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BACKGROUND: Trastuzumab deruxtecan has shown encouraging activity in patients with treatment-refractory HER2-positive, RAS wild-type and BRAF wild-type metastatic colorectal cancer. Dose optimisation and further antitumour assessments in patients with RAS mutations and those with previous anti-HER2 therapy are warranted. We aimed to evaluate two doses of trastuzumab deruxtecan (5·4 mg/kg and 6·4 mg/kg) to establish the recommended dose in patients with pretreated HER2-positive, RAS wild-type or mutant metastatic colorectal cancer. METHODS: DESTINY-CRC02 was a multicentre, randomised, two-stage, two-arm, phase 2 study done in 53 research hospitals and medical centres in Australia, Belgium, France, Italy, Japan, South Korea, Spain, Taiwan, the UK, and the USA. Eligible patients were aged 18 years and older or 20 years and older (depending on region) with pretreated pathologically documented, unresectable, recurrent, or metastatic HER2-positive, and RAS wild-type or mutant colorectal cancer. Patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and have received previous chemotherapy, and anti-EGFR, anti-VEGF, or anti-PD-L1 therapy, if clinically indicated. In stage 1, patients were randomly assigned (1:1), via a secure interactive response technology system, to receive 5·4 mg/kg or 6·4 mg/kg trastuzumab deruxtecan administered intravenously every 21 days. Stratification factors were ECOG performance status, HER2 status, and RAS status. In stage 2, patients were assigned into the 5·4 mg/kg treatment group only. The primary endpoint was confirmed objective response rate by blinded independent central review, assessed in all patients for whom treatment was assigned (full analysis set). Safety was assessed in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, NCT04744831, and is ongoing (not recruiting). FINDINGS: Between March 5, 2021, and March 29, 2022, 135 patients were centrally screened, 122 of whom were enrolled. In stage 1, 40 patients each were randomly assigned to receive trastuzumab deruxtecan 5·4 mg/kg and 6·4 mg/kg. In stage 2, an additional 42 patients were enrolled in the 5·4 mg/kg group. 64 (52%) participants were male and 58 (48%) were female. The median duration of follow-up was 8·9 months (IQR 6·7-10·5) in the 5·4 mg/kg group and 10·3 months (5·9-12·7) in the 6·4 mg/kg group. The confirmed objective response rate by blinded independent central review was 37·8% (31/82 [95% CI 27·3-49·2]) in the 5·4 mg/kg group and 27·5% (11/40 [14·6-43·9]) in the 6·4 mg/kg group. 34 (41%) of 83 patients in the 5·4 mg/kg group and 19 (49%) of 39 in the 6·4 mg/kg group had grade 3 or worse drug-related treatment-emergent adverse events. The most common grade 3 or worse drug-related treatment-emergent adverse events were neutrophil count decreased (13 [16%] of 83 patients), anaemia (six [7%]), nausea (six [7%]), and white blood cell count decreased (five [6%]) in the 5·4 mg/kg group; and were neutrophil count decreased (10 [26%] of 39 patients), anaemia (eight [21%]), platelet count decreased (four [10%]), and white blood cell count decreased (four [10%]) in the 6·4 mg/kg group. Drug-related serious adverse events occurred in 11 (13%) of 83 patients in the 5·4 mg/kg group and six (15%) of 39 patients in the 6·4 mg/kg group; the most common in the 5·4 mg/kg group was nausea (three [4%] patients) and the most common in the 6·4 mg/kg group were fatigue (two [5%] patients), neutropenia (two [5%]), and thrombocytopenia (two [5%]). A drug-related treatment-emergent adverse event related to death occurred in one (1%) patient in the 5·4 mg/kg group (due to hepatic failure). Adjudicated drug-related interstitial lung disease or pneumonitis events were observed in seven (8%) patients in the 5·4 mg/kg group (all grade 1 or 2) and in five (13%) patients in the 6·4 mg/kg group (four grade 1 or 2; one grade 5). INTERPRETATION: The promising antitumour activity and favourable safety profile support trastuzumab deruxtecan 5·4 mg/kg as the optimal single-agent dose for patients with pretreated HER2-positive metastatic colorectal cancer, including those with RAS mutations, previous anti-HER2 therapy, or both. FUNDING: Daiichi Sankyo and AstraZeneca.
