RESUMO
Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated ß-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.
Assuntos
Células Acinares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Estresse Fisiológico , Animais , Apoptose , Autofagia , Proteína 5 Relacionada à Autofagia , Senescência Celular , Citocinas/genética , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Ligadura , Ativação de Macrófagos , Camundongos Knockout , Modelos Biológicos , Especificidade de Órgãos , Fenótipo , Glândula Submandibular/metabolismoRESUMO
Excessive apoptosis has been implicated in a number of acute and chronic human diseases. The activation of caspases has been shown to be critical for the apoptotic process. The objective of this investigation was to evaluate the beneficial effects and mechanism of action of the caspase-8 inhibitor IETD-CHO and the caspase-3 inhibitor DEVD-CHO against tumor necrosis factor (TNF)-induced hepatocellular apoptosis in vivo and compare these results to effects of the same inhibitors against Fas-induced apoptosis. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine/100 microg/kg endotoxin induced parenchymal apoptosis (indicated by caspase-3 activation and morphology) and severe liver injury (indicated by the increase in plasma alanine aminotransferase activities and histology) at 7 h. Treatment with IETD-CHO or DEVD-CHO (10 mg/kg at 3, 4.5, and 5.5 h) significantly attenuated caspase-3 activation and liver injury. Western analysis showed that DEVD-CHO had no effect while IETD-CHO substantially reduced procaspase-3 and procaspase-9 processing. On the other hand, caspase-3 activation and liver injury by the anti-Fas antibody Jo-2 was completely prevented by a single dose of DEVD-CHO and, as previously shown, by IETD-CHO at 90 min. Both inhibitors prevented procaspase-3 and procaspase-9 processing. Thus, there are fundamental differences in the efficacy of caspase inhibitors in these two models. We conclude that Fas may rely exclusively on caspase-8 activation and mitochondria to activate caspase-3, which can process more procaspase-8 and thus propagate the amplification of the apoptotic signal. TNF can activate a similar signaling pathway. However, alternative signaling mechanisms seem to exist, which can compensate if the main pathway is blocked.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Hepatócitos/efeitos dos fármacos , Falência Hepática/prevenção & controle , Oligopeptídeos/farmacologia , Animais , Western Blotting , Caspase 3 , Caspase 8 , Caspase 9 , Inibidores de Caspase , DNA/análise , Fragmentação do DNA , Galactosamina/farmacologia , Hepatócitos/enzimologia , Hepatócitos/patologia , Falência Hepática/induzido quimicamente , Falência Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Modelos Animais , Método Simples-Cego , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/farmacologiaRESUMO
Lymphocytes can kill target cells including hepatocytes during various inflammatory diseases by Fas receptor-mediated apoptosis. Caspase-8 is activated at the receptor level, thereby initiating the processing of downstream effector caspases. The aim of this study was to investigate the time course of caspase-8 activation and to evaluate the efficacy of the caspase-8 inhibitor IETD-CHO in a model of Fas-induced apoptosis in vivo. C3Heb/FeJ mice were treated with the anti-Fas antibody Jo-2 (0.6 mg/kg). Western blot analysis demonstrated increased cytochrome c in the cytosol (20 min), which was followed by the progressive activation of caspase-3, -9 (40-120 min), and caspase-8 (120 min). At 90 and 120 min, extensive hemorrhage was observed, indicating damage to sinusoidal lining cells. In addition, high plasma ALT levels (997 +/- 316 U/L) and histological evaluation indicated severe parenchymal cell injury. Parenchymal and nonparenchymal cells showed a similar increase in caspase-3 activity and DNA fragmentation. Treatment with IETD-CHO (10 mg/kg) attenuated the increase in caspase-3 activity and DNA fragmentation by 80-90% and completely prevented hemorrhage and parenchymal cell damage. IETD-CHO also prevented the early release of mitochondrial cytochrome c and the processing of caspase-3, -8, and -9. Thus, our data support the hypothesis that Fas-mediated apoptosis is dependent on caspase-8 activation in hepatocytes and nonparenchymal cells. However, the bulk of procaspase-8 is processed late, suggesting that only a small amount of procaspase-8 may actually be activated at the Fas receptor. This initial signal may be amplified by further activation of caspase-8 by effector caspases, i.e., after mitochondrial activation. Caspase-8 is a promising therapeutic target for inhibition of Fas-mediated apoptosis.
