Attenuation of Helicobacter pylori-induced gastric inflammation by prior cag− strain (AM1) infection in C57BL/6 mice

Helicobacter pylori, colonize in stomach of ~50% of the world population. cag pathogenicity Island of H. pylori is one of the important virulent factors that attributed to gastric inflammation. Coinfection with H. pylori strain with different genetic makeup alters the degree of pathogenicity and susceptibility towards antibiotics. The present study investigates host immunomodulatory effects of H. pylori infection by both cag+ strain (SS1) and cag− strain (AM1). C57BL/6 mice were infected with AM1 or SS1 strain as well as AM1 followed by SS1 (AM1/SS1) and vice versa. Results: Mice infected with AM1/SS1 strain exhibited less gastric inflammation and reduced proMMP9 and proMMP3 activities in gastric tissues as compared to SS1/SS1 and SS1/AM1 infected groups. The expression of both MMP9 and MMP3 followed similar trend like activity in infected tissues. Both Th1 and Th17 responses were induced by SS1 strain more profoundly than AM1 strain infection which induced solely Th1 response in spleen and gastric tissues. Moreover, IFN-γ, TNF-α, IL-1β and IL-12 were significantly downregulated in mice spleen and gastric tissues infected by AM1/SS1 compared to SS1/SS1 but not with SS1/AM1 coinfection. Surprisingly, IL-17 level was dampened significantly in AM1/ SS1 compared to SS1/AM1 coinfected groups. Furthermore, number of Foxp3+ T-regulatory (Treg) cells and immunosuppressive cytokines like IL-10 and TGF-β were reduced in AM1/SS1 compared to SS1/SS1 and SS1/AM1 coinfected mice gastric tissues. Conclusions: These data suggested that prior H. pylori cag− strain infection attenuated the severity of gastric pathology induced by subsequent cag+ strain in C57BL/6 mice. Prior AM1 infection induced Th1 cytokine IFN-γ, which reduced the Th17 response induced by subsequent SS1 infection. The reduced gastritis in AM1/SS1-infected mice might also be due to enrichment of AM1- primed Treg cells in the gastric compartment which inhibit Th1 and Th17 responses to subsequent SS1 infection. In summary, prior infection by non-virulent H. pylori strain (AM1) causes reduction of subsequent virulent strain (SS1) infection by regulation of inflammatory cytokines and MMPs expressio

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

Trehalose 6,6′-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-μm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis

Catalytic sites for 3'- and 5' incision of Escherichia coli nucleotide excision repair are both located in UvrC.

Medical Biochemistr

The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex - Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site

Bio-organic Synthesi

The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair

Bio-organic Synthesi

Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis

Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

BACKGROUND: Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs(Shh+)) that are able to regulate in vitro angiogenesis.METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated the ability of MPs(Shh+) to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs(Shh+), MPs(Shh+) plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs(Shh+)-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs(Shh+)-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs(Shh+) increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs(Shh+) enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs(Shh+) on eNOS pathway. Quantitative RT-PCR revealed that MPs(Shh+) treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. CONCLUSIONS/SIGNIFICANCE: These findings suggest that MPs(Shh+) may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis

Polyoma Virus-Induced Osteosarcomas in Inbred Strains of Mice: Host Determinants of Metastasis

The mouse polyoma virus induces a broad array of solid tumors in mice of many inbred strains. In most strains tumors grow rapidly but fail to metastasize. An exception has been found in the Czech-II/Ei mouse in which bone tumors metastasize regularly to the lung. These tumors resemble human osteosarcoma in their propensity for pulmonary metastasis. Cell lines established from these metastatic tumors have been compared with ones from non-metastatic osteosarcomas arising in C3H/BiDa mice. Osteopontin, a chemokine implicated in migration and metastasis, is known to be transcriptionally induced by the viral middle T antigen. Czech-II/Ei and C3H/BiDa tumor cells expressed middle T and secreted osteopontin at comparable levels as the major chemoattractant. The tumor cell lines migrated equally well in response to recombinant osteopontin as the sole attractant. An important difference emerged in assays for invasion in which tumor cells from Czech-II/Ei mice were able to invade across an extracellular matrix barrier while those from C3H/BiDa mice were unable to invade. Invasive behavior was linked to elevated levels of the metalloproteinase MMP-2 and of the transcription factor NFAT. Inhibition of either MMP-2 or NFAT inhibited invasion by Czech-II/Ei osteosarcoma cells. The metastatic phenotype is dominant in F1 mice. Osteosarcoma cell lines from F1 mice expressed intermediate levels of MMP-2 and NFAT and were invasive. Osteosarcomas in Czech-II/Ei mice retain functional p53. This virus-host model of metastasis differs from engineered models targeting p53 or pRb and provides a system for investigating the genetic and molecular basis of bone tumor metastasis in the absence of p53 loss