental immunotoxicology (DIT) focuses on the concern that early-life (during pre-/post-natal development) exposure to agents which target the immune system may result in enhanced susceptibility to immune-related disease (e.g., infection, autoimmunity, and cancer, particularly leukemia) compared to adults, unique effects not observed in adults, or more persistent effects in comparison to those following adult exposure. This article provides a substantive review of the literature and presents detailed considerations for DIT testing strategies with a specific focus on pharmaceuticals and biopharmaceuticals. In this regard, differences between small molecule and large molecule therapeutics will be considered, along with recommendations for best practices in the assessment of DIT during drug development. In addition, gaps in the DIT knowledge base and current testing strategies are identified. Finally, a summary of an ILSI-HESI-ITC sponsored Workshop conducted in 2010, entitled 'Developmental Immunotoxicity Testing of Pharmaceuticals' will be presented. This Workshop consisted of participants from the pharmaceutical, biotechnology, academic, and regulatory sectors, where many of the issues relating to DIT outlined in this review were discussed, key points of consensus reached, and current gaps in the science identified.
Assuntos
Anormalidades Induzidas por Medicamentos/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Desenvolvimento Embrionário/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Fatores Imunológicos/toxicidade , Exposição Materna/efeitos adversos , Animais , Educação , Feminino , Diretrizes para o Planejamento em Saúde , Sistema Imunitário/embriologia , Fatores Imunológicos/classificação , Preparações Farmacêuticas/classificação , Testes de ToxicidadeRESUMO
HuR is a member of the Drosophila Elav protein family that binds mRNA degradation sequences and prevents RNase-mediated degradation. Such HuR-mediated mRNA stabilization, which is stimulated by integrin engagement and is controlled at the level of HuR nuclear export, is critically involved in T-cell cytokine production. However, HuR's role in macrophage soluble factor production, in particular in response to angiogenic stimuli, has not yet been established. We show that the labile transcripts that encode vascular endothelial growth factor and matrix metalloproteinase-9 are stabilized when murine macrophages adhere to the ß(2) integrin ligand intercellular adhesion molecule-1. This mRNA stabilization response was absent in bone marrow-derived macrophages obtained from conditional macrophage-specific HuR knockout mice. The microvascular angiogenic response to an inflammatory stimulus (ie, subcutaneous polyvinyl alcohol sponge implantation) was markedly diminished in these macrophage HuR knockout mice despite the equal levels of macrophage localization to those observed in littermate wild-type controls. Furthermore, blood flow recovery and ischemic muscle neovascularization after femoral artery ligation were impaired in the conditional macrophage-specific HuR knockout mice. These results demonstrate that dynamic effects on mRNA, mediated by the RNA-binding and RNA-stabilizing protein HuR, are required for macrophage production of angiogenic factors, which play critical roles in the neovascular responses to a variety of stimuli, including tissue ischemia.
Assuntos
Indutores da Angiogênese/metabolismo , Antígenos CD18/fisiologia , Proteínas ELAV/fisiologia , Macrófagos/metabolismo , Neovascularização Patológica/metabolismo , Animais , Adesão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Proteínas ELAV/deficiência , Proteínas ELAV/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes , Membro Posterior/irrigação sanguínea , Inflamação/complicações , Isquemia/genética , Isquemia/fisiopatologia , Camundongos , Camundongos Knockout , Músculo Esquelético/irrigação sanguínea , Neovascularização Patológica/etiologia , Neovascularização Patológica/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
BACKGROUND: Engagement of the ß2 integrin, lymphocyte function-associated antigen-1 (LFA-1), results in stabilization of T cell mRNA transcripts containing AU-rich elements (AREs) by inducing rapid nuclear-to-cytosolic translocation of the RNA-stabilizing protein, HuR. However, little is known regarding integrin-induced signaling cascades that affect mRNA catabolism. This study examines the role of the GTPases, Rac 1 and Rac 2, and their downstream effectors, in the LFA-1-induced effects on mRNA. METHODOLOGY/PRINCIPAL FINDINGS: Engagement of LFA-1 to its ligand, ICAM-1, in human peripheral T cells resulted in rapid activation of Rac1 and Rac2. siRNA-mediated knockdown of either Rac1 or Rac2 prevented LFA-1-stimulated stabilization of the labile transcripts encoding IFN-γ and TNF-α, and integrin mediated IFN-γ mRNA stabilization was absent in T cells obtained from Rac2 gene-deleted mice. LFA-1 engagement-induced translocation of HuR and stabilization of TNF- α mRNA was lost in Jurkat cells deficient in the Rac guanine nucleotide exchange factor Vav-1 (J.Vav1). The transfection of J.Vav1 cells with constitutively active Rac1 or Rac2 stabilized a labile ß-globin reporter mRNA, in a HuR-dependent manner. Furthermore, LFA-1-mediated mRNA stabilization and HuR translocation in mouse splenic T cells was dependent on the phosphorylation of the mitogen-activated protein kinase kinase, MKK3, and its target MAP kinase p38MAPK, and lost in T cells obtained from MKK3 gene-deleted mice. CONCLUSIONS/SIGNIFICANCE: Collectively, these results demonstrate that LFA-1-induced stabilization of ARE-containing mRNAs in T cells is dependent on HuR, and occurs through the Vav-1, Rac1/2, MKK3 and p38MAPK signaling cascade. This pathway constitutes a molecular switch that enhances immune and pro-inflammatory gene expression in T cells undergoing adhesion at sites of activation and effector function.
