RESUMO
We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24-28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.
Assuntos
Antígenos CD/metabolismo , Plaquetas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Domínios e Motivos de Interação entre Proteínas , Semaforinas/metabolismo , Proteína ADAM17/metabolismo , Antígenos CD/química , Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática , Humanos , Proteólise , Semaforinas/químicaRESUMO
BACKGROUND: In this study, we assessed the prognostic value of the lymph node ratio (LNR), established a hypothetical tumor-ratio-metastasis (TRM) staging system and compared it with the 7th edition International Union Against Cancer pathological N (pN) and tumor-node-metastasis (TNM) system. PATIENTS AND METHODS: A total of 1343 gastric cancer patients undergoing D2 resection were staged using the TRM staging system and the 7th edition TNM system. Optimal cut points of LNR were calculated using X-tile software and validated by bootstrapping. Homogeneity, discriminatory ability, and monotonicity of gradients of the TRM and TNM systems were compared using linear trend χ(2), likelihood ratio χ(2) statistics, and Akaike information criterion (AIC) calculations. RESULTS: Optimal cut points classified patients into LNR0 (0%), LNR1 (1%-30%), LNR2 (31%-60%), and LNR3 (61%-100%) groups. In univariate, multivariate and stratified analyses, the LNR staging showed superiority to the 7th edition pN staging. The TRM staging system had higher linear trend and likelihood ratio χ(2) scores and smaller AIC values compared with those for the TNM system, which represented the optimum prognostic stratification. CONCLUSIONS: The novel TRM staging system predicts survival of gastric cancer more accurately than the 7th edition TNM system. It may be considered as an alternative to TNM system.
Assuntos
Linfonodos/patologia , Linfonodos/cirurgia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Excisão de Linfonodo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Metástase Neoplásica , Estadiamento de Neoplasias/métodos , Estudos Retrospectivos , Análise de Sobrevida , Adulto JovemRESUMO
Kimura disease (KD) is a rare, chronic inflammatory disease of unknown cause and is characterized by painless s.c. swellings and lymphadenopathy commonly affecting the head and neck region. Much therapeutics has been used to treat KD, but is not satisfactory because of frequent relapse. Imatinib has been reported previously to be useful for treatment of hypereosinophilic syndrome and may work by selectively blocking protein-tyrosine kinases, such as platelet-derived growth factor receptor, and c-Kit. We carried out immunohistochemical examination of platelet-derived growth factor receptor-alpha and c-Kit in tissues from patients with KD. The results were positive and suggested that Imatinib might be an effective drug for the treatment of the disease. We have also briefly reviewed the epidemiology, aetiology, clinical manifestations, laboratory and pathological examinations, differential diagnoses, treatment and prognosis of KD in this manuscript.
Assuntos
Hiperplasia Angiolinfoide com Eosinofilia/diagnóstico , Hiperplasia Angiolinfoide com Eosinofilia/tratamento farmacológico , Hiperplasia Angiolinfoide com Eosinofilia/epidemiologia , Hiperplasia Angiolinfoide com Eosinofilia/patologia , Diagnóstico Diferencial , Humanos , PrognósticoRESUMO
BACKGROUND: To analyze the effects of difference antiviral agents and the effects of the treatments on long-term prognosis. METHODS: Retrospective research method was applied. RESULTS: About 40% of the patients were treated with interferon or lamivudine. After the treatment, in lamivudine group, the negative rate of HBV DNA was the highest. In the interferon group, the sero conversion rates of HBeAg/HBeAb were 22.9%. In the antiviral treatment patients, the disease progression and the occurrence of cirrhosis and liver cancer were much lower than those of the control groups. The mortality of cirrhosis and liver cancer in the HBeAg/HBeAb sero converted group was much lower than that of the group without HBeAg/HBeAb sero conversion groups (P less than 0.05). CONCLUSION: The antiviral effects of interferon and lamivudine were better than those of the other drug groups. The antiviral drugs could relieve the disease progression and reduce the mortality of cirrhosis and liver cancer.
Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Antivirais/uso terapêutico , DNA Viral , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Lamivudina/uso terapêutico , Prognóstico , Estudos RetrospectivosRESUMO
The objective of the present study was to determine whether hypoxia/reoxygenation in the absence or presence of intestinal bacteria would affect the integrity of the gut mucosal epithelium (as evidenced by histologic changes) and increase the local production of cytokines (interleukin 6 [IL-6] and tumor necrosis factor [TNF]). Rat ileal mucosal membranes were harvested and their electrophysiologic properties and barrier function were measured ex vivo in the Ussing chamber system. Membranes were exposed to normoxia, normoxia + Escherichia coli, hypoxia for 40 min followed by normoxia, or hypoxia for 40 min + E. coli followed by normoxia for 3 h. IL-6 and TNF levels were measured using cytokine-dependent cellular assays. Morphological changes and the degree of DNA fragmentation were used as quantitative markers of gut mucosal injury. Mucosal integrity was maintained in the normoxia group. The addition of bacteria increased the IL-6 response and reduced mucosal integrity. During the hypoxic period, a transient decline in resistance (R) occurred and cytokine production was reduced. In the hypoxic ileal membranes not exposed to E coli, reoxygenation reversed the change in R and increased IL-6 production. The combination of hypoxia/reoxygenation plus E. coli bacterial challenge resulted in the greatest extent of gut mucosal injury and increase in TNF production. The results of this study support the hypothesis that the combination of increased intestinal bacterial levels superimposed on an ischemia/reperfusion injury increases the magnitude of gut mucosal injury and the production and subsequent release of proinflammatory cytokines.
Assuntos
Citocinas/metabolismo , Fragmentação do DNA , Escherichia coli/fisiologia , Hipóxia/fisiopatologia , Mucosa Intestinal/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Eletrofisiologia , Hipóxia/genética , Hipóxia/metabolismo , Técnicas In Vitro , Mucosa Intestinal/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismoRESUMO
OBJECTIVE: To determine whether hemorrhagic shock-induced bone marrow failure is mediated by the gut through the production of toxic mesenteric lymph and whether shock-induced bone marrow failure could be prevented by division of the mesenteric lymphatics. DESIGN: Prospective, controlled study. SETTING: University surgical research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were divided into five groups: unmanipulated controls (n = 12), hemorrhagic shock with laparotomy (n = 8), hemorrhagic shock with mesenteric lymph duct ligation (n = 10), sham shock with laparotomy (n = 6), and sham shock with mesenteric lymph duct ligation (n = 7). At either 3 or 6 hrs after resuscitation, bone marrow was obtained for determination of early (cobblestone forming cells) and late (granulocyte-macrophage colony forming unit and erythroid burst forming unit) hematopoietic progenitor cell growth. Parallel cultures were plated with plasma (1% and 2% v/v) from all groups to determine the effect of lymphatic ligation on hematopoiesis. MEASUREMENTS AND MAIN RESULTS: Bone marrow cellularity, cobblestone forming cells, granulocyte-macrophage colony forming unit, and erythroid burst forming unit growth in rats subjected to hemorrhagic with lymph duct ligation were similar to those observed in sham-treated animals and significantly greater than in rats subjected to shock and laparotomy without lymphatic duct ligation. Plasma from rats subjected to shock without lymph ligation was inhibitory to hematopoietic progenitor cell growth. In contrast, this shock-induced inhibition was not observed with plasma obtained from shocked rats that underwent mesenteric lymph ligation. CONCLUSIONS: Hemorrhagic shock suppresses bone marrow hematopoiesis as measured by a decrease in early and late progenitor cell growth. This suppression appears mediated through mesenteric lymph as the effect is abrogated by mesenteric lymph duct ligation. These data clearly demonstrate a link between the gut and bone marrow failure after hemorrhagic shock
Assuntos
Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Choque Hemorrágico/metabolismo , Animais , Laparotomia , Ligadura , Linfa/metabolismo , Masculino , Mesentério/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
G-protein coupled (GPC) chemoattractants are important neutrophil (PMN) activators in human shock and sepsis, acting in part by increasing cytosolic calcium ([Ca2+]i). Rats are widely used as laboratory models of shock and sepsis, but reports of [Ca2+]i flux in circulating rat PMN are rare. Moreover, the [Ca2+]i values reported often differ markedly from human systems. We developed study methods where basal [Ca2+]i values in circulating rat PMN were comparable to human PMN, but rat PMN still mobilized calcium poorly after stimulation. Trauma (laparotomy) did not change rat PMN basal [Ca2+]i, but induced brisk [Ca2+]i responses to chemokine and lipid mediators that approximated human PMN responses. This was associated with marked loading of microsomal calcium stores. Formyl peptides still mobilized calcium less well in rat than human PMN. Normal rat PMN appear to circulate in a less mature or primed form than human PMN. A very limited injury rapidly converts rat PMN to a more activated phenotype. PMN thus activated act quite similar to human PMN in terms of GPC receptor-mediated calcium mobilization. Trauma enhances rat PMN responses to GPC agonists at least in part by loading cell calcium stores.
