Activation of type I IFN signaling by NOD1 mediates mucosal host defense against Helicobacter pylori infection

Infection of gastric epithelial cells with Helicobacter pylori (H. pylori) induces a complex array of host protective immune responses. The best known are the adaptive T helper type 1 and type 17 responses that are induced in the gastric lamina propria by antigen-presenting cells via presentation of H. pylori antigens to CD4⁺ T cells. Recently, it has become apparent that innate immune responses are also induced by H.pylori infection, both in epithelial cells and in underlying antigen-presenting cells. One important component of these innate responses involves the activity of NOD1, an intra-cellular sensor of peptides derived from the peptidoglycan component of the bacterial cell wall. In this review, we discuss our recent work showing that the signaling pathway utilized by NOD1 results in the generation of type I interferon and that this cytokine mediates both chemokine and cytokine responses that regulate the severity of gastric H. pylori infection.     

Integrin expression by human articular chondrocytes

Objective. To perform a comprehensive analysis of the integrin forms expressed by normal human articular chondrocytes. Methods. Cartilage sections and collagenase-released chondrocytes were probed with a comprehensive panel of integrin isoform–specific monoclonal antibodies (MAb), using in situ immunohistochemistry techniques, indirect immunofluorescence and flow cytometry, and immunoprecipitation/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Results. Chondrocytes in cartilage sections reacted with MAb specific for the α₅, α_v, and β₁ integrin subunits and the α_vβ₃ and α_vβ₅ heterodimers. They also reacted with a polyclonal antibody specific for the intracytoplasmic portion of the α₁ subunit. MAb specific for the α_v subunit reacted more strongly with chondrocytes near the articular surface than with those in deeper layers of cartilage, and the α_vβ₃-specific MAb reacted exclusively with chondrocytes within the most superficial 30 μm of cartilage. Flow cytometric analysis and SDS-PAGE analysis of immunoprecipitates prepared from extracts of cell-surface radioiodinated chondrocytes confirmed the above observations, and additionally revealed the presence of the α₃β₁ integrin. Conclusion. Normal human articular chondrocytes prominently display substantial quantities of the α₁β₁, α₅β₁, and α_vβ₅ integrin heterodimers, as well as lesser quantities of the α₃β₁ and α_vβ₃ heterodimers, The α_v subunit–containing integrins are detected more readily on the more superficial chondrocytes than on chondrocytes deep within cartilage. These observations provide the basis for analysis of the role of chondrocyte integrins in cartilage homeostasis and in the pathogenesis of joint diseases.     

Characterization of virulence factors of mouse-adapted Helicobacter pylori strain SS1 and effects on gastric hydrophobicity

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 ± 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 ± 410 pg/ml, P < 0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2° ± 2.2° and 41.4° ± 3.2° for C57BL/6 and gld, respectively) compared to uninfected mice (43.2° ± 2.3° and 39.5° ± 1.6°, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.     

Helicobacter pylori directs tolerogenic programming of dendritic cells

Our laboratory has shown that Helicobacter pylori infection in mice triggers an increase in the number of subepithelial lamina propria CD11c⁺ dendritic cells with luminal projections. The physical characteristic of these cells is consistent with their ability to traverse epithelial tight junctions as reported by Maria Recigno (Recigno et al. Nature Immunology 2001; 2:361-7). Gastric CD103⁺ dendritic cells, which are known to induce mucosal regulatory T cells, were also increased in number, raising the question whether H. pylori infection induces a regulatory T cell-skewed response by way of a bacteria-dendritic cell interaction. In fact, bone marrow-derived dendritic cells underwent tolerogenic programming, skewing the balance between effector and regulatory T cell responses towards regulatory T cell differentiation in a transforming growth factor-β- and interleukin-10-dependent manner. Depletion of regulatory T cell numbers augmented H. pylori-specific effector helper T cell responses, which correlated with a lower degree of H. pylori colonization. These results suggest H. pylori is capable of inducing a regulatory T cell-skewed response that limits the host's ability to eradicate the bacteria, allowing the H. pylori infection to persist. To better understand the mechanism of H. pylori tolerogenic programming we compared the differential expressions of 34 genes critical for dendritic cell function in bone marrow-derived dendritic cells pulsed with live H. pylori or other gram-negative bacteria (e.g., Escherichia coli, Acinetobacter lwoffii). Our data imply that H. pylori targets the Toll-like receptor 2 pathway to induce a regulatory T cell-skewed response. In addition, we show that H. pylori-pulsed dendritic cells are capable of inducing the conversion of naïve T cells to regulatory T cells. These observations are evidence of a unique tolerogenic program in dendritic cells that involves active editing of the immune response by H. pylori, favoring its persistence in the gastric mucosa.     

