Pathogenic molecular mechanisms in an animal model of fulminant hepatic failure: rabbit hemorrhagic viral disease

In this study we sought to determine whether molecular mechanisms involved in the pathogenesis of fulminant hepatic failure are present in rabbits experimentally infected with rabbit hemorrhagic disease virus (RHDV). The activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, as well as bilirubin concentration, were found to be significantly increased 36 hours after infection. Infected animals also demonstrated significant decreases in factor VII activity, in the Fischer index, and in the deterioration of prothrombin time. The concentration of reduced glutathione was significantly decreased 36 hours after infection, and we noted a marked increase in the ratio of oxidized to reduced glutathione. Infected animals showed progressive decreases in liver activity of the antioxidant enzyme superoxide dismutase. Expression of hepatocyte growth factor and c-met was found to be progressively reduced from 24 hours after infection, during which time we detected no modification in messenger RNA (mRNA) levels of transforming growth factor (TGF)-alpha. TFG-beta 1 was overexpressed 24 and 36 hours after infection, and 36 hours after infection we detected a significant increase in TNF-alpha mRNA levels. Experimental RHDV infection also induced marked activation of nuclear factor-kappaB and a significant increase in inducible nitric oxide synthase mRNA levels from 24 hours after infection. Data obtained from this animal model support its usefulness in the investigation of potential novel therapeutical modalities aimed at neutralizing reactive oxygen species and hepatocyte growth inhibitors or enhancing hepatocyte responsiveness to mitogens.     

Intramyocardial synthesis of pro- and anti-inflammatory cytokines in infants with congenital cardiac defects

OBJECTIVES: We sought to test the hypothesis that cytokines would be expressed in the myocardium of infants with congenital cardiac defects and to identify the signaling pathways involved. BACKGROUND: Mechanical stress upregulates pro-inflammatory cytokines in the myocardium. METHODS: Fifteen infants with tetralogy of Fallot (TOF) (n = 7) or with ventricular septal defects (VSDs) (n = 8) were investigated. Concentrations of pro- and anti-inflammatory cytokines and of the inducible nitric oxide synthase (iNOS) were measured by enzyme-linked immunosorbent assay and/or Western blotting in the right ventricular myocardium taken during cardiac surgery. Activation of the nuclear factor-kappa-B (NF-kappa-B) and p38 mitogen-activated protein kinase (MAPK) pathways was assessed by electrophoretic mobility shift assay with supershift and/or Western blotting, respectively. RESULTS: The pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, and IL-6 and the anti-inflammatory cytokine IL-10 were detected in the myocardium of all patients. Concentrations of the pro-inflammatory cytokines and also of phosphorylated p38 MAPK were higher in patients with TOF than in those with VSD and correlated with the degree of pressure overload of the right ventricle. Levels of phosphorylated I-kappa-B-alpha, iNOS, and IL-10 were similar in patients with TOF and in those with VSD. CONCLUSIONS: Our results show intramyocardial synthesis of pro-inflammatory cytokines in infants with congenital cardiac defects. This is associated with activation of both the NF-kappa-B and p38 MAPK pathways. The latter could be particularly important for the transduction of mechanical signals in the infant's myocardium. Synthesis of IL-10 indicates an intramyocardial anti-inflammatory potential in this age group.     

Cyclic AMP enhances resolution of allergic pleurisy by promoting inflammatory cell apoptosis via inhibition of PI3K/Akt and NF-κB

Selective and timely induction of apoptosis is an effective means of resolving inflammation. The effects and putative mechanisms by which cyclic AMP (cAMP) modulates leukocyte apoptosis in vivo are still unclear. The present study aims at identifying intracellular pathways underlying the ability of cAMP elevating agents to resolve eosinophilic inflammation in a model of allergic pleurisy in mice. Ovalbumin (OVA) challenge of immunized mice induced eosinophil recruitment that peaked at 24 h and persisted till 48 h. Treatment with the PDE4 inhibitor rolipram, cAMP mimetic db-cAMP or adenylate cyclase activator forskolin, at 24 h after antigen-challenge resulted in profound resolution of eosinophilic inflammation, without a decrease of mononuclear cell numbers. There was a concomitant increase in number of apoptotic cells in the pleural cavity. The effects of rolipram and db-cAMP were inhibited by the PKA inhibitor H89. Inhibition of PI3K/Akt or NF-κB induced resolution of inflammation that was associated with increased apoptosis. OVA-challenge resulted in a time-dependent activation of Akt and NF-κB, which was blocked by treatment with rolipram or PI3K/Akt pathway inhibitors. Thus, cAMP elevating agents resolve established eosinophilic inflammation by inducing leukocyte apoptosis. Mechanistically, the actions of cAMP are dependent on PKA and target a PI3K/Akt-dependent NF-κB survival pathway.     

大蒜素对人白血病Jurkat细胞生长和凋亡的影响

目的探讨大蒜素对人白血病Jurkat细胞生长抑制和诱导凋亡的作用。方法用凝胶直流法(EMSA)分析大蒜素处理前后Jurkat细胞核中NF-κB的DNA结合活性。MTT比色分析法检测大蒜素对Jurkat细胞生长的抑制作用,流式细胞术分析大蒜素对Jurkat细胞凋亡的影响。结果大蒜素处理后NF-κB的DNA结合活性降低,各浓度大蒜素均能抑制Jurkat细胞生长,其抑制作用呈时间浓度依赖性。结论大蒜素对Jurkat细胞生长有抑制作用,其抑制作用与诱导Jurkat细胞凋亡有关,可能是通过NF-κB信号途径起作用。     

MZF-2转录因子与甲基化后的DNA结合基序结合活性检测

 目的 探讨MZF-2转录因子与甲基化后的DNA结合基序结合活性。方法 常规培养CNE-2细胞株,提取核蛋白;合成、标记和纯化MZF-2探针,其中一组用M.SssI甲基化酶处理,进行凝胶迁移分析（EMSA）。结果 EMSA图中单独加入甲基化探针组无结合条带,单独加入原样未甲基化探针组有结合条带,同时加入标记和未标记原样未甲基化探针组结合条带强度减弱。结论 甲基化可以抑制MZF-2转录因子与其DNA结合基序的结合活性。