Assuntos
Neoplasias Colorretais , Receptor ErbB-2 , Trastuzumab , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Trastuzumab/uso terapêutico , Trastuzumab/administração & dosagem , Feminino , Masculino , Receptor ErbB-2/genética , Pessoa de Meia-Idade , Idoso , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Camptotecina/administração & dosagem , Adulto , Antineoplásicos Imunológicos/uso terapêutico , Antineoplásicos Imunológicos/efeitos adversos , Mutação , ImunoconjugadosRESUMO
Gonadotropin-releasing hormone (GnRH) neurons are master regulators of the reproductive axis in vertebrates. During early mammalian embryogenesis, GnRH1 neurons emerge in the nasal/olfactory placode. These neurons undertake a long-distance migration, moving from the nose to the preoptic area and hypothalamus. While significant advances have been made in understanding the functional importance of the GnRH1 neurons in reproduction, where GnRH1 neurons come from and how are they specified during early development is still under debate. In addition to the GnRH1 gene, most vertebrate species including humans have one or two additional GnRH genes. Compared to the GnRH1 neurons, much less is known about the development and regulation of GnRH2 neuron and GnRH3 neurons. The objective of this article is to review what is currently known about GnRH neuron development. We will survey various cell autonomous and non-autonomous factors implicated in the regulation of GnRH neuron development. Finally, we will discuss emerging tools and new approaches to resolve open questions pertaining to GnRH neuron development.
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Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Vertebrados/metabolismo , Animais , Hormônio Liberador de Gonadotropina/genética , Humanos , Modelos Biológicos , NeurogêneseRESUMO
Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.
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Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Animais , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Humanos , Camundongos , Relação Estrutura-Atividade , Linfócitos T Reguladores/metabolismoRESUMO
BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Asma/terapia , Modelos Animais de Doenças , Hipersensibilidade/terapia , Inflamação/terapia , Receptores de Citocinas/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Asma/imunologia , Cricetinae , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Humanos , Hipersensibilidade/imunologia , Inflamação/imunologia , Macaca fascicularis/imunologia , Receptores de Citocinas/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Betaína-Homocisteína S-Metiltransferase/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Falência Hepática Aguda/genética , Vitamina U/metabolismo , Acetaminofen/metabolismo , Acetaminofen/farmacocinética , Animais , Betaína-Homocisteína S-Metiltransferase/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Dieta , Perfilação da Expressão Gênica , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Falência Hepática Aguda/prevenção & controle , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNARESUMO
Lysophosphatidic acid (LPA) is a class of bioactive lyso-phospholipid that mediates most of its biological effects through a family of G protein-coupled receptors of which six have been identified. The role of the LPA pathway in driving chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) has gained considerable academic and industry attention. Modulation of the pulmonary artery endothelial barrier function by the LPA1 receptor has been shown to drive pulmonary fibrosis in murine models of disease. The purpose of this study was (i) to assess the effect of LPA on the barrier function of human pulmonary arterial (HPAEC) and microvascular (HMVEC) endothelial cells and (ii) to identify the LPA receptor subtype(s) responsible for changes in human pulmonary endothelial cell permeability using LPA receptor antagonists and siRNA technology. Analysis of the LPA receptor subtype expression demonstrated predominant expression of LPA2 and LPA6 receptor subtypes in both HPAECs and HMVECs. HPAECs also exhibit low expression of LPA1, LPA3, and LPA4 receptor subtypes. Treatment of cells with increasing concentrations of LPA caused loss of barrier function in HPAECs but not HMVECs, despite both cell types exhibiting very similar LPA receptor expression profiles. The LPA-mediated loss of barrier function in HPAECs appears to be independent of the LPA1 receptor and likely to be mediated via the LPA6 receptor although we cannot exclude an additional role for the LPA2 and LPA4 receptors in mediating these effects. These results suggest cell-specific mechanisms exist in human pulmonary endothelial cells to permit regulation of barrier function downstream of LPA receptors. More importantly, our data indicate that selective LPA1 receptor antagonism may be insufficient for therapeutic use in pulmonary diseases where impaired endothelial barrier function is related to disease initiation and progression.
