Biomedical subjects

Thomas G Blanchard

Publications and source records attributed to Thomas G Blanchard.

11 recordsLinked to original sources

PubMed2006

Induction of CTLA-4-mediated anergy contributes to persistent colonization in the murine model of gastric Helicobacter pylori infection.

Helicobacter pylori infection induces gastric inflammation but the host fails to generate protective immunity. Therefore, we evaluated the immunologic mechanisms that contribute to the failure of the T cells to promote active immunity to H. pylori in the mouse model of H. pylori infection. Spleen cells from infected C57BL/6 mice underwent significantly less proliferation and cytokine production than cells from immune mice upon in vitro stimulation with H. pylori lysate. Similar results were observed when stimulating with Ag-pulsed macrophages demonstrating that hyporesponsiveness was not due to a direct effect of H. pylori virulence factors on the T cells. Ag-specific hyporesponsiveness could be reversed by the addition of high-dose IL-2 but not by removal of CD4(+)CD25(+) T cells, indicating that hyporesponsiveness was due to anergy and not due to active suppression. Cells from infected mice lacked significant suppressor activity as shown by the failure to reduce the recall response of cells from immune mice in coculture at physiologic ratios. Direct blockade of CTLA-4 using anti-CTLA-4 Fabs or indirect blockade using CTLA-4 Ig plus anti-CD28 Ab resulted in significantly increased T cell activation in vitro. The importance of CTLA-4 in establishing anergy was confirmed in an in vivo model of H. pylori infection in which mice that received anti-CTLA-4 Fabs responded to H. pylori challenge with significantly greater inflammation and significantly reduced bacterial load. These results suggest that CTLA-4 engagement induces and maintains functional inactivation of H. pylori-specific T cells during H. pylori infection resulting in a reduced immune response.

Animals↗

PubMed2006

Regulation of murine dendritic cell immune responses by Helicobacter felis antigen.

Helicobacter infections are present in approximately 50% of humans, causing severe illnesses such as gastritis and malignancies. Dendritic cells (DC) are critical antigen-presenting cells which link innate and adaptive immune responses. The mechanism of dendritic cell regulation in Helicobacter-induced gastritis is poorly understood. These studies characterized DC isolated from the lamina propria of Helicobacter-infected mice and analyzed innate and adaptive immune responses elicited by Helicobacter antigen (Ag)-pulsed DC. The presence of DC was elevated in the gastric lamina propria infiltrate of infected mice in comparison with controls. After treatment with Helicobacter felis Ag, DC were polarized to secrete interleukin-6 as the dominant cytokine. In the presence of DC and Helicobacter Ag, responder allogeneic T cells in culture exhibited limited cell division. We suggest that the response of DC and T cells to Helicobacter Ag is critical to the chronic persistence of Helicobacter-induced gastritis.

Animals↗

PubMed2005

Colon lamina propria dendritic cells induce a proinflammatory cytokine response in lamina propria T cells in the SCID mouse model of colitis.

Intestinal immune responses are normally regulated to maintain a state of immune balance. Dendritic cells (DC) are antigen-presenting cells, which induce immune responses against microbes and other stimuli and are key players in the regulation of tolerance in the gut. These cells influence the differentiation of cytokine responses in T cells, and in the gut, in particular, such interactions may be critical to the course of inflammatory bowel disease (IBD). Using the CD45RBhi CD4+ T cell-reconstituted severe combined immunodeficient mouse model of colitis, we investigated the ability of isolated colon DC to stimulate immune responses in syngeneic and allogeneic spleen CD4+ T cells, as well as in colon T cells isolated from the same tissue as DC in IBD mice. We found that the frequency of DC in IBD mice colons and spleens was elevated in comparison with control mice, but colon and spleen DC exhibited different phenotypic and functional properties. Colon DC stimulated significantly higher levels of interferon-gamma and interleukin-6 when cocultured with autologous colon T cells than in cocultures with syngeneic or allogeneic spleen T cells. These data suggest that in the IBD colon, DC-T cell interactions may create conditions with an abundance of proinflammatory cytokines, which favor the inflammatory state.

Animals↗

PubMed2005

Eradication of Helicobacter pylori and resolution of gastritis in the gastric mucosa of IL-10-deficient mice.