Assuntos
Cálcio/metabolismo , Quimiocinas CXC , Peptídeos e Proteínas de Sinalização Intercelular , Neutrófilos/metabolismo , Ferimentos e Lesões/metabolismo , Animais , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas/metabolismo , Fatores Quimiotáticos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Substâncias de Crescimento/metabolismo , Humanos , Laparotomia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Post-shock mesenteric lymph kills and injures endothelial cells (ECs), but neither the mechanism nor the mediators of lymph's toxic effect are known. Thus, in these studies we investigated and characterized potential factors that may be involved in lymph's toxic effect on ECs. METHODS: Lymph was collected hourly from rats before shock, during the shock period, and for 6 hours post-shock and processed in several ways-including removal of cellular elements, freezing, heating, or separation by molecular weight-after which they were tested for toxicity (lactate dehydrogenase as a marker of cell injury and trypan blue as a marker of cell viability). RESULTS: Controls consisting of medium, pre-shock lymph, and post-shock portal vein plasma had no EC toxicity. Lymph collected 1 to 3 hours post-shock resulted in the death of 90% to 95% of ECs and caused an 8- to 10-fold increase in lactate dehydrogenase release; however, this toxic effect waned by 4 hours post-shock. Endotoxin neutralization and immune cell removal did not decrease lymph cytotoxicity but complement inactivation did. By fractionating the toxic lymph samples by size, it appears that the putative EC cytotoxic mediator(s) is larger than 100,000 d. CONCLUSIONS: Mesenteric lymph collected 1 to 3 hours after hemorrhagic shock is toxic to ECs, but this effect is lost by 4- to 5-hours post-shock and is not dependent on the presence of immune cells or endotoxin but does involve complement and other putative mediators of greater than 100,000 d.
Assuntos
Endotélio Vascular/fisiopatologia , Linfa/química , Linfa/fisiologia , Mesentério/fisiopatologia , Choque Hemorrágico/fisiopatologia , Morte Celular/fisiologia , Células Cultivadas , Proteínas do Sistema Complemento/fisiologia , Endotélio Vascular/patologia , Humanos , L-Lactato Desidrogenase/metabolismo , Peso Molecular , Fatores de TempoRESUMO
BACKGROUND: We have previously documented that lymphatic duct division protects against shock-induced lung injury when tested 3 hours post-shock and that lymph collected at 3 hours post-shock increases endothelial cell monolayer permeability. However, whether lymph collected at other time points post-shock also increases endothelial cell permeability is not known. We tested the protective effects of lymphatic division on lung permeability at 6, 12, and 24 hours post-shock and the ability of lymph collected before, during, and hourly (up to 6 hours) after shock to increase endothelial cell monolayer permeability. METHODS: At 3, 6, 12, or 24 hours after sham or actual shock (30 mm Hg for 90 min), lung permeability was measured by using Evans blue dye in rats subjected to sham or actual mesenteric duct ligation. In separate experiments, the ability of lymph collected from rats subjected to shock or sham shock to increase human umbilical vein endothelial cell (HUVEC) monolayer permeability to a 40 kd dextran rhodamine permeability probe. Lymph was tested at 10% and 1% concentrations. RESULTS: Hemorrhagic shock induced a 3- to 4-fold increase in lung permeability compared with sham-shock rats when tested at 3, 6, 12, or 24 hours post-shock. Lymphatic division prevented this increase in lung permeability at each of these time points. Sham shock lymph did not increase HUVEC permeability, while lymph from the shocked rats did, whether tested at 1% or 10%. Lymph samples collected during the shock period and hourly for 6 hours post-shock all increased HUVEC permeability; however, the greatest relative increase in HUVEC permeability was observed in the 3- and 6- hour post-shock samples. CONCLUSIONS: Lung injury after hemorrhagic shock appears to be caused by toxic factors carried in the mesenteric lymph, and factors capable of increasing HUVEC permeability initially appear in the lymph during the shock period and increase over time.