Role of αv integrin in osteoprotegerin-induced endothelial cell migration and proliferation

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor κB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin α_vβ₃ or integrin α_vβ₅ blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-α_vβ₃ antibody or anti-α_vβ₅ antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin α_v activity. Taken together, these results suggest that integrins α_vβ₃ and/or α_vβ₅ contribute to endothelial cell proliferation and migration induced by OPG.     

Ethanol, Flunitrazepam, and Pentobarbital Modulation of GABAA Receptors Expressed in Mammalian Cells and Xenopus Oocytes

GABA_A receptors composed of human α1β2γ2L, α1β2γ2S, α1 β3γ2S, α6β3γ2S, and αβ3γ3 subunits as well as bovine α1 β1 γ2L and α1β1 subunits were stably expressed in mammalian L(tk^?) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a ³⁶CI^? flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk^?) cells than oocytes; this difference ranged from 2.6–to 26–fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk^?) cells, low (5–50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the γ2L subunit. In oocytes, concentrations of 30–100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except α6β3γ2S and α1β1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk^?) cells and oocytes, except for α6β3γ2S where the pentobarbital enhancement was much greater in oocytes than cells. The α6β3γ2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABA_A receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.     

The vast majority of gastric T cells are polarized to produce T helper 1 type cytokines upon antigenic stimulation despite the absence of Helicobacter pylori infection

Helicobacter pylori infection is associated with chronic infiltration by various cell types, including T cells, whose cytokine production may regulate the inflammatory reaction as well as local immune response to the bacterium. We prospectively analyzed the constituents of the cellular infiltrates and the cytokines produced by T cells in antral biopsies obtained from 73 subjects with and without H. pylori infection, before and after eradication therapy, and compared them with a histological grade of gastritis. We found that T cells predominated in cell number, followed by granulocytes/monocytes and plasma cells in both H. pylori-infected and H. pylori-uninfected subjects. Despite the absence of H. pylori infection, more than 70% of gastric CD4-positive T cells obtained from uninfected tissue produced interferon-γ (IFN-γ) in the cytosol. Upon receptor cross-linking of a CD3 and a CD28 molecule, T cells in both infected and uninfected tissue continuously secreted a far greater amount of IFN-γ than those in peripheral blood mononuclear cell controls for a period of cell culture, whereas the increase in interleukin-4 (IL-4) was very small, and no increase in IL-2 secretion was seen. In H. pylori-infected patients, IFN-γ secretion was correlated with the grade of mononuclear cell infiltration and decreased to an uninfected control level after eradication therapy. We did not see the effect of eradication on IL-4 secretion. Anti-H. pylori antibody of the IgG2 subclass was remarkably increased in H. pylori-infected subjects. These results together suggest that gastric T cells are already differentiated to produce a large amount of IFN-γ by a mechanism unrelated to H. pylori infection. H. pylori infection appeared to activate T cells to secrete even more IFN-γ, which may contribute to maintaining a perpetual inflammation in H. pylori-infected stomach. Received: January 15, 1999 / Accepted: May 28, 1999     

CD4+ and CD8+ T cell expansions using selected TCR V and J gene segments at the onset of giant cell arteritis