Assuntos
Apoptose , Caspases/metabolismo , Inibidores Enzimáticos/farmacologia , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Falência Hepática/prevenção & controle , Mitocôndrias Hepáticas/enzimologia , Oligopeptídeos/farmacologia , Receptor fas/metabolismo , Animais , Western Blotting , Caspase 8 , Caspase 9 , Inibidores de Caspase , Grupo dos Citocromos c/metabolismo , Hepatócitos/enzimologia , Hepatócitos/patologia , Células de Kupffer/enzimologia , Células de Kupffer/patologia , Falência Hepática/enzimologia , Falência Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mitocôndrias Hepáticas/efeitos dos fármacos , Processamento de Proteína Pós-TraducionalRESUMO
We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.
Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Eosinófilos/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Pulmão/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Antígenos/imunologia , Células da Medula Óssea , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Imunoglobulina A/análise , Interleucina-5/análise , Leucócitos/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Eosinofilia Pulmonar/imunologia , Ratos , Linfócitos T/metabolismoRESUMO
Polymorphonuclear leukocytes (neutrophils) can cause hepatic parenchymal cell injury during endotoxin (ET) shock. Because adhesion molecules are critical for inflammatory cell damage, the role of vascular cell adhesion molecule-1 (VCAM-1) was studied in the pathophysiology of ET shock. ET-sensitive mice (C3Heb/FeJ) were treated with 700 mg/kg galactosamine in combination with 100 microg/kg Salmonella abortus equi ET, 15 microg/kg TNF-alpha, or 13 to 23 microg/kg IL-1. VCAM-1 mRNA formation was strongly activated in animals treated with ET, TNF-alpha, or IL-1. In contrast, only TNF-alpha and IL-1, not ET, induced VCAM-1 gene transcription in livers of ET-resistant mice (C3H/HeJ). Immunohistochemistry and isolation of liver cells during endotoxemia indicated that VCAM-1 mRNA and protein were only formed in endothelial cells and Kupffer cells, not in hepatocytes. Galactosamine/ET induced neutrophil accumulation in sinusoids (515 +/- 30 neutrophils/50 high power fields) followed by transmigration at 7 h. At that time, severe liver injury was observed (necrosis, 53 +/- 5%). An anti-VCAM-1 Ab (3 mg/kg) attenuated the area of necrosis by 60%. The Ab reduced neutrophil transmigration by 84%, but had no effect on the total number of cells in the liver vasculature. Flow cytometric analysis identified the presence of very late Ag-4 on mouse peripheral neutrophils. Our data demonstrated cytokine-dependent VCAM-1 gene transcription and protein expression in the liver during endotoxemia. Neutrophils were able to use very late Ag-4/VCAM-1 interactions to transmigrate into liver parenchyma in vivo. Preventing transmigration by blocking VCAM-1 protected hepatocytes against neutrophil-induced injury.
Assuntos
Hepatopatias/fisiopatologia , Neutrófilos/imunologia , Choque Séptico/fisiopatologia , Ativação Transcricional , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Necrose , RNA Mensageiro/análiseRESUMO
The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.
Assuntos
Antígenos CD/fisiologia , Leucócitos/fisiologia , Pulmão/fisiopatologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Animais , Anticorpos Monoclonais , Brônquios/patologia , Movimento Celular , Feminino , Imunização , Imuno-Histoquímica/métodos , Integrina alfa4 , Leucócitos/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Pneumonia/imunologia , Pneumonia/patologia , Ratos , Coloração e RotulagemRESUMO
We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.
