RESUMO
Individuals who spontaneously clear hepatitis C virus (HCV) infection have demonstrated evidence of partial protective immunity, whereas treatment-induced clearance provides little or no protection against reinfection. We aimed to investigate whether treatment of acute HCV infection with direct-acting antivirals (DAA) prevents establishment of, or reverses, T-cell exhaustion, leading to a virus-specific T-cell immune profile more similar to that seen in spontaneous clearance. The magnitude and breadth of HCV-specific T-cell responses before and after DAA or interferon-based therapy in acute or chronic HCV were compared to those of participants with spontaneous clearance of infection, using Enzyme-linked Immunospot (ELISPOT). PBMCs were available for 55 patients comprising 4 groups: spontaneous clearance (n = 17), acute interferon (n = 14), acute DAA (n = 13) and chronic DAA (n = 11). After controlling for sex, the magnitude of post-treatment HCV-specific responses after acute DAA treatment was greater than after chronic DAA or acute IFN treatment and similar to those found in spontaneous clearers. However, spontaneous clearers responded to more HCV peptide pools indicating greater breadth of response. In conclusion, early treatment with DAAs may prevent or reverse some degree of immune exhaustion and result in stronger HCV-specific responses post-treatment. However, individuals with spontaneous clearance had broader HCV-specific responses.
Assuntos
Hepatite C Crônica , Hepatite C , Humanos , Hepacivirus , Antivirais/uso terapêutico , Antivirais/farmacologia , Hepatite C Crônica/tratamento farmacológico , Interferons/uso terapêutico , ImunidadeRESUMO
UNLABELLED: Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE: Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.
Assuntos
Endopeptidases/genética , Hepatócitos/imunologia , Hepatócitos/metabolismo , Interferon-alfa/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina Tiolesterase/genética , Animais , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Hepacivirus/fisiologia , Hepatite C Crônica/imunologia , Hepatócitos/virologia , Humanos , Imunidade Inata , Inflamação/virologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interleucina-10/farmacologia , Interleucina-6/farmacologia , Isquemia/sangue , Fígado/irrigação sanguínea , Fígado/lesões , Fígado/patologia , Camundongos , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Ubiquitina Tiolesterase/metabolismoRESUMO
UNLABELLED: Systemic levels of interferon-gamma-inducible protein-10 (IP-10) are predictive of treatment-induced clearance in chronic hepatitis C virus (HCV). In the present study, factors associated with plasma IP-10 levels at the time of acute HCV detection and the association between IP-10 levels and spontaneous clearance were assessed in three cohorts of acute HCV infection. Among 299 individuals, 245 (181 male, 47 human immunodeficiency virus-positive [HIV+]) were HCV RNA+ at acute HCV detection. In adjusted analysis, factors independently associated with IP-10 levels ≥150 pg/mL (median level) included HCV RNA levels >6 log IU/mL, HIV coinfection and non-Aboriginal ethnicity. Among 245 HCV RNA+ at acute HCV detection, 214 were untreated (n = 137) or had persistent infection (infection duration ≥26 weeks) at treatment initiation (n = 77). Spontaneous clearance occurred in 14% (29 of 214). Individuals without spontaneous clearance had significantly higher mean plasma IP-10 levels at the time of acute HCV detection than those with clearance (248 ± 32 versus 142 ± 22 pg/mL, P = 0.008). The proportion of individuals with spontaneous clearance was 0% (0 of 22, P = 0.048) and 16% (27 of 165) and in those with and without plasma IP-10 levels ≥380 pg/mL. In adjusted analyses, favorable IL28B genotype was associated with spontaneous clearance, while higher HCV RNA level was independently associated with lower odds of spontaneous clearance. CONCLUSION: High IP-10 levels at acute HCV detection were associated with failure to spontaneously clear HCV. Patients with acute HCV and high baseline IP-10 levels, particularly >380 pg/mL, should be considered for early therapeutic intervention, and those with low levels should defer therapy for potential spontaneous clearance. (HEPATOLOGY 2013;).
