Human metapneumovirus infection of airway epithelial cells is associated with changes in core metabolic pathways.

Human metapneumovirus (hMPV) is an important cause of acute lower respiratory tract infections in infants, elderly and immunocompromised individuals. Ingenuity pathway analysis of microarrays data showed that 20% of genes affected by hMPV infection of airway epithelial cells (AECs) were related to metabolism. We found that levels of the glycolytic pathway enzymes hexokinase 2, pyruvate kinase M2, and lactate dehydrogenase A were significantly upregulated in normal human AECs upon hMPV infection, as well as levels of enzymes belonging to the hexosamine biosynthetic and glycosylation pathways. On the other hand, expression of the majority of the enzymes belonging to the tricarboxylic acid cycle was significantly diminished. Inhibition of hexokinase 2 and of the glycosylating enzyme O-linked N-acetylglucosamine transferase led to a significant reduction in hMPV titer, indicating that metabolic changes induced by hMPV infection play a major role during the virus life cycle, and could be explored as potential antiviral targets.

Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt-NOS signaling.

Ischemia/reperfusion (I/R) injury in cardiomyocytes is related to excess reactive oxygen species (ROS) generation and can be modulated by nitric oxide (NO). We have previously shown that grape seed proanthocyanidin extract (GSPE), a naturally occurring antioxidant, decreased ROS and may potentially stimulate NO production. In this study, we investigated whether GSPE administration at reperfusion was associated with cardioprotection and enhanced NO production in a cardiomyocyte I/R model. GSPE attenuated I/R-induced cell death [18.0 +/- 1.8% (GSPE, 50 microg/ml) vs. 42.3 +/- 3.0% (I/R control), P < 0.001], restored contractility (6/6 vs. 0/6, respectively), and increased NO release. The NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 200 microM) significantly reduced GSPE-induced NO release and its associated cardioprotection [32.7 +/- 2.7% (GSPE + L-NAME) vs. 18.0 +/- 1.8% (GSPE alone), P < 0.01]. To determine whether GSPE induced NO production was mediated by the Akt-eNOS pathway, we utilized the Akt inhibitor API-2. API-2 (10 microM) abrogated GSPE-induced protection [44.3% +/- 2.2% (GSPE + API-2) vs. 27.0% +/- 4.3% (GSPE alone), P < 0.01], attenuated the enhanced phosphorylation of Akt at Ser473 in GSPE-treated cells and attenuated GSPE-induced NO increases. Simultaneously blocking NOS activation (L-NAME) and Akt (API-2) resulted in decreased NO levels similar to using each inhibitor independently. These data suggest that in the context of GSPE stimulation, Akt may help activate eNOS, leading to protective levels of NO. GSPE offers an alternative approach to therapeutic cardioprotection against I/R injury and may offer unique opportunities to improve cardiovascular health by enhancing NO production and increasing Akt-eNOS signaling.

Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium.

The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS)3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 microM H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY-294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor Nomega-nitro-L-arginine methyl ester (200 microM) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.

Biomechanical signals suppress TAK1 activation to inhibit NF-kappaB transcriptional activation in fibrochondrocytes.

Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1beta, TNF-alpha, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1beta, TNF-alpha, and LPS converge to initiate NF-kappaB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1beta-induced proinflammatory cascade downstream of IkappaB kinase activation to regulate IkappaBalpha and IkappaBbeta degradation and synthesis, and promote IkappaBalpha shuttling to export nuclear NF-kappaB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-kappaB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription.

Helicobacter pylori HopH (OipA) and bacterial pathogenicity: genetic and functional genomic analysis of hopH gene polymorphisms.

BACKGROUND: Expression of the Helicobacter pylori outer membrane protein HopH is regulated by phase variation within a CT dinucleotide repeat motif of the hopH gene. METHODS: To investigate the importance of HopH for bacterial pathogenicity, we performed a detailed functional genomic and population-based genetic characterization of this contingency locus. RESULTS: Sequencing of hopH in H. pylori strains from 58 patients revealed that the hopH "on" genotype is linked to bacterial virulence determinants, such as the vacAs1, vacAm1, babA2, and, most strongly, cagA genotypes. hopH mutagenesis resulted in reduced bacterial adherence to gastric epithelia in vitro. Complementation of hopH in trans restored the adherence properties of hopH mutants. Although HopH has been previously linked to proinflammatory epithelial signaling, hopH mutagenesis did not alter epithelial interleukin-8 secretion in vitro. Comparative epithelial gene-expression profiling by cDNA microarrays revealed no significant differences between the wild-type-specific and hopH mutant-specific transcriptomes. By contrast, a large set of genes was differentially regulated in a cag pathogenicity island-dependent manner. CONCLUSION: An in-frame hopH gene may be linked to gastroduodenal diseases because of its association with other virulence factors or increased bacterial adherence and colonization. The strong linkage with cagA indicates that HopH may contribute to the fitness of cagA-positive strains in vivo.

CD25+/Foxp3+ T cells regulate gastric inflammation and Helicobacter pylori colonization in vivo.

BACKGROUND & AIMS: Helicobacter pylori infects more than half of the world's population. In contrast to most other pathogens, the microbe persists for the virtual life of its host. It is unclear why the immune system is unable to eliminate the infection, but recent studies suggested that CD4+/CD25+/Foxp3+ regulatory T cells may be involved in this process. METHODS: By using a mouse model of infection and gastric biopsies from 108 patients, we performed a detailed descriptive and functional characterization of the Helicobacter-induced CD25+/Foxp3+ T-cell response. RESULTS: In C57BL/6 mice, H pylori induced a marked gastric Foxp3+ T-cell response, which increased over several months together with the severity of inflammation, until a stable homeostatic situation became established. Accordingly, in Helicobacter-infected patients, but not in uninfected individuals, large numbers of gastric Foxp3+ T cells were detected immunohistochemically. To define the functional in vivo relevance of this response, CD25+ cells were depleted systemically in mice by using an anti-CD25 monoclonal antibody (PC61). Already 4 weeks after infection, PC61-treated mice, but not untreated animals, developed a severe gastritis with heightened cytokine expression and increased numbers of mucosal T cells, B cells, and macrophages. This was accompanied by increased titers of H pylori-specific IgG1 and IgG2c antibodies in the sera of PC61-treated mice. This increased gastric inflammatory response in CD25-depleted mice was associated with reduced bacterial loads. CONCLUSIONS: CD25+/Foxp3+ T cells actively participate in the immune response to H pylori. In vivo depletion of these cells in infected mice leads to increased gastric inflammation and reduced bacterial colonization.