Assuntos
Antialérgicos/imunologia , Eosinófilos/imunologia , Integrinas/fisiologia , Pulmão/imunologia , Receptores de Retorno de Linfócitos/fisiologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Eosinófilos/citologia , Citometria de Fluxo , Imunofenotipagem , Integrina alfa4beta1 , Contagem de Leucócitos , Pulmão/citologia , Subpopulações de Linfócitos/imunologia , Tecido Linfoide/citologia , Masculino , Camundongos , Ovalbumina/imunologia , Pneumonia/imunologia , Pneumonia/patologia , Ratos , Ratos Endogâmicos BN , Hipersensibilidade Respiratória/imunologia , Linfócitos T/citologiaRESUMO
We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype. 1A29 treatment produced dose-related increases in circulating 1A29 and blood neutrophils. In the BAL fluid of 1A29-treated animals, significant (P < 0.05) reductions in the numbers of eosinophils and lymphocytes, but not neutrophils or alveolar macrophages, were observed in association with a reduced inflammatory pathology in lung tissue. 1A29 administration reduced the number of detectable ICAM-1 binding sites on T cells in peripheral blood and BAL fluid examined ex vivo by flow cytometry. We conclude that ICAM-1 is critically important for the antigen-specific recruitment of eosinophils and lymphocytes into the lungs.
Assuntos
Antígenos/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Pneumonia/imunologia , Animais , Anticorpos Monoclonais , Células Sanguíneas/fisiologia , Líquido da Lavagem Broncoalveolar/citologia , Imunização , Pulmão/imunologia , Pulmão/patologia , Tecido Linfoide/patologia , Ovalbumina/imunologia , Fenótipo , Pneumonia/patologia , Ratos , Ratos Endogâmicos BN , Linfócitos T/fisiologiaRESUMO
In order to investigate whether the pulmonary response to helminth antigens mimics that seen in allergic inflammation of the airways, we have examined the phenotypic characteristics of lymphocytes and eosinophils recruited to the airways following Nippostrongylus brasiliensis (N.b.) infection. Specifically, the cellular response was divided into an early and a late phase. During the early response there was a small but significant increase in neutrophil numbers recovered by bronchoalveolar lavage (BAL). Phenotypic analysis of BAL leukocytes revealed an early rise in the percentage of BAL lymphocytes expressing the naive T cell markers CD45RB and L-selectin, and the activation marker IL-2R. In addition, during the early response, there was an increased percentage of lymphocytes expressing the gamma delta TCR, but not the alpha beta TCR. In contrast, the late response was marked by a much larger accumulation, in the lungs and BAL, of memory CD4+ T lymphocytes and an influx of small, hypodense eosinophils which produced LTB4 and LTC4 on stimulation with calcium ionophore. At this time there was a substantial increase in the number of T lymphocytes and eosinophils expressing ICAM-1 and the integrins VLA-4 and LFA-1, implicating these adhesion molecules in inflammatory cell recruitment to the airways. We conclude that the pattern and phenotypic characteristics of the cellular recruitment seen following N.b. infection resemble those seen in early- and late-phase allergic inflammation of the airways in asthma, and therefore N.b. may be used to model these aspects of the disease.