BACKGROUND: Helicobacter pylori has been shown to induce pronounced gastric inflammation in the absence of interleukin-10 (IL-10) by 6 weeks post inoculation. The ability of IL-10(-/-) mice to eradicate H. pylori has not been demonstrated, possibly due to early sacrifice. Therefore, the long-term effect of enhanced gastritis on H. pylori colonization was determined in IL-10(-/-) mice. METHODS: C57BL/6 and IL-10(-/-) mice were infected with H. pylori and assessed for the degree of gastritis, bacterial load, and in vitro T-cell recall response at 4 and 16 weeks of infection. RESULTS: Infection of IL-10(-/-) mice resulted in significantly more severe gastritis than wild-type control mice and eradication of H. pylori by 4 weeks post inoculation. By 16 weeks, the level of gastritis in IL-10(-/-) was reduced to the levels observed in wild-type mice. Splenocytes from IL-10(-/-) mice were prone to produce significantly greater amounts of IFN-gamma than wild-type mice when stimulated with bacterial antigens. CONCLUSIONS: These results indicate that the host is capable of spontaneously eradicating H. pylori from the gastric mucosa when inflammation is elevated beyond the chronic inflammation induced in wild-type mice, and that the gastritis dissipates following bacterial eradication. Additionally, these data provide support for a model of gastrointestinal immunity in which naturally occurring IL-10-producing regulatory T cells modulate the host response to gastrointestinal bacteria.

Animals↗

PubMed2004

Molecular analysis of Helicobacter pylori-associated gastric inflammation in naïve versus previously immunized mice.

To identify mechanisms of immunity against Helicobacter pylori, we performed microarray analysis on gastric tissue from infected mice and mice vaccinated prior to challenge. RNA from gastric tissue was used to screen over 10,000 genes. MHC antigens and GTP binding proteins were upregulated in both groups. Infected mice were characterized by expression of innate host defense markers while immune mice expressed many IFN-gamma response genes and T cell markers. Results were confirmed for several genes by RT-PCR. CD4+ spleen cells from immune mice produced significantly more IFN-gamma than from infected mice. These results support a role for T cell regulated inflammation in H. pylori immunity.

Animals↗

PubMed2004

Clearance of Helicobacter pylori infection through immunization: the site of T cell activation contributes to vaccine efficacy.

Helicobacter pylori vaccine development has progressed rapidly in animal models. Both H. pylori-associated pathogenesis and protective immunity are CD4+ T cell dependent, with no discernable phenotypic difference to distinguish pathogenic T cells from protective T cells. Functionally however, protective T cells promote enhanced inflammation upon H. pylori challenge. Additionally, only mouse models such as phagocyte oxidase- or IL-10-deficient mice that respond to H. pylori infection with intense gastritis are capable of demonstrating spontaneous eradication of the bacteria. These data, combined with recent descriptions of down-regulatory T cells in infected humans and mice, support an emerging model of H. pylori pathogenesis in which H. pylori induces inflammation that is limited by regulatory T cells in the stomach. Immunization therefore may succeed by activating T cells in peripheral lymph nodes that are capable of promoting qualitatively or quantitatively different inflammation when recruited to the stomach. Evidence in support of this model will be discussed.

Animals↗

PubMed2004

Helicobacter pylori gamma-glutamyltranspeptidase upregulates COX-2 and EGF-related peptide expression in human gastric cells.

Gastric mucosa responds to Helicobacter pylori-induced cell damage by increasing the expression of COX-2 and EGF-related peptides. We sought to investigate the bacterial virulence factor/s and the host cellular pathways involved in the upregulation of COX-2, HB-EGF and amphiregulin in MKN 28 and AGS gastric mucosal cells. H. pylori strain CCUG 17874 was grown in Brucella broth supplemented with 0.2% (2,6-dimethyl)-beta-cyclodextrins. The soluble proteins released in the culture medium by the bacterium were fractionated by exclusion size and anion exchange chromatography. A single peak retaining the ability to upregulate COX-2 and HB-EGF mRNA and protein expression was obtained. SDS-PAGE analysis of the peak showed two peptides with an apparent molecular weight of 38 and 22 kDa, which were identified by automated Edman degradation analysis as the N-terminal and C-terminal peptides of H. pylori gamma-glutamyltranspeptidase respectively. Acivicin, a selective gamma-glutamyltranspeptidase inhibitor, counteracted H. pylori-induced upregulation of COX-2 and EGF-related peptide mRNA expression. An H. pylori isogenic mutant gamma-glutamyltranspeptidase-deficient strain did not exert any effect on COX-2, HB-EGF and amphiregulin mRNA expression. Blockade of phosphatidylinositol-3 kinase and p38 kinase, but not MAP kinase kinase, inhibited H. pylori gamma-glutamyltranspeptidase-induced upregulation of COX-2 and EGF-related peptide mRNA expression.

Cell Line↗

PubMed2004

Helicobacter infection: pathogenesis.