Assuntos
Células Endoteliais/citologia , Pulmão/citologia , Lisofosfolipídeos/metabolismo , Artérias/patologia , Cálcio/química , Primers do DNA/genética , Relação Dose-Resposta a Droga , Endotélio/citologia , Humanos , Fibrose Pulmonar Idiopática/patologia , Microcirculação , Microscopia de Fluorescência/métodos , Permeabilidade , Reação em Cadeia da Polimerase/métodos , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fatores de TempoRESUMO
Microarray analysis of multiple sclerosis (MS) lesions obtained at autopsy revealed increased transcripts of genes encoding inflammatory cytokines, particularly interleukin-6 and -17, interferon-gamma and associated downstream pathways. Comparison of two poles of MS pathology--acute lesions with inflammation versus 'silent' lesions without inflammation--revealed differentially transcribed genes. Some products of these genes were chosen as targets for therapy of experimental autoimmune encephalomyelitis (EAE) in mice. Granulocyte colony-stimulating factor is upregulated in acute, but not in chronic, MS lesions, and the effect on ameliorating EAE is more pronounced in the acute phase, in contrast to knocking out the immunoglobulin Fc receptor common gamma chain where the effect is greatest on chronic disease. These results in EAE corroborate the microarray studies on MS lesions. Large-scale analysis of transcripts in MS lesions elucidates new aspects of pathology and opens possibilities for therapy.
Assuntos
Encefalomielite Autoimune Experimental/genética , Interferon gama/genética , Interleucina-17/genética , Interleucina-6/genética , Esclerose Múltipla/genética , Análise de Sequência com Séries de Oligonucleotídeos , Doença Aguda , Animais , Autopsia , Doença Crônica , Encefalomielite Autoimune Experimental/patologia , Feminino , Fator Estimulador de Colônias de Granulócitos/fisiologia , Humanos , Inflamação/genética , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Receptores Fc/fisiologia , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
[This corrects the article DOI: 10.3389/fendo.2020.00100.].
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Insulin-like growth factor (IGF) signaling is regulated by a conserved family of IGF binding proteins (IGFBPs) in vertebrates. Among the six distinct types of IGFBPs, IGFBP-5 is the most highly conserved across species and has the broadest range of biological activities. IGFBP-5 is expressed in diverse cell types, and its expression level is regulated by a variety of signaling pathways in different contexts. IGFBP-5 can exert a range of biological actions including prolonging the half-life of IGFs in the circulation, inhibition of IGF signaling by competing with the IGF-1 receptor for ligand binding, concentrating IGFs in certain cells and tissues, and potentiation of IGF signaling by delivery of IGFs to the IGF-1 receptor. IGFBP-5 also has IGF-independent activities and is even detected in the nucleus. Its broad biological activities make IGFBP-5 an excellent representative for understanding IGFBP functions. Despite its evolutionary conservation and numerous biological activities, knockout of IGFBP-5 in mice produced only a negligible phenotype. Recent research has begun to explain this paradox by demonstrating cell type-specific and physiological/pathological context-dependent roles for IGFBP-5. In this review, we survey and discuss what is currently known about IGFBP-5 in normal physiology and human disease. Based on recent in vivo genetic evidence, we suggest that IGFBP-5 is a multifunctional protein with the ability to act as a molecular switch to conditionally regulate IGF signaling.
Assuntos
Doença/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Animais , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Camundongos , Camundongos KnockoutRESUMO
Staff and provider engagement leads to better quality and experience of care and less turnover and burnout. In this program, we describe an approach to better understand underlying factors that lead to low staff and provider engagement and address such factors by creating actionable plans that drive improved engagement measures. Focus groups were conducted with staff, advance practice providers, and faculty to better understand low scored areas in an annual third-party engagement survey. Focus group results were analyzed, and thematic action plans were then developed by a leadership team. These plans and the status of addressing the identified issues were published and disseminated back to all staff and providers using a "stoplight report." The leadership team met every 2 to 4 weeks until all issues were addressed and communicated back to the department. The subsequent year's engagement scores statistically increased across all engagement score domains for both staff and faculty. We conclude that using a qualitative approach to understanding low-scored engagement domains will allow a deeper and authentic understanding of the root factors that drive low engagement scores. This approach allows teams to develop responsive action plans, resulting in higher engagement scores, which will eventually lead to better service and care to patients.