Assuntos
Eosinófilos/patologia , Pneumonia/imunologia , Linfócitos T/patologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Moléculas de Adesão Celular/análise , Modelos Animais de Doenças , Eicosanoides/biossíntese , Eicosanoides/imunologia , Eosinófilos/metabolismo , Eosinófilos/microbiologia , Feminino , Citometria de Fluxo , Imunofluorescência , Imunofenotipagem , Contagem de Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nippostrongylus/imunologia , Pneumonia/microbiologia , Pneumonia/patologia , Ratos , Ratos Sprague-Dawley , Infecções por Strongylida/imunologia , Linfócitos T/química , Linfócitos T/microbiologia , Fatores de TempoRESUMO
Neutrophil-induced liver injury during endotoxemia is dependent on the adhesion molecules Mac-1 (CD11b/CD18) on neutrophils and its counterreceptor on endothelial cells and hepatocytes, intercellular adhesion molecule 1 (ICAM-1). To investigate a potential release of a soluble form of ICAM-1 (sICAM-1), animals received 100 micrograms/kg Salmonella abortus equi endotoxin alone or in combination with 700 mg/kg galactosamine. In endotoxin-sensitive mice (C3Heb/FeJ), injection of endotoxin did not cause liver injury but induced a time-dependent increase of sICAM-1 in serum (300%) and in bile (615%) without affecting bile flow. In galactosamine/endotoxin-treated animals, which developed liver injury, the increase in both compartments was only 97% and 104%, respectively. In either case, the increase in sICAM-1 concentrations paralleled the enhanced ICAM-1 expression in the liver. The endotoxin-resistant strain (C3H/HeJ) did not show elevated sICAM-1 levels in serum or bile after endotoxin administration. In contrast, the intravenous injection of murine tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) or IL-1 beta (13-23 micrograms/kg) into endotoxin-resistant mice induced a 225% to 364% increase in serum sICAM-1 and a 370% elevation of the biliary efflux of sICAM-1, again independent of changes in bile flow. These data indicate that cytokines are major inducers of sICAM-1 formation during endotoxemia in vivo. The described experimental model can be used to investigate the role of sICAM-1 in the pathophysiology of inflammatory liver disease.
Assuntos
Bile/imunologia , Endotoxinas/sangue , Molécula 1 de Adesão Intercelular/metabolismo , Choque Séptico/imunologia , Análise de Variância , Animais , Bile/metabolismo , Bile/fisiologia , Galactosamina/toxicidade , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/sangue , Interleucina-1/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Choque Séptico/metabolismo , Solubilidade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
To facilitate studies of the individual viral proteins, two Borna disease virus proteins, p24 and p38/40, were synthesized in vitro by means of a baculovirus expression system and examined for antigenic identity to viral proteins from BDV-infected cells. Recombinant proteins p24 and p38/40 were nearly identical in size to the viral proteins from BDV-infected cells. Immunoblot and immunocytochemistry analysis of BDV proteins from infected tissue culture cells and rat brain showed binding of antisera directed against the recombinant proteins. Specific recognition of the recombinant proteins by Borna disease virus-specific convalescent antisera and monoclonal antibodies further demonstrated that the antigenic characters of the p24 and p38/40 had been conserved. Polyclonal antibody directed against either of the recombinant proteins recognized only the protein used as immunogen, without cross reactivity with the other recombinant protein, indicating no common epitopes. Moreover, these data confirmed the proposed gene coding assignments of ORF I and II of BDV p38/40 and p24, respectively. Both of the recombinant proteins were secreted into the media of insect cells in tissue culture, but secretion of recombinant p24 was evident only as a dimeric form and not with the monomeric form. Immunoprecipitation studies performed with monoclonal antibodies and BDV proteins from infected rat brain suggested that a heterodimer forms via binding of p40 to the p24.