Objective. To investigate T cell receptor (TCR) V_α/V_β (and in selected cases, J_β) usage in CD4+ and CD8+ peripheral blood lymphocytes of patients with giant cell arteritis (GCA), before and after treatment, as well as to analyze the HLA types of these patients. Methods. Flow cytometry, with 10 anti-TCR V-specific monoclonal antibodies (MAb), was used. To analyze J_β usage by cell populations expressing certain V_β, we used the polymerase chain reaction (PCR) technique, with V_β- and C_β-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabeled J_β-specific probes. HLA typing was performed using the microlymphocytotoxicity technique. Results. Seven of the 9 GCA patients had increased anti-TCR V MAb reactivities (interpreted as T cell expansions), which in many cases, correlated with clinical signs of disease. A strict preference for particular J_β segments was found in 3 of 3 expanded CD4+ T cell populations. Conclusion. T lymphocytes expressing specific antigen receptors are implicated in the pathogenesis of GCA.     

T cell receptor vβ repertoire of double-negative α/β t cells in patients with systemic sclerosis

Objective. To analyze the T cell receptor V_β gene on double-negative (DN) α/β T cells, which are increased in number, on peripheral blood lymphocytes (PBL) from patients with systemic sclerosis (SSc). Methods. The DN α/β T cells were sorted by flow cytometry from PBL obtained from 3 patients with SSc. The V_β repertoire was analyzed by polymerase chain reaction. Results. Only 1 or 2 V_β genes (V_β5/7, 5, or 17) were predominantly expressed on DN α/β T cells from these 3 patients. Conclusion. The V_β repertoire on DN α/β T cells in PBL from patients with SSc is rather restricted.     

Olfactomedin 4 down-regulates innate immunity against Helicobacter pylori infection

Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 KO mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with WT littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1α was increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF--κB pathway and has a negative feedback effect on NF-κB activation induced by H. pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H. pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization.     

Gastric mucosal cytokine levels in relation to host interleukin-1 polymorphisms and Helicobacter pylori cagA genotype

Objective The outcome of a Helicobacter pylori infection is related in part to interrelationships among H. pylori virulence factors and the H. pylori-induced mucosal response. The host inflammatory response is partly governed by polymorphisms in pro-inflammatory genes.

Material and methods Cytokine levels (interleukin (IL)-1β, IL-6 and IL-8) were examined in H. pylori-infected and uninfected normal-appearing mucosa from patients with non-ulcer dyspepsia (NUD), margins of gastric ulcers and cancer tissues. Cytokine levels were compared with cagA genotypes and host interleukin (IL)-1 polymorphisms.

Results The study comprised 168 Thai patients. All infected patients possessed anti-CagA antibody. Gastric mucosal IL-8 levels were significantly higher in H. pylori-positive cases than in -negative cases in all three tissue types (e.g. 1115 versus 217?pg/mg protein for NUD) (p<0.001). Normal-appearing but H. pylori-infected antral mucosa of patients with cagA type 1a strains had higher IL-8 levels than those with type 2a strains (2632 versus 1036 pg/mg protein) (p<0.005). IL-1B-511T/T carriers had higher antral mucosal IL-1β levels versus non-carriers (pg/mg protein) (T/T=221, T/C=178, C/C=70) (p=0.005). IL-1B-511T/T carriers also had higher IL-1β levels versus non-carriers in H. pylori-negative patients.

Conclusions It was found that both the host factors (IL-1 polymorphisms) and bacterial factors (cagA type 1a versus type 2a) influenced gastric mucosal cytokine levels. Future studies should concentrate on interactions among host factors (e.g. genetics and tissue responses) and bacterial and environmental factors.     