PURPOSE OF REVIEW: Helicobacter pylori remains one of the world's most prevalent bacterial pathogens, often causing gastritis, peptic ulcer disease, gastric mucosa-associated lymphatic tissue lymphoma, or gastric adenocarcinoma. Elucidation of H. pylori virulence mechanisms and characteristics of the host that contribute to pathogenesis will facilitate the development of both pharmacologic and immunologic therapies. RECENT FINDINGS: The functional status of the outer inflammatory protein A may have predictive value for duodenal ulcer, and host alleles for interleukin-1beta, interleukin-1R, tumor necrosis factor-alpha, and interleukin-10 correlate with increased risk for gastric cancer. H. pylori vacuolating cytotoxin A and cytotoxin-associated gene A protein interact with multiple host proteins, although downstream signaling events need further characterization. It does appear however, that CagA may participate in a negative feedback loop on Src family kinases to prevent further phosphorylation of CagA. Several models, including delayed type hypersensitivity in immune mice, and spontaneous clearance of H. pylori from interleukin-10 and phagocyte oxidase mice, provide evidence that severe inflammation may be sufficient to eradicate H. pylori. The strategies used by H. pylori to avoid the inflammatory response are also becoming clearer. H. pylori remains viable when internalized by epithelial cells, and it remains viable in macrophage phagosomes by inhibiting phagosome maturation. Additionally, H. pylori may regulate the host immune response through activation of dendritic cells and CD25 regulatory T cells, and it may direct immunosuppression of T cells. SUMMARY: Helicobacter pylori virulence is accomplished through many mechanisms, including vacuolating cytotoxin A and CagA activities, and may be predicted based on bacterial and host genotypes. Ultimately, H. pylori persistence may depend on its success in downregulating the inflammatory response.

Journal Article↗

PubMed2004

Absence of catalase reduces long-term survival of Helicobacter pylori in macrophage phagosomes.

BACKGROUND: Some Helicobacter pylori strains can survive within macrophage phagosomes for up to 24 hours. The factors that play a role in this survival remain ill-defined. Therefore, the contribution of catalase in mediating the survival of H. pylori following phagocytosis was investigated in vitro. METHODS: An isogenic, catalase-deficient strain of H. pylori was generated and tested for sensitivity to hydrogen peroxide and susceptibility to macrophage-mediated killing. RESULTS: The isogenic, catalase-deficient strain of H. pylori was effectively killed by hydrogen peroxide within 3 minutes compared to wild-type H. pylori which maintained 100% survival up to 21 minutes. The catalase-deficient mutant was also significantly more susceptible to macrophage-mediated killing than the parent strain, even when the ratio of bacteria to macrophage was increased. CONCLUSION: These results indicate that although some strains of H. pylori are capable of survival within the macrophage phagosome, survival is dependent on virulence factors such as catalase for evasion of innate host defense.

Animals↗

PubMed2003

Severe inflammation and reduced bacteria load in murine helicobacter infection caused by lack of phagocyte oxidase activity.

The vaccine-induced immune mechanisms that protect against Helicobacter pylori infection in the mouse model have not been identified. This study investigated the contribution of reactive oxygen and nitrogen intermediates to Helicobacter pathogenesis and immunity. Mice deficient in nicotinamide-adenine dinucleotide phosphate oxidase activity (gp91(phox-/-)), nitric oxide synthase activity (NOS2(-/-)), or both (gp91(phox-/-)/NOS2(-/-)) were infected with Helicobacter organisms and evaluated for inflammation and bacteria load. Infection of all 3 transgenic strains resulted in significantly more inflammation than found in infected C57BL/6 wild-type mice. However, only gp91(phox-/-) and gp91(phox-/-)/NOS2(-/-) mice had significantly reduced numbers of infected gastric glands. Intranasal immunization of NOS2(-/-) or gp91(phox-/-)/NOS2(-/-) mice against H. pylori resulted in protective immunity comparable to that seen in C57BL/6 control mice. Therefore, reactive oxygen species may play a role in limiting the inflammatory response associated with H. pylori infection of the gastric mucosa but may also limit the host's ability to eradicate Helicobacter organisms.

Animals↗

PubMed2003

Protective efficacy of anti-Helicobacter pylori immunity following systemic immunization of neonatal mice.

Helicobacter pylori infection of the gastric mucosa is a significant cause of morbidity and mortality because of its etiologic role in symptomatic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Infection occurs in young children; therefore, a prophylactic vaccine would have to be administered within the first year of life, a period thought to be immunologically privileged. We investigated vaccine formulations administered by different routes to confer protective anti-H. pylori immunity in neonatal mice. Neonatal mice immunized with a single dose of vaccine in complete Freund's adjuvant (CFA) generated antigen-specific gamma interferon-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in numbers similar to those in immunized adult mice, while vaccine administered to neonates in incomplete Freund's adjuvant (IFA) induced such cells in reduced numbers compared to those in adult mice. Both IFA and CFA, however, provided partial protection from a challenge with infectious H. pylori when the vaccine was administered subcutaneously. Neonatal immunized mice also had reduced bacterial loads when immunized intraperitoneally with CFA. In all cases, protection was equivalent to that achieved when adult counterparts were immunized. These studies suggest that an efficacious vaccine might be successfully administered to very young children to prevent perinatal infection of H. pylori.

Aluminum Hydroxide↗