Assuntos
Vírus da Doença de Borna/imunologia , Proteínas Virais/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Baculoviridae/genética , Células Cultivadas , Epitopos , Immunoblotting , Coelhos , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Spodoptera , Proteínas Virais/biossíntese , Proteínas Estruturais Virais/biossínteseRESUMO
The protective effect of the 21-aminosteroid tirilazad mesylate (U-74006F) was investigated in an experimental model of endotoxin shock and acute liver failure. In male Fischer rats subjected to 20 min of hepatic no-flow ischemia followed by reperfusion and injection of 0.5 mg/kg of Salmonella enteritidis endotoxin, severe hepatic injury developed, as indicated by a histological evaluation and liver enzyme release. Treatment with U-74006F (two bolus doses of 3 mg/kg each; the first dose was injected i.v. 30 min before ischemia and the second dose, at the time of reflow) reduced the hepatic injury by 60% at 4 hr of reperfusion, improved the survival rate from 18% to 55% and decreased the degree of hepatic injury at 48 hr of reperfusion. U-74006F treatment did not affect the extent of complement activation during reperfusion, the Kupffer cell-induced oxidant stress, or tumor necrosis factor-alpha formation in this model. U-74006F did not significantly reduce superoxide formation of Kupffer cells and neutrophils in vitro or in vivo. The substantial neutrophil infiltration in the liver during the pathogenesis was not affected at 4 hr of reperfusion but was attenuated by 70% at 48 hr. It was therefore concluded that, in the sequence of pathophysiological events, U74006F acted at a site distal to inflammatory cell activation and the generation of cytotoxic mediators. The protection against the initial endotoxin-enhanced reperfusion injury in the liver strongly inhibited the progression of the inflammatory response and subsequent liver failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antioxidantes/farmacologia , Falência Hepática Aguda/prevenção & controle , Pregnatrienos/farmacologia , Choque Séptico/prevenção & controle , Animais , Peroxidação de Lipídeos , Masculino , Neutrófilos/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Traumatismo por Reperfusão/prevenção & controle , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Plasma levels of glutathione disulfide (GSSG) as an indicator of a vascular oxidant stress, tumor necrosis factor-alpha (TNF-alpha) formation, and liver injury (alanine aminotransferase activity, histology) were monitored in male Fischer rats after 30 min of hepatic ischemia followed by up to 4 hr of reperfusion. The injection of 1 mg/kg Salmonella enteritidis endotoxin at 30 min of reflow potentiated the postischemic oxidant stress and liver injury. TNF-alpha levels increased from 10 +/- 7 pg/ml (baseline) to 3,553 +/- 738 pg/ml after ischemia-reperfusion followed by endotoxin, or to 3,670 +/- 508 pg/ml after endotoxin alone. Depletion of serum complement before ischemia attenuated the endotoxin-mediated increase of reactive oxygen formation by 70% but did not affect TNF-alpha levels. Complement activation with cobra venom factor (CVF) during reperfusion had an effect similar to that of endotoxin on the oxidant stress and liver injury. CVF did not increase TNF-alpha formation during reperfusion. Kupffer cells and neutrophils isolated from the postischemic liver 2.5 hr after endotoxin injection generated 600% and 400% more superoxide, respectively, than cells isolated from control livers. The results demonstrate a substantial priming of hepatic phagocytes for reactive oxygen production but not TNF-alpha formation, even after short periods of hepatic ischemia, and the vulnerability of the postischemic liver to severe endotoxin-induced injury. Activated complement seems to be mainly responsible for the effects. These results may explain the high risk for hepatic failure after extensive liver resection and hypovolemic shock.
Assuntos
Endotoxinas/toxicidade , Hepatopatias/etiologia , Fígado/irrigação sanguínea , Fagócitos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão , Trifosfato de Adenosina/metabolismo , Alanina Transaminase/sangue , Animais , Ativação do Complemento , Venenos Elapídicos/farmacologia , Glutationa/sangue , Isquemia , Células de Kupffer/metabolismo , Masculino , Neutrófilos/metabolismo , Oxirredução , Ratos , Salmonella enteritidis , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
An enzyme immunoassay that uses easily regenerated reagents was developed and evaluated for the ability to detect group B rotaviruses (GBR) in fecal specimens. Homologous rat GBR and heterologous porcine and bovine GBR were detected by this immunoassay, although a human GBR isolate was not. This immunoassay should prove useful in studies of GBR infections of animals.