Modulation of GABAA Receptor Function by Alcohols: Effects of Subunit Composition and Differential Effects of Ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABA_A receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABA_A receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABA_A receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA_A in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 μM GABA in oocytes expressing the α₁β₂γ_2L and α₂β₂γ_2L, receptor isoforms. In the α₁β₂γ_2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABA, receptor function, but 2-butanol had a greater efficacy. In the α₂β₂ receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the a2p2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 μM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a γ_2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the α₁β₂γ_2L receptor construct. Ethanol potentiation of GABA_A receptor function was completely blocked by the benrodiazepine receptor partial inverse agonist R015-4513 at a concentration (0.5 μM) that did not alter the control GABA response. In contrast, R015-4513 did not block potentiation of GABA_A receptor activity induced by npropanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,0-diisopropylphenol). These results suggest that alcohols have specific interactions with GABA_A receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Synovial distribution of αd/CD18, a novel leukointegrin. Comparison with other integrins and their ligands

Objective. To define the synovial distribution of the novel leukointegrin α_d/CD18, and compare this with other members of the β₂-integrin family of adhesion molecules, and their counter-receptors. Methods. Monoclonal antibodies to the CD3, CD14, CD29, CD68, β₂-integrin, and immunoglobulin supergene families were used to immunohistologically define the distribution of these molecules in synovial tissue samples from normal subjects and osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Results. The normal synovial lining cell layer (SLC) expresses CD68, vascular cell adhesion molecule 1, β₁-integrin (CD29), the β₂-integrins CD11b/CD18 (α_m/β₂, Mac-1), and α/CD18, whereas CD11a/CD18 (α_L/β₂, lymphocyte function-associated antigen 1) and CD11c/CD18 (α_x/β₂, gp150/95) expression is generally absent. In RA synovitis, expression of β₂-integrins in the SLC increases in proportion to the degree of hyperplasia. The ratio of cells in the SLC which express CD11c/CD18 increases substantially, approaching that of CD11b/CD18 and α_d/CD18, while there is minimal increase in CD11a/CD18 expression. In the sublining areas of the tissues, aggregates and diffuse infiltrates of CD3/CD11a/ICAM-3+ lymphocytes are interspersed among CD68/CD14/CD11b/α_d+ macrophages. A number of aggregates demonstrate intense α_d staining of the lymphocytes. The synovial endothelium variably expresses intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule 1 (VCAM-1), with minimal evidence of ICAM-3 expression. Conclusion. The leukointegrin α_d/CD18 is expressed constitutively by synovial macrophages and macrophage-like lining cells. In rheumatoid synovitis, the intense coexpression of this integrin and its known counter-receptor, ICAM-3, in the inflammatory infiltrates, suggests a potential role for this adhesion pathway in cellular interactions occurring the synovium.     

Quantitative measurement of nitric oxide and hydrogen peroxide in Helicobacter pylori-infected Mongolian gerbils in vivo

Objective. Peroxynitrite formation, as reflected by nitrotyrosine expression, is low in Helicobacter pylori-infected Mongolian gerbils despite pronounced expression of radical-forming enzymes. The aim of the present study was to investigate in vivo whether H. pylori inhibits either one or both of the nitro- and oxyradical formation pathways. Material and methods. Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. Six months after inoculation, direct measurement of NO and H₂O₂ was performed in vivo using electrochemical microsensors positioned in close proximity to the gastric mucosa. Results. In the TN2GF4-infected animals the level of NO was significantly lower than that in controls. No significant difference in NO levels was detected between the SS1-infected group and the controls. H₂O₂ was significantly increased in the SS1 animals compared with that in controls after 6 months. The H₂O₂ level in the TN2GF4 group did not differ from that in controls. Conclusions. The results indicate that H. pylori infection is associated with strain-dependent functional inhibition of both the NO and oxyradical formation pathways in the gastric mucosa.     