Assuntos
Antígenos de Superfície/metabolismo , Citocinas/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , MAP Quinase Quinase 3/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas ELAV , Proteína Semelhante a ELAV 1 , GTP Fosfo-Hidrolases/metabolismo , Humanos , Integrinas/metabolismo , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Little is known about the tyrosine kinase c-Src's function in the systemic circulation, in particular its role in arterial responses to hormonal stimuli. In human aortic and venous endothelial cells, c-Src is indispensable for 17beta-estradiol (E2)-stimulated phosphatidylinositol 3-kinase/Akt/endothelial NO synthase (eNOS) pathway activation, a possible mechanism in E2-mediated vascular protection. Here we show that c-Src supports basal and E2-stimulated NO production and is required for E2-induced vasorelaxation in murine aortas. Only membrane c-Src is structurally and functionally involved in E2-induced eNOS activation. Independent of c-Src kinase activity, c-Src is associated with an N-terminally truncated estrogen receptor alpha variant (ER46) and eNOS in the plasma membrane through its "open" (substrate-accessible) conformation. In the presence of E2, c-Src kinase is activated by membrane ER46 and in turn phosphorylates ER46 for subsequent ER46 and c-Src membrane recruitment, the assembly of an eNOS-centered membrane macrocomplex, and membrane-initiated eNOS activation. Overall, these results provide insights into a critical role for the tyrosine kinase c-Src in estrogen-stimulated arterial responses, and in membrane-initiated rapid signal transduction, for which obligate complex assembly and localization require the c-Src substrate-accessible structure.
Assuntos
Aorta/enzimologia , Receptor alfa de Estrogênio/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Tirosina Quinases/metabolismo , Vasodilatação , Animais , Aorta/efeitos dos fármacos , Proteína Tirosina Quinase CSK , Membrana Celular/química , Membrana Celular/metabolismo , Ativação Enzimática , Estradiol/farmacologia , Receptor alfa de Estrogênio/análise , Receptor alfa de Estrogênio/genética , Humanos , Camundongos , Fosforilação , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/química , Splicing de RNA , Quinases da Família srcRESUMO
Lymphokine gene expression is a precisely regulated process in T cell-mediated immune responses. In this study we demonstrate that engagement of the beta(2) integrin LFA-1 in human peripheral T cells markedly extends the half-life of TNF-alpha, GM-CSF, and IL-3 mRNA, as well as a chimeric beta-globin mRNA reporter construct containing a strongly destabilizing class II AU-rich element from the GM-CSF mRNA 3'-untranslated region. This integrin-enhanced mRNA stability leads to augmented protein production, as determined by TNF-alpha ELISPOT assays. Furthermore, T cell stimulation by LFA-1 promotes rapid nuclear-to-cytoplasmic translocation of the mRNA-stabilizing protein HuR, which in turn is capable of binding an AU-rich element sequence in vitro. Abrogation of HuR function by use of inhibitory peptides, or marked reduction of HuR levels by RNA interference, prevents LFA-1 engagement-mediated stabilization of T cell TNF-alpha or IFN-gamma transcripts, respectively. Thus, HuR-mediated mRNA stabilization, stimulated by integrin engagement and controlled at the level of HuR nuclear export, is critically involved in T cell activation.
Assuntos
Antígenos de Superfície/metabolismo , Núcleo Celular/metabolismo , Citocinas/genética , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/farmacologia , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos de Superfície/genética , Sequência de Bases , Antígenos CD28/farmacologia , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Antígeno-1 Associado à Função Linfocitária/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
17beta-Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17beta-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17beta-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17beta-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17beta-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17beta-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17beta-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.