Assuntos
Lesão Pulmonar , Sistema Linfático/cirurgia , Choque Hemorrágico/complicações , Animais , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/fisiologia , Humanos , Ligadura , Pulmão/irrigação sanguínea , Pulmão/fisiopatologia , Linfa/fisiologia , Masculino , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.
Assuntos
Antígeno HLA-A2/genética , Hepatite C/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Anticorpos Antivirais/imunologia , Linfócitos B/citologia , Linhagem Celular , Mapeamento de Epitopos , Antígeno HLA-A2/imunologia , Hepacivirus/imunologia , Humanos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Valor Preditivo dos Testes , Linfócitos T Citotóxicos/virologia , Proteínas do Core Viral/imunologiaRESUMO
Of the estimated 50 million new cases of hepatitis B virus (HBV) infection diagnosed annually, 5-10% of adults and up to 90% of infants will become chronically infected, 75% of these in Asia where hepatitis B is the leading cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). In Indonesia, 4.6% of the population was positive for HBsAg in 1994 and of these, 21% were positive for HBeAg and 73% for anti-HBe; 44% and 45% of Indonesian patients with cirrhosis and HCC, respectively, were HBsAg positive. In the Philippines, there appear to be two types of age-specific HBsAg prevalence, suggesting different modes of transmission. In Thailand, 8-10% of males and 6-8% of females are HBsAg positive, with HBsAg also found in 30% of patients with cirrhosis and 50-75% of those with HCC. In Taiwan, 75-80% of patients with chronic liver disease are HBsAg positive, and HBsAg is found in 34% and 72% of patients with cirrhosis and HCC, respectively. In China, 73% of patients with chronic hepatitis and 78% and 71% of those with cirrhosis and HCC, respectively, are HBsAg positive. In Singapore, the prevalence of HBsAg has dropped since the introduction of HBV vaccination and the HBsAg seroprevalence of unvaccinated individuals over 5 years of age is 4.5%. In Malaysia, 5.24% of healthy volunteers, with a mean age of 34 years, were positive for HBsAg in 1997. In the highly endemic countries in Asia, the majority of infections are contracted postnatally or perinatally. Three phases of chronic HBV infection are recognized: phase 1 patients are HBeAg positive with high levels of virus in the serum and minimal hepatic inflammation; phase 2 patients have intermittent or continuous hepatitis of varying degrees of severity; phase 3 is the inactive phase during which viral concentrations are low and there is minimal inflammatory activity in the liver. In general, patients who clear HBeAg have a better prognosis than patients who remain HBeAg-positive for prolonged periods of time. The outcome after anti-HBe seroconversion depends on the degree of pre-existing liver damage and any subsequent HBV reactivation. Without pre-existing cirrhosis, there may be only slight fibrosis or mild chronic hepatitis, but with pre-existing cirrhosis, further complications may ensue. HBsAg-negative chronic hepatitis B is a phase of chronic HBV infection during which a mutation arises resulting in the inability of the virus to produce HBeAg. Such patients tend to have more severe liver disease and run a more rapidly progressive course. The annual probability of developing cirrhosis varies from 0.1 to 1.0% depending on the duration of HBV replication, the severity of disease and the presence of concomitant infections or drugs. The annual incidence of hepatic decompensation in HBV-related cirrhosis varies from 2 to 10% and in these patients the 5-year survival rate drops dramatically to 14-35%. The annual risk of developing HCC in patients with cirrhosis varies between 1 and 6%; the overall reported annual detection rate of HCC in surveillance studies, which included individuals with chronic hepatitis B and cirrhosis, is 0.8-4.1%. Chronic hepatitis B is not a static disease and the natural history of the disease is affected by both viral and host factors. The prognosis is poor with decompensated cirrhosis and effective treatment options are limited. Prevention of HBV infection thorough vaccination is still, therefore, the best strategy for decreasing the incidence of hepatitis B-associated cirrhosis and HCC.