Assuntos
Quimiocina CXCL10/sangue , Hepatite C/sangue , RNA Viral/sangue , Adulto , Biomarcadores/sangue , Feminino , Hepatite C/virologia , Humanos , Modelos Logísticos , Masculino , Adulto JovemRESUMO
BACKGROUND & AIMS: Less than half of patients infected with hepatitis C virus (HCV) achieve sustained viral clearance after pegylated interferon (peginterferon) and ribavirin therapy. S-adenosyl methionine (SAMe) improves interferon signaling in cell culture. We assessed the effect of SAMe on the kinetics of the early antiviral response and interferon signaling in nonresponders to previous antiviral therapy and investigated the mechanisms involved. METHODS: Nonresponders with HCV genotype 1 were given peginterferon alfa-2a and ribavirin for 2 weeks (course A, baseline/control). After 1 month, patients received SAMe (1600 mg daily) for 2 weeks and then peginterferon and ribavirin for 48 weeks (course B; completed by 21 of 24 patients). Viral kinetics and interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) were compared between courses. RESULTS: The decrease in HCV RNA from 0 to 48 hours (phase 1) was similar with and without SAMe. However, the second phase slope of viral decline was improved with SAMe (course A, 0.11 ± 0.04 log(10) IU/mL/wk; course B, 0.27 ± 0.06; P = .009); 11 patients (53%) achieved an early virological response, and 10 (48%) had undetectable HCV RNA by week 24. Induction of ISGs in PBMCs was significantly greater during course B. In cultured cells, SAMe increased induction of ISGs and the antiviral effects of interferon by increasing STAT1 methylation, possibly affecting STAT1-DNA binding. CONCLUSIONS: The addition of SAMe to peginterferon and ribavirin improves the early viral kinetics and increases ISG induction in nonresponders to previous therapy. SAMe might be a useful adjunct to peginterferon-based therapies in chronic HCV infection.
Assuntos
Antivirais/administração & dosagem , Hepatite C/tratamento farmacológico , Interferon-alfa/administração & dosagem , Leucócitos Mononucleares/efeitos dos fármacos , Polietilenoglicóis/administração & dosagem , Ribavirina/administração & dosagem , S-Adenosilmetionina/administração & dosagem , Adulto , Idoso , Linhagem Celular Tumoral , Citocinas/genética , Esquema de Medicação , Quimioterapia Combinada , Feminino , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/genética , Humanos , Interferon alfa-2 , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Fosforilação , Proteínas/genética , RNA Mensageiro/metabolismo , RNA Viral/sangue , Proteínas Recombinantes , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Falha de Tratamento , Ubiquitinas/genética , Carga ViralRESUMO
MEG2, a protein tyrosine phosphatase with a unique NH2-terminal lipid-binding domain, binds to and is modulated by the polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3. Recent data implicate MEG2 in vesicle fusion events in leukocytes. Through the genesis of Meg2-deficient mice, we demonstrate that Meg2-/- embryos manifest hemorrhages, neural tube defects including exencephaly and meningomyeloceles, cerebral infarctions, abnormal bone development, and >90% late embryonic lethality. T lymphocytes and platelets isolated from recombination activating gene 2-/- mice transplanted with Meg2-/- embryonic liver-derived hematopoietic progenitor cells showed profound defects in activation that, in T lymphocytes, was attributable to impaired interleukin 2 secretion. Ultrastructural analysis of these lymphocytes revealed near complete absence of mature secretory vesicles. Taken together, these observations suggest that MEG2-mediated modulation of secretory vesicle genesis and function plays an essential role in neural tube, vascular, and bone development as well as activation of mature platelets and lymphocytes.
Assuntos
Desenvolvimento Embrionário/fisiologia , Ativação Linfocitária/fisiologia , Ativação Plaquetária/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Desenvolvimento Ósseo/fisiologia , Infarto Cerebral/genética , Infarto Cerebral/patologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Hemorragia/genética , Hemorragia/patologia , Fígado/embriologia , Fígado/patologia , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/ultraestrutura , Proteínas Tirosina Fosfatases/deficiência , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não Receptoras , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Linfócitos T/metabolismo , Linfócitos T/ultraestruturaRESUMO
The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.
