Helicobacter felis�CAssociated Gastric Disease in Microbiota-Restricted Mice

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis–specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.     

Inflammation-associated S100 proteins: new mechanisms that regulate function

This review focuses on new aspects of extracellular roles of the calgranulins. S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and induced in several cell types. The S100A8 and S100A9 genes are regulated by pro- and anti-inflammatory mediators and their functions may depend on cell type, mediators within a particular inflammatory milieu, receptors involved in their recognition and their post-translational modification. The S100A8 gene induction in macrophages is dependent on IL-10 and potentiated by immunosuppressive agents. S100A8 and S100A9 are oxidized by peroxide, hypochlorite and nitric oxide (NO). HOCl generates intra-chain sulfinamide bonds; stronger oxidation promotes cross-linked forms that are seen in human atheroma. S100A8 is >200-fold more sensitive to oxidative cross-linking than low-density lipoprotein and may reduce oxidative damage. S100A8 and S100A9 can be S-nitrosylated. S100A8–SNO suppresses mast cell activation and inflammation in the microcirculation and may act as an NO transporter to regulate vessel tone in inflammatory lesions. S100A12 activates mast cells and is a monocyte and mast cell chemoattractant; a G-protein-coupled mechanism may be involved. Structure–function studies are discussed in relation to conservation and divergence of functions in S100A8. S100A12 induces cytokines in mast cells, but not monocytes/macrophages. It forms complexes with Zn²⁺ and, by chelating Zn²⁺, S100A12 significantly inhibits MMPs. Zn²⁺ in S100A12 complexes co-localize with MMP-9 in foam cells in atheroma. In summary, S100A12 has pro-inflammatory properties that are likely to be stable in an oxidative environment, because it lacks Cys and Met residues. Conversely, S100A8 and S100A9 oxidation and S-nitrosylation may have important protective mechanisms in inflammation.     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.     

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.     

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-β and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.     

Regulation of airway tight junctions by proinflammatory cytokines

Epithelial tight junctions (TJs) provide an important route for passive electrolyte transport across airway epithelium and provide a barrier to the migration of toxic materials from the lumen to the interstitium. The possibility that TJ function may be perturbed by airway inflammation originated from studies reporting (1) increased levels of the proinflammatory cytokines interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and IL-1beta in airway epithelia and secretions from cystic fibrosis (CF) patients and (2) abnormal TJ strands of CF airways as revealed by freeze-fracture electron microscopy. We measured the effects of cytokine exposure of CF and non-CF well-differentiated primary human airway epithelial cells on TJ properties, including transepithelial resistance, paracellular permeability to hydrophilic solutes, and the TJ proteins occludin, claudin-1, claudin-4, junctional adhesion molecule, and ZO-1. We found that whereas IL-1beta treatment led to alterations in TJ ion selectivity, combined treatment of TNF-alpha and IFN-gamma induced profound effects on TJ barrier function, which could be blocked by inhibitors of protein kinase C. CF bronchi in vivo exhibited the same pattern of expression of TJ-associated proteins as cultures exposed in vitro to prolonged exposure to TNF-alpha and IFN-gamma. These data indicate that the TJ of airway epithelia exposed to chronic inflammation may exhibit parallel changes in the barrier function to both solutes and ions.     

The α promoter regulator-ovalbumin chimeric gene resident in human cells is regulated like the authentic α 4 gene after infection with herpes simplex virus 1 mutants in α 4 gene

Human TK^? 143 cells were converted to TK⁺ phenotype with a plasmid containing the native herpes simplex virus 1 (HSV-1), thymidine kinase, a β gene, and a chimeric ovalbumin gene consisting of the coding sequences of the ovalbumin gene linked to the promoter-regulatory region of the HSV-1 α 4 gene. Comparison of the synthesis of ovalbumin and the α 4 gene product in the converted cells infected with ts mutants in α 4 gene and incubated at the permissive (33°C) and nonpermissive (39°C) temperatures revealed the following. (i) The synthesis of both ovalbumin and α 4 gene product was transiently induced at the permissive temperature but continued at elevated levels for many hours at the nonpermissive temperature. (ii) The synthesis of both ovalbumin and α 4 gene products resumed when the infected cells were shifted from permissive to nonpermissive temperature after the shut-off of α protein synthesis. (iii) Although both the β-TK and α 4-ovalbumin chimeric genes were covalently linked on the same plasmid, each was regulated independently. We conclude that α gene regulation is determined solely by (a) the inducer and (b) the induction sequence contained in the promoter-regulatory region and not by the location or the higher order structure of the immediate environment of the gene.     

Identification of Novel Cryptosporidium Genotypes from the Czech Republic

Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.