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Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.
Assuntos
Farmacogenética/métodos , Varfarina/farmacologia , Animais , Biotransformação , Mapeamento Cromossômico , Sistema Enzimático do Citocromo P-450/genética , Variação Genética , Haplótipos , Masculino , Camundongos , Camundongos Endogâmicos , Isoformas de Proteínas , Especificidade da Espécie , Varfarina/metabolismoRESUMO
Insulin-like growth factors (IGFs) are key growth-promoting peptides that act as both endocrine hormones and autocrine/paracrine growth factors. In the bloodstream and in local tissues, most IGF molecules are bound by one of the members of the IGF-binding protein (IGFBP) family, of which six distinct types exist. These proteins bind to IGF with an equal or greater affinity than the IGF1 receptor and are thus in a key position to regulate IGF signaling globally and locally. Binding to an IGFBP increases the half-life of IGF in the circulation and blocks its potential binding to the insulin receptor. In addition to these classical roles, IGFBPs have been shown to modulate IGF signaling locally under various conditions. Although members of the IGFBP family share significant sequence homology, they each have unique structural features and play distinct roles. These IGFBP genes also have different modes of regulation and distinct expression patterns. Some IGFBPs have been found to bind to their own receptors or to translocate into the interior compartments of cells where they may execute IGF-independent actions. In spite of this functional and regulatory diversity, it has been puzzling that loss-of-function studies have yielded relatively little information about the physiological functions of IGFBPs. In this review, we suggest that evolution has tended to retain an array of IGFBPs in order to facilitate fine-tuning of IGF signaling. We explore the emerging explanation that many IGFBP functions have evolved to allow the targeted adjustment of IGF signaling under stressful or irregular conditions, which would likely not be revealed in a standard laboratory setting.
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Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.
Assuntos
Bronquiolite Viral/genética , Canais de Cloreto/genética , Mucoproteínas/genética , Doenças Respiratórias/genética , Animais , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/metabolismo , Bronquiolite Viral/metabolismo , Bronquiolite Viral/patologia , Canais de Cloreto/metabolismo , Cruzamentos Genéticos , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Metaplasia/genética , Metaplasia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucoproteínas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/patologia , Vírus SendaiRESUMO
The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes. For this reason, 5-LO activity is important for immune defense, whereas improper regulation contributes to pathogenesis, including chronic inflammation, asthma and atherosclerosis. Like all lipoxygenases, the 5-LO protein consists of two domains, a regulatory domain and a catalytic domain. Naturally, the regulatory domain determines catalytic activity and controls leukotriene synthesis. This domain shares features with classical C2 domains in that it has a beta-sandwich structure and binds calcium, nucleotides and phospholipids. However, important structural features place this domain in a distinct family, the PLATs (for Polycystin-1, Lipoxygenase, alpha-Toxin). In this review, we summarize our current understanding of the three dimensional organization of this important component of the 5-LO molecule. In addition, we point to findings from structural analyses of related proteins to suggest further details relating 5-LO structure to function.
Assuntos
Araquidonato 5-Lipoxigenase/química , Sinalização do Cálcio/fisiologia , Sequência de Aminoácidos , Araquidonato 5-Lipoxigenase/metabolismo , Cálcio/metabolismo , Humanos , Leucotrienos/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFß was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Bleomicina/efeitos adversos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Bleomicina/administração & dosagem , Análise por Conglomerados , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Imidazóis/farmacologia , Inflamação/induzido quimicamente , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Mitose/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinoxalinas/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Anilidas/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Calcineurina/metabolismo , Inibidores de Calcineurina , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Ciclosporina/farmacologia , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Ratos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Molécula 1 de Interação Estromal , Tacrolimo/farmacologia , Tiadiazóis/farmacologiaRESUMO
Hormones regulate growth, development, metabolism, and other complex processes in multicellular animals. For many years it has been suggested that hormones may also influence the rate of the aging process. Aging is a multifactorial process that causes biological systems to break down and cease to function in adult organisms as time passes, eventually leading to death. The exact underlying causes of the aging process remain a topic for debate, and clues that may shed light on these causes are eagerly sought after. In the last two decades, gene mutations that result in delayed aging and extended longevity have been discovered, and many of the affected genes have been components of endocrine signaling pathways. In this review we summarize the current knowledge on the roles of endocrine signaling in the regulation of aging and longevity in various animals. We begin by discussing the notion that conserved systems, including endocrine signaling pathways, "regulate" the aging process. Findings from the major model organisms: worms, flies, and rodents, are then outlined. Unique lessons from studies of non-traditional models: bees, salmon, and naked mole rats, are also discussed. Finally, we summarize the endocrinology of aging in humans, including changes in hormone levels with age, and the involvement of hormones in aging-related diseases. The most well studied and widely conserved endocrine pathway that affects aging is the insulin/insulin-like growth factor system. Mutations in genes of this pathway increase the lifespan of worms, flies, and mice. Population genetic evidence also suggests this pathway's involvement in human aging. Other hormones including steroids have been linked to aging only in a subset of the models studied. Because of the value of comparative studies, it is suggested that the aging field could benefit from adoption of additional model organisms.