Assuntos
Técnicas Imunoenzimáticas , Rotavirus/isolamento & purificação , Animais , Antígenos Virais/análise , Bovinos , Diarreia/microbiologia , Estudos de Avaliação como Assunto , Fezes/microbiologia , Humanos , Técnicas Imunoenzimáticas/estatística & dados numéricos , Indicadores e Reagentes , Ratos , Rotavirus/classificação , Rotavirus/imunologia , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/microbiologia , Sensibilidade e Especificidade , SuínosRESUMO
The second largest genomic segment of the IDIR strain (infectious diarrhea of infant rats) of group B rotavirus (GBR) was completely sequenced, cloned, and expressed in insect cells, and its gene coding assignment was determined. The sequence of IDIR virus gene 2 (IDIRg2) contained 2847 bp with a single, long open reading frame that encoded a deduced polypeptide of 934 amino acids (M(r) 106 kDa, pI 5.325). BestFit homology indicated that the predicted amino acid sequence of IDIRg2 shared 46.5% similar and 19.8% identical sequences with VP2 of the SA11 strain of group A rotavirus. The polypeptide product encoded by this gene was synthesized in insect cells by means of a baculovirus expression vector and employed to elucidate the corresponding gene to protein coding assignment. Recombinant IDIRg2 product maintained virion antigenic epitopes as evidenced by reactivity with convalescent antisera from infant rat pups infected with the IDIR agent. Reactivity with antisera from heterologous human and porcine GBR strains was also observed. Antibody directed against recombinant IDIRg2 product specifically reacted with VP2 of IDIR virus and a human strain of GBR (ADRV) obtained from fecal specimens. These experiments identified the IDIRg2 product as the VP2 core protein of the IDIR virus strain of GBR.
Assuntos
Capsídeo/genética , Rotavirus/genética , Sequência de Aminoácidos , Animais , Capsídeo/biossíntese , Proteínas do Capsídeo , Linhagem Celular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Éxons , Genes Virais , Immunoblotting , Dados de Sequência Molecular , Mariposas , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Rotavirus/classificação , Homologia de Sequência de AminoácidosRESUMO
The polypeptide encoded by gene 3 of the IDIR strain of group B rotavirus (GBR) was synthesized by means of a baculovirus expression system. Immunoblot analysis identified the IDIR virus gene 3 product as analogous to the outer capsid protein, VP4, encoded by gene 4 of group A rotaviruses (GAR). This coding assignment had previously been difficult to establish by sequence comparisons, since IDIR virus gene 3 shared < 20% identical amino acid sequences with any GAR protein. Trypsin digestion of the gene 3 protein resulted in the appearance of a product indistinguishable in size from the VP5* outer capsid protein of IDIR agent virions. The recombinant IDIR virus VP4 maintained at least some antigenic epitopes of the native virion protein, as evidenced by reactivity with convalescent antibody obtained following infection of infant rat pups with the IDIR agent. Reactivity was also demonstrated with antisera directed against bovine and porcine isolates of GBR as well as with ADRV, a human strain of GBR.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Genes Virais/genética , Rotavirus/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Baculoviridae/genética , Capsídeo/biossíntese , Capsídeo/efeitos dos fármacos , Células Cultivadas , Reações Cruzadas , Epitopos/imunologia , Variação Genética , Dados de Sequência Molecular , Mariposas , Proteínas Recombinantes/biossíntese , Rotavirus/imunologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tripsina/farmacologiaRESUMO
A purification scheme was developed that allowed for the partial purification of complete, double-shelled particles of the infectious diarrhea of infant rats (IDIR) virus, a group B rotavirus (GBR). Structural proteins from the isolated, complete viral particles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and evaluated by silver staining and by immunoblotting using antisera obtained from rats that had recovered from IDIR virus infection. Analysis of the stained gels and immunoblots demonstrated the presence of IDIR virus structural proteins having estimated molecular weights of 130, 100, 88, 80, 61, 44, 32, and 25 kDa. Treatment of complete viral particles with EDTA removed the outer capsid layer producing single-shelled particles that lacked the 80, 61, 32, and 25 kDa proteins seen in the double-shelled particles. The 44-kDa protein was most abundant and was recognized by a mouse monoclonal antibody and hyperimmune guinea pig serum prepared against IDIR virus and by hyperimmune guinea pig serum prepared against adult diarrhea rotavirus (ADRV), a human strain of GBR. Convalescent serum obtained from a piglet inoculated with a porcine GBR reacted with the 61 kDa, outer capsid protein of IDIR virus. This information on the structural nature of IDIR virus should be helpful in establishing the genetic and structural relationships among the GBR and to further exploit the potential of IDIR virus as a tool for understanding GBR infections in human and animal populations.