Assuntos
Hepatite B Crônica/epidemiologia , Ásia , Carcinoma Hepatocelular/virologia , DNA Viral/fisiologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/complicações , Hepatite B Crônica/fisiopatologia , Hepatite B Crônica/virologia , Humanos , Neoplasias Hepáticas/virologia , Prevalência , Ativação Viral , Replicação ViralRESUMO
BACKGROUND: Previously, we showed that mesenteric lymph generated following hemorrhagic shock increases endothelial cell permeability and contributes to lung injury. It has also been shown that lymph produced at the site of burn injury plays a role in altering pulmonary vascular hemodynamics. In addition, previous experimental work has suggested that organs and tissues distant from the injury site may contribute to pulmonary dysfunction. One explanation would be that gut-derived inflammatory factors (in addition to those produced locally at the site of injury) are reaching the pulmonary circulation, where they exert their effects via the gut lymphatics. HYPOTHESES: The 2 hypotheses herein were that (1) gut-derived factors carried in the mesenteric lymph of rats generated following thermal injury will contribute to lung injury and (2) intestinal bacterial overgrowth will potentiate the degree of burn-induced lung injury. These hypotheses were tested by examining the effect of mesenteric lymph flow interruption prior to thermal injury on burn-induced lung injury in rats with a normal intestinal bacterial flora and in rats with intestinal Escherichia coli overgrowth. These rats were termed E. coli-monoassociated rats. METHODS: Normal intestinal bacterial flora and monoassociated male Sprague-Dawley rats were subjected to sham burn, 40% total body surface area burn, or lymphatic division plus burn. After 3 hours, 10 mg of Evans blue was injected to measure lung permeability. After the rats were killed, a bronchoalveolar lavage was performed and the fluid analyzed spectrophotometrically. Bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase activity, and alveolar apoptosis served to further quantitate lung injury. RESULTS: Both normal intestinal bacterial flora and monoassociated-burned rats exhibited significant increases in lung permeability, bronchoalveolar lavage fluid protein content, myeloperoxidase activity, and alveolar apoptosis. The combination of monoassociation and thermal injury resulted in even further increases in lung injury over thermal injury alone. Lymphatic division prior to thermal injury ameliorated burn-induced increases in lung permeability, bronchoalveolar lavage fluid protein content, pulmonary myeloperoxidase accumulation, and alveolar apoptosis in both normal intestinal bacterial flora and monoassociated rats. CONCLUSIONS: The results of this study support the hypothesis that gut-derived factors carried in the mesenteric lymph contribute to burn-induced lung injury and may therefore play a role in postburn respiratory failure and suggest that intestinal bacterial overgrowth primes the host such that when animals are exposed to a second stimulus (such as thermal injury) an exaggerated response occurs.