T cells increase with gastric mucosal interleukin (IL)-7, IL-1, and Helicobacter pylori urease specific immunoglobulin levels via CCR2 upregulation in Helicobacter pylori gastritis

Background and Aims: The purpose of this study was to investigate possible factors that could impact on γδ T cell accumulation in the gastric mucosa. Method: Subjects were 22 Helicobacter pylori (H. pylori)‐free and 75 H. pylori‐infected mucosa biopsies classified into grades I～III gastritis as per our previous study. The number of γδ‐ and 45 RO‐positive T cells were determined by immunostaining. Gastric mucosal anti‐H. pylori urease specific antibodies and interleukin (IL)‐1β, IL‐2, 4, 7, 10 and IL‐12 levels were assayed by enzyme‐linked immunosorbent assay (ELISA). CC chemokine receptor 2 (CCR2) expression levels, migration, and cytokine production in γδ T cells stimulated by H. pylori urease were also evaluated. Results: The γδ T cell count was significantly higher in grade III gastritis which exhibits strong immunoglobulin (Ig)A and IgG responses to H. pylori urease with lymphoid follicles than in other groups. γδ T cell count was significantly correlated with IL‐1β and interleukin‐7 (IL‐7) levels in the gastric mucosa. H. pylori urease immunoreactivity was detected in lamina propria of grade III gastritis, along with many γδ T cells. After H. pylori eradication therapy, the γδ T cell count in grade III gastritis significantly decreased. H. pylori urease stimulated significant increases in CCR2 expression levels, although to a lesser degree than those induced by IL‐7 stimulation in both peripheral and mucosal γδ T cells. Interferon (IFN)‐γ and IL‐10 production was also stimulated by H. pylori urease in peripheral γδ T cells. Conclusions: Gastric mucosal increases in IL‐7 and IL‐1β closely corresponded to the accumulation of γδ T cells in gastric mucosa. An association was also seen between γδ T cell accumulation and H. pylori urease‐specific Ig levels.     

Upregulated Cyclooxygenase-2 Inhibits Apoptosis of Human Gastric Epithelial Cells Infected with Helicobacter pylori

Helicobacter pylori induces apoptosis and alters the proliferation of gastric mucosal epithelial cells. Cyclooxygenase-2 (COX-2), the inducible form of prostaglandin (PG) synthesis, is known to cause alteration in epithelial cell growth. The goal of this study was to determine whether COX-2 gene expression by H. pylori infection could influence gastric epithelial cell apoptosis. Expression of COX-2 mRNA and proteins was up-regulated in Hs746T gastric epithelial cell lines infected with H. pylori, when assessed by quantitative RT-PCR and western blot. Inhibition of COX-2 expression using NS-398, a specific COX-2 inhibitor, showed a significant increase of gastric epithelial cell apoptosis and caspase-3 activation in Hs746T cells infected with H. pylori. Moreover, the effect of NS-398 on H. pylori-induced apoptosis was reversed by the addition of PGE₂. These results suggest that up-regulated COX-2 expression by H. pylori infection can inhibit apoptosis of gastric epithelial cells.     

Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.     

Ultrastructural analysis of the distribution of the vitronectin receptor (αvβ3) in human platelets and megakaryocytes reveals an intracellular pool and labelling of the α-granule membrane

The vitronectin receptor (VnR or α_vβ₃) belongs to the cytoadhesin subclass of the integrin family. This subclass consists of two receptors which have the β₃ subunit in common: GP IIb–IIIa complexes (or α_IIbβ₃) and VnR. We report the subcellular distribution of VnR within human platelets as determined by immunogold staining of ultrathin frozen sections and transmission electron microscopy. Monoclonal antibodies directed against: (i) the α_v subunit (LM142, AMF7, CLB-706), or (ii) an epitope specific to the complex (LM609) were used. Although VnR is present on platelets, it is a minor component. We therefore first compared several different staining procedures to detect this integrin. Optimal localization of VnR was obtained using a multistep procedure in which biotinylated anti-mouse IgG and a monoclonal anti-biotin antibody provided staining enhancement. Results showed that although present on the surface, α_vβ₃ was mostly detected in internal membrane systems including those of α-granules. Occasionally, platelet sections showed special vesicular structures covered by gold particles. These were often localized at the edge or immediately under the plasma membrane and their origin remains unclear. An internal pool of α_vβ₃ was confirmed by flow cytometry and by using platelets from a patient with type I Glanzmann's thrombasthenia arising from a GP IIb gene defect. We also investigated the presence of VnR in megakaryocytes (MK) obtained from normal human bone marrow. A fluorescence study showed VnR in small MK with unilobulated nuclei, suggesting that synthesis of this integrin occurs early during megakaryocytopoiesis. In mature cells, VnR expression had decreased relative to GP IIb–IIIa, although intracellular staining was present in EM and α-granules were again labelled.     