Assuntos
Fusão de Membrana , Proteínas Tirosina Fosfatases/fisiologia , Vesículas Secretórias/fisiologia , Humanos , Membranas Intracelulares/fisiologia , Células Jurkat , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Proteínas Sensíveis a N-Etilmaleimida , Fosforilação , Fosfotirosina , Ligação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases não Receptoras , Proteínas Qa-SNARE , Vesículas Secretórias/enzimologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
The Type I Interferon family of cytokines all act through the same cell surface receptor and induce phosphorylation of the same subset of response regulators of the STAT family. Despite their shared receptor, different Type I Interferons have different functions during immune response to infection. In particular, they differ in the potency of their induced anti-viral and anti-proliferative responses in target cells. It remains not fully understood how these functional differences can arise in a ligand-specific manner both at the level of STAT phosphorylation and the downstream function. We use a minimal computational model of Type I Interferon signaling, focusing on Interferon-α and Interferon-ß. We validate the model with quantitative experimental data to identify the key determinants of specificity and functional plasticity in Type I Interferon signaling. We investigate different mechanisms of signal discrimination, and how multiple system components such as binding affinity, receptor expression levels and their variability, receptor internalization, short-term negative feedback by SOCS1 protein, and differential receptor expression play together to ensure ligand specificity on the level of STAT phosphorylation. Based on these results, we propose phenomenological functional mappings from STAT activation to downstream anti-viral and anti-proliferative activity to investigate differential signal processing steps downstream of STAT phosphorylation. We find that the negative feedback by the protein USP18, which enhances differences in signaling between Interferons via ligand-dependent refractoriness, can give rise to functional plasticity in Interferon-α and Interferon-ß signaling, and explore other factors that control functional plasticity. Beyond Type I Interferon signaling, our results have a broad applicability to questions of signaling specificity and functional plasticity in signaling systems with multiple ligands acting through a bottleneck of a small number of shared receptors.
Assuntos
Interferon-alfa/fisiologia , Interferon beta/fisiologia , Modelos Imunológicos , Receptor Cross-Talk/fisiologia , Receptor de Interferon alfa e beta/fisiologia , Transdução de Sinais/fisiologia , Animais , Simulação por Computador , Dimerização , Retroalimentação Fisiológica , Feminino , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Mapeamento de Interação de Proteínas , Fatores de Transcrição STAT/metabolismo , Baço/citologia , Proteína 1 Supressora da Sinalização de Citocina/fisiologia , Linfócitos T/imunologia , Ubiquitina TiolesteraseRESUMO
OBJECTIVES: Preconditioning of cells or organs by transient sublethal ischemia-reperfusion (IR), termed ischemic preconditioning (IPC), protects the cell or organ from a subsequent prolonged ischemic insult. The mechanisms of this effect remain to be fully elucidated. We have recently reported that IPC of a forearm results in alterations in gene expression profiles of circulating polymorphonuclear leukocytes. The goal of the current study was to determine if the observed changes in gene expression lead to functional changes in neutrophils. METHODS: We examined the effect of repetitive transient human forearm ischemia (three cycles of 5 min ischemia, followed by 5 min of reperfusion) on the function of circulating neutrophils. Neutrophil functions were examined before, after 1 d, and after 10 d of daily transient forearm ischemia. To modulate IR-induced inflammation the neutrophils were stimulated with N-formyl-methionyl-leucyl phenylalanine (FMLP) and lipopolysaccharide (LPS). RESULTS: Neutrophil adhesion was significantly decreased on day 1 and remained low on day 10 (P = 0.0149) without significant change in CD11b expression. Phagocytosis was significantly suppressed on day 10 compared with day 0 (P < 0.0001). Extracellular cytokine levels were low in the absence of an exogenous stimulus but stimulation with LPS induced significant changes on day 10. We observed a trend in reduction of apoptosis on day 1 and day 10 that did not reach statistical significance (P < 0.08). CONCLUSION: This study indicates that repetitive IPC of the forearm results in substantial alterations in neutrophil function, including reduced adhesion, exocytosis, phagocytosis, and modified cytokine secretion.