Assuntos
Queimaduras/fisiopatologia , Mucosa Intestinal/fisiopatologia , Linfa/fisiologia , Mesentério/fisiopatologia , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Apoptose/fisiologia , Permeabilidade Capilar/fisiologia , Escherichia coli/patogenicidade , Mediadores da Inflamação/sangue , Mediadores da Inflamação/fisiologia , Pulmão/irrigação sanguínea , Masculino , Alvéolos Pulmonares/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Previously, using in vivo models, we have demonstrated that ischemia/reperfusion can increase intestinal mucosal permeability, promote bacterial translocation, and induce gut cytokine production. Because of the cellular heterogeneity of the gut, however, studies investigating the direct effects of hypoxia/reoxygenation on intestinal epithelial cells are confounded in in vivo model systems. Consequently, this study examines oxidant-mediated enterocyte injury using an in vitro intestinal enterocyte hypoxia/reoxygenation model system. METHODS: Two intestinal epithelial cell lines, IEC-6 and Caco-2, were seeded onto 3-microm filters in a Transwell bicameral system and grown until tight junction integrity was established. Cells were subjected to hypoxia in a sealed chamber with 95% nitrogen and 5% carbon dioxide and incubated at 37 degrees C for 60 or 90 minutes. Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon dioxide. Permeability and bacterial translocation were assayed by measuring the phenol red concentration and culturing the bacteria that crossed the cell monolayer and reached the basal chamber of the bicameral system. Monolayer tight junction integrity was monitored by serial measurements of transepithelial electrical resistance (TEER), and cell viability was assessed by trypan blue dye. RESULTS: IEC-6 cell monolayers subjected to 60 or 90 minutes of hypoxia showed significantly higher permeability to phenol red, with 54+/-5% and 57+/-5% of the dye crossing the monolayers, respectively, compared with normoxic control (38+/-2%; p < 0.01). Caco-2 cell monolayers also had increased permeability to phenol red, with 24+/-6% and 20+/-4% of the phenol red crossing the monolayer after 60 or 90 minutes of hypoxia, respectively, compared with 8+/-3% in the normoxic controls (p < 0.01). At 3 hours after challenge with Escherichia coli, the monolayers subjected to 60 or 90 minutes of hypoxia had significantly increased bacterial translocation (IEC-6 cells, p < 0.05; Caco-2 cells, p < 0.01) compared with controls. The increased permeability of the hypoxic Caco-2 and IEC-6 monolayers was associated with a decrease in TEER beginning as early as 1 hour after reoxygenation (p < 0.01). Cell viability, however, was not decreased. CONCLUSION: These results indicate that hypoxia/reoxygenation can directly impair cellular function as manifested by increased monolayer permeability to phenol red, increased E. coli bacterial translocation, and a decrease in TEER values.
Assuntos
Células CACO-2/fisiologia , Hipóxia Celular/fisiologia , Células Epiteliais/fisiologia , Hipóxia/fisiopatologia , Intestinos/citologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Translocação Bacteriana/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Sobrevivência Celular , Citocinas/fisiologia , Modelos Animais de Doenças , Impedância Elétrica , Humanos , RatosRESUMO
We constructed two fusion proteins of scFv linked to biotin mimetic sequence (BMS) via different linkers, and expressed them in the Pichia pastoris expression/secretion system. We found that both bi-functional scFv proteins exhibited their intrinsic binding activities to antigen CA125 determined in competitive radioimmunoassay experiments, but the fusion protein with a spacer between the scFv and BMS (scFv-spacer-BMS) showed higher binding activity of streptavidin than the one with c-Myc peptide as a linker.
Assuntos
Anticorpos Biespecíficos/imunologia , Biotina/metabolismo , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Biotina/química , Antígeno Ca-125/imunologia , Humanos , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Proto-Oncogênicas c-myc , Radioimunoensaio/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Estreptavidina/metabolismoRESUMO
OBJECTIVE: To determine whether gut-derived factors leading to organ injury and increased endothelial cell permeability would be present in the mesenteric lymph at higher levels than in the portal blood of rats subjected to hemorrhagic shock. This hypothesis was tested by examining the effect of portal blood plasma and mesenteric lymph on endothelial cell monolayers and the interruption of mesenteric lymph flow on shock-induced lung injury. SUMMARY BACKGROUND DATA: The absence of detectable bacteremia or endotoxemia in the portal blood of trauma victims casts doubt on the role of the gut in the generation of multiple organ failure. Nevertheless, previous experimental work has clearly documented the connection between shock and gut injury as well as the concept of gut-induced sepsis and distant organ failure. One explanation for this apparent paradox would be that gut-derived inflammatory factors are reaching the lung and systemic circulation via the gut lymphatics rather than the portal circulation. METHODS: Human umbilical vein endothelial cell monolayers, grown in two-compartment systems, were exposed to media, sham-shock, or postshock portal blood plasma or lymph, and permeability to rhodamine (10K) was measured. Sprague-Dawley rats were subjected to 90 minutes of sham or actual shock and shock plus lymphatic division (before and after shock). Lung permeability, pulmonary myeloperoxidase levels, alveolar apoptosis, and bronchoalveolar fluid protein content were used to quantitate lung injury. RESULTS: Postshock lymph increased endothelial cell monolayer permeability but not postshock plasma, sham-shock lymph/plasma, or medium. Lymphatic division before hemorrhagic shock prevented shock-induced increases in lung permeability to Evans blue dye and alveolar apoptosis and reduced pulmonary MPO levels. In contrast, division of the mesenteric lymphatics at the end of the shock period but before reperfusion ameliorated but failed to prevent increased lung permeability, alveolar apoptosis, and MPO accumulation. CONCLUSIONS: Gut barrier failure after hemorrhagic shock may be involved in the pathogenesis of shock-induced distant organ injury via gut-derived factors carried in the mesenteric lymph rather than the portal circulation.