Inhibition of plasma-mediated adherence of sickle erythrocytes to microvascular endothelium by conformationally constrained RGD-containing peptides

Adherence of sickle erythrocytes to vascular endothelium likely initiates or participates in microvascular occlusion, leading to ischemic tissue and organ damage characteristic of sickle-cell pain episodes. In vitro, sickle-cell adherence to endothelium involves adhesive plasma proteins and integrin and nonintegrin receptors on sickle cells and endothelial cells. The involvement of arginine-glycine-aspartic acid (RGD) sequences in adhesive plasma proteins and integrin receptors suggests that RGD-containing peptides may inhibit sickle-cell/endothelial-cell adherence. In the present study, inhibition of plasma-mediated sickle-erythrocyte adherence to endothelium using conformationally constrained RGD-containing peptides was quantified in vitro under continuous flow at a shear stress of 1.0 dyn/cm². Two conformationally constrained RGD peptides were investigated: 6Z (which has high affinity for α₅β₁, α_vβ₃, and α_IIIbβ₃ integrin receptors), and TP9201 (which preferentially binds to α_IIbβ₃). Peptide 6Z at 50 μM inhibited plasma-mediated sickle-cell adherence to microvascular endothelium 70% when incubated with sickle red cells, and 63% when incubated with endothelium. Under similar conditions, peptide TP9201 inhibited plasma-mediated sickle-cell adherence up to 85% at concentrations from 250 to 500 μM TP9201. The inhibition of plasma-mediated adherence by conformationally constrained RGD peptides, but not by linear or circular constructs, suggests that the tertiary structure of the peptide containing the binding sequence is important. Inhibition of plasma-mediated sickle-cell adhesion with these peptides in vitro suggests that such conformationally constrained RGD peptides could provide therapeutic interventions in the course of the disease by inhibiting receptor-ligand interactions. © 1996 Wiley-Liss, Inc.     

Gastric expression of inducible nitric oxide synthase and myeloperoxidase in relation to nitrotyrosine in Helicobacter pylori-infected Mongolian gerbils

Objective. For obscure reasons Helicobacter pylori infection of the gastric mucosa is maintained despite a pronounced host defence response. The present study elucidates possible H. pylori-related interference in the oxy- and nitro-radical formation pathways. Material and methods. Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. At 3, 6, 12 or 18 months after inoculation, gastric expressions of myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and nitrotyrosine were assessed by Western blotting. Results. Expression of both iNOS and MPO was markedly up-regulated in the H. pylori-infected animals compared with non-infected controls. The TN2GF4-infected animals initially (at 3 and 6 months) demonstrated pronounced expression of both iNOS and MPO. The SSI-infected animals exhibited a slower onset with significantly increased iNOS after 12 and 18 months. Nitrotyrosine expression was slightly elevated in the infected groups at 3 and 6 months compared with that in the controls. Nitrotyrosine levels then decreased and were no longer significantly different from those of controls (TN2GF4-infected animals) or were lower (SS1-infected animals) than in the controls. Conclusions. The results indicate that peroxynitrite formation as reflected by nitrotyrosine expression is low or even inhibited in chronic H. pylori infection despite pronounced expression of enzymes representing both the oxy- and nitro-radical formation pathways. The results support the theory that H. pylori survival is related to functional inhibition of mucosal enzymatic NO and/or oxy-radical formation.