Passive oral immunization by egg yolk immunoglobulin (IgY) to Vibrio cholerae effectively prevents cholera.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease.

BACKGROUND: Since the discovery of Helicobacter pylori, various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown. MATERIALS AND METHODS: We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis. RESULTS: Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher (p = .029) prevalence of H. hepaticus infection than samples from patients with other diseases. CONCLUSION: Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.

Autoimmunity, infectious immunity, and atherosclerosis.

INTRODUCTION: Vascular inflammation is common in certain systemic autoimmune diseases and contributes to the oxidation of low-density lipoprotein (oxLDL) and oxLDL/beta2-glycoprotein I (beta2GPI) complex formation. These complexes have been implicated as proatherogenic autoantigens that participate in the development of atherosclerotic disease. DISCUSSION: We have demonstrated that the in vitro macrophage uptake of oxLDL/beta2GPI complexes increases in the presence of IgG anti-beta2GPI antibodies and that IgG immune complexes containing oxLDL/beta2GPI upregulate the expression of both scavenger and Fcgamma receptors to activate beta2GPI specific T cells. Some persistent infections may cause immune responses that promote atherogenesis. Cellular immunity (Th1) against Helicobacter pylori (H. pylori) derived heat shock protein 60 (Hp-HSP60) cross-reacts with endogenous HSP60 to cause cardiovascular disease likely by molecular mimicry. CONCLUSION: Infectious cellular response may be proatherogenic,while the humoral response (antibody production) maybe protective. We review the recent progress in our understanding of autoimmunity and infectious immunity that promote atherosclerosis.

Immune reactions against elongation factor 2 kinase: specific pathogenesis of gastric ulcer from Helicobacter pylori infection.

Helicobacter pylori (H. pylori) infection is a definite causative factor for gastric ulcers (GUs). In the present study we detected a specific antigen of gastric epithelial cells (HGC-27) using cell ELISA, which was recognized by the sera of GU patients (n = 20) but not in patients with chronic gastritis (CG; n = 20) or in healthy volunteers (HC; n = 10). This antigen was over-expressed by a stressful (heat-stressed) environment, and was identified as elongation factor 2 kinase (EF-2K) by western blotting. The GU patients' lymphocytes stimulated by H. pylori specifically disrupted heat-stressed HGC-27 cells in a cytotoxic assay. In flow cytometry, the effector cells (lymphocytes) from GU patients were significantly differentiated to T helper type 1 lymphocyte (Th1) and cytotoxic T lymphocyte (CTL) as opposed to those from CG patients. The target cells (HGC-27) expressed EF-2K and MHC-class I together with costimulatory molecules from heat stress. This antigen specific immune mechanism could have a prominent role in the pathogenesis of GU.

Chronic infections and atherosclerosis.

The immune response against heat shock protein 60 (HSP60) derived from pathogens causing chronic infections is thought to be an important pro-atherogenic mechanism because high serum levels of antibodies against HSP60 have been associated with atherosclerotic diseases, such as coronary artery diseases, or cerebro-vascular events. Furthermore, the presence of HSP60-specific T lymphocytes in circulation may increase the risk of atherosclerosis. Our recent in vitro and in vivo studies have also shown an association of Helicobacter pylori-HSP60 (Hp-HSP60) specific Th1 immune responses elicited by H. pylori infection with the progression of atherosclerosis in a hyperlipidemic mouse model. These Th1 dominant immune responses may cross-react with endogenous HSP60 expressed on stressed cells of the vascular endothelium, likely due to molecular mimicry. However, the exact mechanisms by which endothelial cells display their HSP60 molecule or present HSP60 antigenic epitopes on the surface are still unclear.

Chronic infections and atherosclerosis.

Immunoinflammatory processes due to chronic infection are thought to be one of the definitive atherogenetic processes. Especially, anti-heat shock protein antibodies have been related to the prevalence of disease such as coronary artery disease or cerebral infarction, etc., resulted from atherosclerosis. Furthermore, the presence of HSP60-specific T lymphocytes in circulation may increase the risk of atherosclerosis. We have recently demonstrated the evidences that Helicobacter pylori infection induced atherosclerosis in apoe+/- ldlr+/- mice and that Hp-anti-heat-shock protein specific Th1-dominant immune responses had a major involvement in the progression of atherosclerosis. These cellular immune responses caused autoimmunity against endogenous HSP60 (expressed on the stressed cells of vascular endothelium), due to the molecular mimicry. Therefore, an appropriate treatment with antibiotics or with anti-HSP60 antibodies, which regulates the Th1 induction, could sufficiently reduce the progression of atherosclerosis.

Antibodies against heat shock protein 60 derived from Helicobacter pylori: diagnostic implications in cardiovascular disease.