Assuntos
Precondicionamento Isquêmico , Neutrófilos/fisiologia , Adulto , Apoptose , Antígeno CD11b/análise , Adesão Celular , Citocinas/biossíntese , Grânulos Citoplasmáticos/metabolismo , Exocitose , Humanos , Estudos Longitudinais , Masculino , FagocitoseRESUMO
RATIONALE: A well-known clinical paradox is that severe bacterial infections persist in the lungs of patients with cystic fibrosis (CF) despite the abundance of polymorphonuclear neutrophils (PMN) and the presence of a high concentration of human neutrophil peptides (HNP), both of which are expected to kill the bacteria but fail to do so. The mechanisms remain unknown. OBJECTIVES: This study examined several possible mechanisms to understand this paradox. METHODS: PMN were isolated from sputum and blood of subjects with and without CF or non-CF bronchiectasis for phagocytic assays. HNP isolated from patients with CF were used to stimulate healthy PMN followed by phagocytic tests. MEASUREMENTS AND MAIN RESULTS: PMN isolated from the sputum of the bronchiectatic patients display defective phagocytosis that correlated with high concentrations of HNP in the lung. When healthy PMN were incubated with HNP, decreased phagocytic capacity was observed in association with depressed surface Fc gamma RIII, actin-filament remodeling, enhanced intracellular Ca(2+), and degranulation. Treatment of PMN with an intracellular Ca(2+) blocker or alpha1-proteinase inhibitor to attenuate the activity of HNP largely prevented the HNP-induced phagocytic deficiency. Intratracheal instillation of HNP in Pallid mice (genetically deficient in alpha1-proteinase inhibitor) resulted in a greater PMN lung infiltration and phagocytic deficiency compared with wild-type mice. CONCLUSIONS: HNP or PMN alone exert antimicrobial ability, which was lost as a result of their interaction. These effects of HNP may help explain the clinical paradox seen in patients with inflammatory lung diseases, suggesting HNP as a novel target for clinical therapy.
Assuntos
Bronquiectasia/metabolismo , Bronquiectasia/patologia , Fibrose Cística/patologia , Neutrófilos/fisiologia , Fagocitose/fisiologia , alfa-Defensinas/metabolismo , Adolescente , Adulto , Animais , Bronquiectasia/complicações , Estudos de Casos e Controles , Técnicas de Cultura de Células , Fibrose Cística/complicações , Fibrose Cística/metabolismo , Feminino , Humanos , Elastase de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Infiltração de Neutrófilos/fisiologia , Receptores de IgG/metabolismo , Adulto JovemRESUMO
Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development.
Assuntos
Lesão Pulmonar Aguda/cirurgia , Hepacivirus/efeitos da radiação , Hepatite C/prevenção & controle , Transplante de Pulmão , Pulmão/virologia , Complicações Pós-Operatórias/prevenção & controle , Inativação de Vírus/efeitos da radiação , Animais , Modelos Animais de Doenças , Hepacivirus/fisiologia , Hepatite C/virologia , Humanos , Masculino , Fototerapia , Complicações Pós-Operatórias/virologia , Suínos , Doadores de TecidosRESUMO
In order to expand hepatitis C virus (HCV) screening, a change in the diagnostic paradigm is warranted to improve accessibility and decrease costs, such as utilizing dried blood spot (DBS) collection. In our study, blood from 68 patients with chronic HCV infection was spotted onto DBS cards and stored at the following temperatures for one week: -80 °C, 4 °C, 21 °C, 37 °C, and alternating 37 °C and 4 °C; to assess whether temperature change during transportation would affect sensitivity. Sample was eluted from the DBS cards and tested for HCV antibodies (HCV-Ab) and HCV core antigen (core-Ag). HCV-Abs were detected from 68/68 DBS samples at -80 °C, 4 °C, 21 °C, and 67/68 at 37 °C and alternating 37 °C and 4 °C. Sensitivity of core-Ag was as follows: 94% (-80 °C), 94% (4 °C), 91% (21 °C), 93% (37 °C), and 93% (37 °C/4 °C). Not only did temperature not greatly affect sensitivity, but sensitivities are higher than previously reported, and support the use of this assay as an alternative to HCV RNA. We then completed a head-to-head comparison (n = 49) of venous versus capillary samples, and one versus two DBS. No difference in core-Ag sensitivity was observed by sample type, but there was an improvement when using two spots. We conclude that HCV-Abs and core-Ag testing from DBS cards has high diagnostic accuracy and could be considered as an alternative to HCV RNA in certain settings.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Hepacivirus/imunologia , Antígenos da Hepatite C/análise , Hepatite C/sangue , Adulto , Idoso , Feminino , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Hepatite C/virologia , Anticorpos Anti-Hepatite C/análise , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.