Assuntos
Fatores Biológicos/fisiologia , Permeabilidade da Membrana Celular , Endotélio/fisiologia , Pulmão/metabolismo , Linfa/fisiologia , Choque Hemorrágico/metabolismo , Animais , Fenômenos Fisiológicos Sanguíneos , Morte Celular , Humanos , Pulmão/patologia , Masculino , Mesentério , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Stress-gene responses, including the heat shock (HS) response and the acute phase response, are protective mechanisms for cells after exposure to stress. Both responses cannot occur simultaneously, and, in endothelial cells, the sequence of stress-gene expression seems to be a critical factor in whether cellular protection or injury occurs. OBJECTIVE: To determine if the sequence of stress-gene expression affects cellular protection or injury in epithelial cells. DESIGN: Randomized controlled in vitro study. SETTING: University research laboratory. SUBJECTS: Rat intestinal epithelial cell-6 (IEC-6) cells were grown on 35-mm culture dishes, chamber slides, or in a bicameral system to confluence or until tight junction integrity was established. INTERVENTIONS: Rat IEC-6 cells were examined for viability, apoptosis, and bacterial translocation (BT) after exposure to 25-micrograms/mL lipopolysaccharide (LPS) for 18 hours to HS (43 degrees C) for 90 minutes, to LPS followed by HS, or to HS followed by LPS. MAIN OUTCOME MEASURES: The IEC-6 cells were stained for viability and apoptosis using trypan blue and a direct immunoperoxidase detection of digoxigenin-labeled genomic DNA (Apop Tag Plus In Situ Apoptosis Detection Kit, Oncor, Gaithersburg, Md), respectively. Bacterial translocation was measured by culturing the bacteria (ie, Escherichia coli) that crossed the IEC-6 cell monolayer in the bicameral system. RESULTS: Control cells (medium only) and cells exposed to LPS alone, HS alone, or HS followed by LPS had a viability from 92% to 98%, and the percentage of apoptotic cells ranged from 2.2% to 5.7%. In contrast, IEC-6 cells exposed to LPS followed by HS had a significantly lower viability (83%, P < .05 vs all other groups) and a higher percentage of apoptotic cells (12.2%, P < .01). At 3 hours after challenge with E coli, the LPS-exposed IEC-6 cell monolayers had significantly increased BT vs control monolayers (P < .05), while the IEC-6 cell monolayers exposed to HS followed by LPS had decreased BT (P < .05). Conversely, cells exposed to LPS followed by HS had the highest magnitude of BT (P < .01 vs all other groups). CONCLUSIONS: These results indicate that preinduction of HS response can diminish LPS-induced cell injury, while induction of HS response after the LPS challenge (ie, the acute phase response) may lead to decreased enterocyte viability, increased apoptosis, and cellular dysfunction as manifested by BT.