RESUMO
UNLABELLED: The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus. IMPORTANCE: Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Carga Viral , Animais , Citocinas/análise , Modelos Animais de Doenças , Mediadores da Inflamação/análise , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Pulmão/química , Pulmão/patologia , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/mortalidade , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de SobrevidaRESUMO
The glycol ether solvents 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) produce testicular toxicity characterized by spermatocyte degeneration, while a similar glycol ether, 2-butoxyethanol (2-BE), has no testicular effects. The goal of the current study was to better understand the mechanism of glycol ether testicular toxicity through gene expression profiling and functional classification of differentially expressed genes. Male rats were administered 2-ME (150 and 50mg/kg/day), 2-EE (500 mg/kg/day), 2-BE (125 mg/kg/day), or vehicle for 3 days, and testes were collected for histopathological and gene expression analysis. Histopathological changes in the testes were observed only in animals given 150 mg/kg/day 2-ME, consisting of degeneration and necrosis of spermatocytes and reductions in spermatocyte numbers. Microarray analysis of testicular samples from these animals revealed a large number of differentially expressed genes from animals exposed to 2-EE or to 50mg/kg or 150 mg/kg 2-ME (>900 each at >1.5-fold changed), compared to 28 genes from 2-BE treated animals. Expression Analysis Systematic Explorer (EASE) analysis of these genes demonstrated statistical enrichment in genes in categories including protein transport, endocytosis, protein kinase activity, cell cycle, and meiosis. Quantitative PCR confirmation of select genes confirmed increased expression of the actin binding protein cortactin and the transcription factor Wilm's tumor 1 (Wt1) following 2-ME exposure. Increased localization of cortactin in abnormal spermatocytes was also observed by immunohistochemistry, consistent with a possible role for this protein in the mechanism of toxicity.
Assuntos
Cortactina/biossíntese , Etilenoglicóis/toxicidade , Espermatócitos/metabolismo , Teratogênicos/toxicidade , Testículo/metabolismo , Animais , Cortactina/genética , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Imuno-Histoquímica , Masculino , RNA/biossíntese , RNA/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatócitos/efeitos dos fármacos , Testículo/efeitos dos fármacos , Proteínas WT1/biossíntese , Proteínas WT1/genéticaRESUMO
To understand the pathogenesis of chronic inflammatory disease, we analyzed an experimental mouse model of chronic lung disease with pathology that resembles asthma and chronic obstructive pulmonary disease (COPD) in humans. In this model, chronic lung disease develops after an infection with a common type of respiratory virus is cleared to only trace levels of noninfectious virus. Chronic inflammatory disease is generally thought to depend on an altered adaptive immune response. However, here we find that this type of disease arises independently of an adaptive immune response and is driven instead by interleukin-13 produced by macrophages that have been stimulated by CD1d-dependent T cell receptor-invariant natural killer T (NKT) cells. This innate immune axis is also activated in the lungs of humans with chronic airway disease due to asthma or COPD. These findings provide new insight into the pathogenesis of chronic inflammatory disease with the discovery that the transition from respiratory viral infection into chronic lung disease requires persistent activation of a previously undescribed NKT cell-macrophage innate immune axis.