Assuntos
Hepatócitos/virologia , Vírus Oncolíticos/fisiologia , Orthoreovirus Aviário/fisiologia , Replicação Viral , Morte Celular , Linhagem Celular Tumoral , Efeito Citopatogênico Viral , Humanos , Modelos Moleculares , Proteínas Estruturais Virais/químicaRESUMO
Remote ischemic preconditioning (IPC) reduces tissue injury caused by ischemia-reperfusion (IR) in distant organs. We tested the hypothesis that remote IPC (rIPC) modifies inflammatory gene transcription in humans. Using a microarray method, we demonstrated that a simple model of brief forearm ischemia suppresses proinflammatory gene expression in circulating leukocytes. Genes encoding key proteins involved in cytokine synthesis, leukocyte chemotaxis, adhesion and migration, exocytosis, innate immunity signaling pathways, and apoptosis were all suppressed within 15 min (early phase IPC) and more so after 24 h (second window IPC). Changes in leukocyte CD11b expression measured by flow cytometry mirrored this pattern, with there being a significant (P = 0.01) reduction at 24 h. The results of this study show that the rIPC stimulus modifies leukocyte inflammatory gene expression. This effect may contribute to the protective effect of IPC against IR injury and may have broader implications in other inflammatory processes. This is the first study of human gene expression following rIPC stimulus. rIPC stimulus suppressed proinflammatory gene transcription in human leukocytes.
Assuntos
Regulação da Expressão Gênica/genética , Inflamação/genética , Precondicionamento Isquêmico , Adulto , Antígeno CD11b/genética , Feminino , Citometria de Fluxo , Humanos , Inflamação/imunologia , Inflamação/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Ativação de Neutrófilo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Molecular analysis of liver biopsy samples from patients requires ideal tissue preservation and handling to yield suitable material for laboratory analysis. Biopsy size, tissue handling and preservation method all may affect the quality and quantity of DNA, RNA and protein that can be extracted from liver biopsy samples. METHOD: In the present study, murine liver biopsies were performed and stored under various conditions: snap-freezing, RNAlater and Allprotect. Yield was compared to fresh biopsy tissue. RESULTS: Fresh tissue generated the highest yield of RNA while samples subjected to the snap-freezing generated the lowest yield of RNA. Preservation in RNAlater yielded higher quantities of RNA than storage in Allprotect, particularly with larger biopsy samples. There was a non-significant trend toward improved RNA quality with RNAlater (p = 0.35). DNA and protein yield were similar with RNAlater and Allprotect under a number of handling condition. Errors in tissue handling such as delays in tissue submersion or freezing did not significantly affect tissue yields in either preservation solution. Tissue yield was unchanged with up to three freeze-thaw cycles in both solutions. Biopsy size (5 vs 2 mm) and width (15 vs 18 g) had a marked effect on tissue yield. CONCLUSION: Ideally 5-mm biopsies with 15-gauge needles should be used to maximize yield. RNAlater provided higher RNA yield with similar yields of DNA and protein and was notably cheaper and easier to handle.
RESUMO
BACKGROUND: High plasma levels of interferon-gamma inducible protein-10 (IP-10) have been shown to be associated with impaired treatment response in chronic hepatitis C virus (HCV) infection. Whether IP-10 levels predict treatment in acute HCV infection is unknown. METHODS: Patients with acute or early chronic HCV infection from the Australian Trial in Acute Hepatitis C (ATAHC) cohort were evaluated. Baseline and on-treatment plasma IP-10 levels were measured by ELISA. IL28B genotype was determined by sequencing. RESULTS: Overall, 74 HCV mono-infected and 35 HIV/HCV co-infected patients were treated in ATAHC, of whom 89 were adherent to therapy and were included for analysis. IP-10 levels correlated with HCV RNA levels at baseline (râ=â0.48, P<0.001) and during treatment. Baseline IP-10 levels were higher in patients who failed to achieve rapid virological response (RVR). Only one patient with a plasma IP-10 level >600 pg/mL achieved RVR. There was no association with IP-10 levels and early virological response (EVR) or sustained virological response (SVR). CONCLUSIONS: Baseline IP-10 levels are associated with early viral kinetics but not ultimate treatment outcome in acute HCV infection. Given previous data showing that patients with high baseline IP-10 are unlikely to spontaneously clear acute HCV infection, they should be prioritized for early antiviral therapy.