Assuntos
Resposta ao Choque Térmico , Mucosa Intestinal/patologia , Lipopolissacarídeos/efeitos adversos , Estresse Fisiológico/patologia , Reação de Fase Aguda/genética , Reação de Fase Aguda/patologia , Reação de Fase Aguda/fisiopatologia , Animais , Apoptose , Translocação Bacteriana , Sobrevivência Celular , Células Cultivadas , Corantes , DNA/análise , Digoxigenina , Epitélio/patologia , Escherichia coli , Regulação da Expressão Gênica , Genoma , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Técnicas Imunoenzimáticas , Mucosa Intestinal/microbiologia , Mucosa Intestinal/fisiopatologia , Ratos , Estresse Fisiológico/fisiopatologia , Azul TripanoRESUMO
Although certain defined diets have been shown to promote bacterial translocation (BT), the ability to reverse diet-induced BT has not previously been investigated. Furthermore, little is known about the effects of defined diets on host immune defenses. To address these questions, we measured BT and immune reactivity in rats fed a normal diet or enteral elemental (ORAL-TPN) diet. After 7 days on the elemental or normal diet, the rats were killed, and BT and mitogen responsiveness to concanavalin A and phytohemagglutinin were measured. In separate experiments, the effects of these diets on in vivo host defenses was measured with a Staphylococcus aureus abscess model. Additional experiments were performed to determine the time required to reverse ORAL-TPN-induced BT and impairment of host immune defenses by reinstituting normal feedings. Administration of the ORAL-TPN diet for 7 days was associated with BT to the mesenteric lymph node complex of all animals, decreased blastogenic response of blood and splenic lymphocytes to mitogens, and decreased ability to control an in vivo infectious challenge with S. aureus. Each of the derangements was reversed by the reinstitution of normal feedings. In summary, the enteral administration of an elemental diet for 7 days is associated with disruption of the gut microflora, BT, and the development of an immunocompromised state, all of which can be reversed by refeeding the animals a normal diet.
Assuntos
Fenômenos Fisiológicos Bacterianos , Nutrição Enteral , Alimentos Formulados , Nutrição Parenteral Total/efeitos adversos , Análise de Variância , Animais , Ceco/microbiologia , Movimento Celular , Feminino , Linfonodos/microbiologia , Masculino , Ratos , Ratos Endogâmicos , Infecções Estafilocócicas/etiologiaRESUMO
The goal of this study was to determine the influence of bacterial translocation on systemic immunity, since bacteria and their products play a major role in the development and maintenance of the host's immune system. To test this hypothesis, we measured the blastogenic response of mononuclear cells harvested from the blood, spleen, Peyer's patches, and mesenteric lymph nodes of control and Escherichia coli C25 monoassociated mice to a battery of mitogens. The E. coli C25 monoassociation model was used because this bacterial translocation model is not associated with experimental manipulations that are likely to affect the systemic immune system. The mitogenic response of lymphocytes isolated from the E. coli C25 monoassociated mice was significantly depressed compared to the control groups (p less than 0.01). Since the biologic significance of depressed in vitro mitogen responsiveness is difficult to determine, we assessed the ability of the mice to control a bacterial challenge using an in vivo Staphylococcus aureus abscess model. It appears that the observed changes in mitogen responsiveness may be of biologic significance, since the ability of the E. coli C25 monoassociated mice to control the injected S. aureus was impaired (p less than 0.01). These results suggest that an association exists between bacterial translocation and decreased systemic immune responsiveness.
Assuntos
Infecções por Escherichia coli/imunologia , Intestinos/microbiologia , Ativação Linfocitária , Linfócitos/imunologia , Animais , Concanavalina A , Feminino , Intestinos/efeitos dos fármacos , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Penicilinas/farmacologia , Nódulos Linfáticos Agregados/imunologia , Valores de Referência , Baço/imunologia , Estreptomicina/farmacologiaRESUMO
Because of the association between the development of an immunocompromised state and an increased risk of infection, increasing attention has been focused on describing and characterizing the immune consequences of thermal injury. Results of human studies are largely based on the in vitro responsiveness of peripheral blood leukocytes, while splenocytes are generally used in the animal studies. Because the response of lymphocytes from different lymphocyte compartments may vary, we compared the responses of murine peripheral blood, splenic, Peyer's patch, and mesenteric lymph node lymphocytes to a battery of mitogens after thermal injury. Burn-induced immunosuppression was maximal in the splenic lymphocyte compartment, where the responses to all three test mitogens were depressed throughout the 28-day postburn study period. Although the PHA-induced mitogen response of lymphocytes from the other three lymphoid compartments remained suppressed throughout the study period, the response to the mitogens Con-A and PWM generally returned to normal or supranormal levels by the seventh postburn day, Therefore it appears that the effect of a thermal injury on lymphocyte function varies according to the lymphocyte compartment examined and the mitogen tested. These results raise the question of whether animal studies using splenic lymphocytes can be correlated with human studies performed on circulating blood lymphocytes.