Assuntos
Diarreia/microbiologia , Rotavirus/química , Rotavirus/isolamento & purificação , Proteínas Estruturais Virais/análise , Animais , Animais Lactentes , Anticorpos Antivirais/imunologia , Immunoblotting , Intestinos/microbiologia , Microscopia Eletrônica , Coloração Negativa , Ratos , Rotavirus/ultraestrutura , Proteínas Estruturais Virais/imunologiaRESUMO
The polypeptide product of gene 6 of the IDIR strain of group B rotavirus was synthesized by means of a baculovirus expression system in order to confirm the coding assignment of the gene and to develop reagents broadly reactive with heterologous strains of Group B rotaviruses (GBR). Earlier experiments indicated that IDIR virus gene 6 encoded the group-specific, major inner capsid protein, but direct confirmation of this coding assignment was not previously reported. The expression of IDIR virus gene 6 from baculovirus recombinants resulted in production of a protein with an apparent molecular weight equivalent to that deduced from the gene sequence (44 kDa). In addition, larger recombinant proteins were also observed, and these appeared to be consistent in size with oligomers of the primary VP6 product. The expressed protein reacted with antibody directed against the IDIR agent and other strains of GBR, but no reaction was observed with antibody directed against group A rotavirus. Serologic reactivity was also observed between the gene 6 product and a monoclonal antibody directed against the GBR group-specific antigen. Antibody directed against the recombinant gene 6 product specifically reacted with the IDIR virus major inner capsid protein in an immunoblot format. These experiments conclusively demonstrated that IDIR virus gene 6 encoded the major inner capsid protein and confirmed the presence of group B-specific antigenic epitopes on the protein.
Assuntos
Capsídeo/genética , Genes Virais/genética , Rotavirus/genética , Animais , Anticorpos Monoclonais , Baculoviridae , Capsídeo/biossíntese , Linhagem Celular , Expressão Gênica , Vetores Genéticos , Immunoblotting , Lepidópteros , Peso Molecular , Proteínas Recombinantes/biossínteseRESUMO
An enteropathogenic Enterococcus-like agent was isolated from a spontaneous outbreak of diarrhea that occurred in a colony containing neonatal rats. Diarrhea was experimentally reproduced in virus-antibody-free neonatal rats inoculated with this purified "enterococcus." Gram-positive cocci were adhered to the small intestinal villi of affected animals from which the organism was reisolated. The isolate's classification in the genus Enterococcus was confirmed by genetic probe; however, because of its unique fermentation pattern, it could not be definitively speciated. Indirect immunofluorescence assays indicate that this strain of enterococcus and Enterococcus hirae, another strain pathogenic for neonatal rats, differ antigenically. Enterococci should be considered as potential etiologic agents in outbreaks of diarrhea involving neonatal rats and future efforts directed to increasing our understanding of the pathophysiology of this disease.
Assuntos
Animais Recém-Nascidos/microbiologia , Diarreia/veterinária , Enterococcus/crescimento & desenvolvimento , Infecções por Bactérias Gram-Positivas/veterinária , Ratos/microbiologia , Doenças dos Roedores/microbiologia , Animais , Diarreia/microbiologia , Diarreia/patologia , Enterococcus/isolamento & purificação , Imunofluorescência/veterinária , Infecções por Bactérias Gram-Positivas/patologia , Doenças dos Roedores/patologiaRESUMO
Terminal nucleic acid sequences were determined for all 11 segments of the IDIR strain of group B rotavirus. Consensus sequences were defined at both ends of the (+) RNA strands as 5' GGN(A/U)NA(A/U)(A/U)(A/U)---and---(A/U)NA(A/G)N(A/C)(C/A)CC3 '. The 5' and 3' terminal sequences of the (+) strand IDIR RNA were not complementary to one another. The IDIR terminal sequences and those of group A rotaviruses (GAR) were similar in that each of the (+) strands began with "GG" and ended with "CC." Otherwise, the IDIR terminal sequences did not match the consensus sequences that have been reported for the ends of the GAR genomic segments.