Assuntos
Antivirais/uso terapêutico , Quimiocina CXCL10/sangue , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Doença Aguda , Adulto , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por HIV/complicações , Hepatite C Crônica/sangue , Hepatite C Crônica/complicações , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Proteínas Recombinantes/uso terapêutico , Resultado do Tratamento , Carga ViralRESUMO
Diverse receptors, including Fcgamma receptors and beta(2) integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44-coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg(2+) but not Ca(2+). In addition, buffering of intracellular Ca(2+) did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.
Assuntos
Receptores de Hialuronatos/imunologia , Antígeno de Macrófago 1/imunologia , Macrófagos/imunologia , Fagocitose/imunologia , Animais , Antígeno CD11b , Antígenos CD18 , Camundongos , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.
Assuntos
Fibrose Cística/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Neutrófilos/imunologia , Mucosa Respiratória/imunologia , Brônquios/citologia , Células Cultivadas , Fibrose Cística/microbiologia , Células Epiteliais/microbiologia , Escherichia coli/crescimento & desenvolvimento , Exocitose , Humanos , Viabilidade Microbiana , Concentração Osmolar , Fagocitose , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/isolamento & purificação , Explosão Respiratória , Mucosa Respiratória/microbiologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificaçãoRESUMO
The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Fosfolipases A/fisiologia , Fator de Ativação de Plaquetas/biossíntese , Animais , Ácido Araquidônico/metabolismo , Líquido da Lavagem Broncoalveolar , Antígeno CD11b/biossíntese , Citosol/enzimologia , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Fosfolipases A2 do Grupo IV , Humanos , Inflamação , Ionomicina/farmacologia , Leucotrieno B4/metabolismo , Camundongos , Camundongos Transgênicos , Neutrófilos/citologia , Neutrófilos/microbiologia , Oxigênio/metabolismo , Fagocitose , Fosfolipases A/metabolismo , Fosfolipases A2 , Pneumonia/metabolismo , Pirrolidinas/farmacologia , Receptores de IgG/biossíntese , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Signaling pathways involving reversible tyrosine phosphorylation are essential for neutrophil antimicrobial responses. Using reverse transcriptase PCR, expression of the protein-tyrosine phosphatase MEG2 by peripheral neutrophilic polymorphonuclear leukocytes (PMN) was identified. Polyclonal antibodies against MEG2 were developed that confirmed expression of MEG2 protein by PMN. Through a combination of immunofluorescence and cell fractionation followed by immunoblotting, we determined that MEG2 is predominantly cytosolic with components present in secondary and tertiary granules and secretory vesicles. MEG2 activity, as determined by immunoprecipitation and in vitro phosphatase assays, is inhibited after exposure of cells to the particulate stimulant opsonized zymosan or to phorbol 12-myristate 13-acetate but largely unaffected by the chemoattractant N-formyl-methionyl-leucyl-phenyalanine. Studies using bacterially expressed glutathione S-transferase MEG2 fusion protein indicate that cysteine 515 is essential for catalytic activity, whereas the noncatalytic (N-terminal) domain of MEG2 negatively regulates the enzymatic activity of the C-terminal phosphatase domain. The activity of MEG2 is enhanced by specific polyphosphoinositides with the order of potency being phosphatidylinositol (PI) 4,5-diphosphate > PI 3,4,5-triphosphate > PI 4-phosphate. MEG2 associates at an early stage with nascent phagosomes. Taken together, our results indicate that MEG2 is a polyphosphoinositide-activated tyrosine phosphatase that may be involved in signaling events regulating phagocytosis, an essential antimicrobial function in the innate immune response.
Assuntos
Neutrófilos/enzimologia , Fagossomos/enzimologia , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/química , Citoplasma/enzimologia , Citosol/enzimologia , Ativação Enzimática , Vetores Genéticos , Glutationa Transferase/metabolismo , Células HL-60 , Hemaglutininas/química , Hemaglutininas/metabolismo , Humanos , Immunoblotting , Lipossomos/metabolismo , Microscopia de Fluorescência , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Testes de Precipitina , Proteínas Tirosina Fosfatases não Receptoras , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações Subcelulares , Fatores de Tempo , Transfecção , Células U937RESUMO
During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.