Immune responses against heat shock protein 60 (HSP60) of pathogen-origin are thought to be defensive events which, due to molecular mimicry, misdirect to a human counterpart. Therefore, atherosclerosis may be serologically predicted by anti-HSP60 antibodies (Abs). In the present study, we analyzed the clinical prevalence of the serum IgG Abs against Helicobacter pylori (Hp)-derived HSP60 (Hp-HSP60) or its peptide fragments in patients with cardiovascular disease (CVD; n=250), as compared to those in age- and gender-matched non-CVD patients (n=293). Anti-Hp cell lysate Abs frequently appeared in Hp-infected patients who were not associated with CVD. In contrast, Abs against the particular amino acid sequence Hp-HSP60(II3) (II3 region, Glu(141)-Leu(160), in Hp-HSP60) predominantly appeared in CVD patients, as well as IgG anti-human HSP60 (Hu-HSP60(w)). Furthermore, neither titer of anti-Hp-HSP60(II3) nor anti-Hu-HSP60(w) Abs was correlated with the levels of high sensitivity C-reactive protein (hsCRP). This data strongly suggested that IgG anti-Hp-HSP60(II3) Abs cross-reacted with Hu-HSP60(w) were independent diagnostic markers relevant to CVD. Further, the 20 amino acid residues (Glu(141)-Leu(160)) might be predominant CVD-associated epitopes that induce anti-Hu-HSP60 auto-Abs, whose location was predicted in the tertiary structure of Hu-HSP60.

Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR)2 and mitogen-activated protein (MAP) kinase pathway in human monocytes.

Previous reports have indicated that Helicobacter pylori heat-shock protein 60 (H. pylori-HSP60), as an immunodominant antigen, induces interleukin (IL)-8 production in human monocytes. The exact mechanism by which H. pylori-HSP60 induces IL-8 production in monocytes has not been fully elucidated. In the present study, the downstream pathway by which H. pylori-HSP60 induces IL-8 secretion in human monocytic cell lines was investigated. Intact H. pylori, heat-killed H. pylori and H. pylori recombinant HSP60 (rHpHSP60) all induced the secretion of IL-8 and the activation of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal kinase (JNK), up to 24 h in NOMO1 cells. The specific inhibitors PD98059 and U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling) down-regulated IL-8 secretion from rHpHSP60-treated NOMO1 cells. An anti-Toll-like receptor (TLR)2 antibody or TLR2 small interfering RNA (siRNA) partially inhibited the secretion of IL-8, and anti-TLR2 antibody also suppressed activation of ERK and p38 MAPK in rHpHSP60-treated NOMO1 cells. These reactions were associated with nuclear factor-kappaB (NF-kappaB)-mediated transcriptional activation, since U0126, SB203580 and the anti-TLR2 antibody decreased NF-kappaB activation. Taken together, the results suggest that ERK and p38 MAPK signalling linked to the TLR2 recognition receptor in human monocytes may be an important pathway in H. pylori-HSP60-induced IL-8 secretion.

Helicobacter pylori heat-shock protein 60 induces production of the pro-inflammatory cytokine IL8 in monocytic cells.

Interleukin 8 (IL8) is usually produced in both epithelial and monocytic cells during bacterial infections, causing inflammation. Helicobacter pylori induces production of IL8 from gastric epithelial cells via its cag pathogenicity island (cag PAI) system, LPS and outer-membrane protein. In some bacteria, heat-shock protein 60 (HSP60) also elicits a strong pro-inflammatory response in cells of the innate immune system. Three recombinant H. pylori HSP60 (rHSP60) proteins of different sizes were produced and one of these was used to raise two monoclonal antibodies (2E7 and 7B5). IL8 production was found to be induced in cultured monocytic cells treated with H. pylori cells or rHSP60 proteins, as measured by ELISA, and the amount of IL8 produced was dose-dependent. Pre-incubation of H. pylori cells or rHSP60 preparations with the antibody 2E7 significantly inhibited IL8 production from monocytic cells. These results indicated that HSP60 is closely associated with IL8 production in monocytic cells.

Helicobacter pylori heat-shock protein 60 induces inflammatory responses through the Toll-like receptor-triggered pathway in cultured human gastric epithelial cells.

Contact between Helicobacter pylori and gastric epithelial cells results in activation of NF-kappaB followed by secretion of interleukin (IL)-8. However, host-cell receptor(s) and their ligands involved in H. pylori-related IL-8 production have yet to be fully defined. In this study, the interaction between Toll-like receptors (TLRs), which are host receptors for pathogens involved in the innate immune response, and heat-shock protein (HSP) 60, an immune-potent antigen of H. pylori, was examined during H. pylori-induced IL-8 secretion in vitro. Recombinant H. pylori HSP60 (rHpHSP60) was prepared and added to cultured KATO III human gastric epithelial cells with or without pre-incubation with mouse monoclonal anti-TLR2 or anti-TLR4 antibodies. IL-8 mRNA expression and IL-8 protein release were analysed by Northern blotting and immunoassay. Involvement of NF-kappaB activation was analysed immunocytochemically by anti-NF-kappaB p65 antibody and ammonium pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-kappaB-mediated transcriptional activation. rHpHSP60 induced IL-8 mRNA expression and IL-8 secretion in a dose-dependent manner in KATO III cells. Anti-TLR2 antibody inhibited rHpHSP60-induced IL-8 secretion by 75 %, and anti-TLR4 antibody inhibited it by 30 %. rHpHSP60 induced nuclear translocation of NF-kappaB p65, which was inhibited by pretreatment with anti-TLR2 antibody. Treatment with PDTC significantly decreased the secretion of IL-8 induced by rHpHSP60. These findings suggest that H. pylori HSP60 activates NF-kappaB and induces IL-8 production through TLR-triggered pathways in gastric epithelial cells. Thus, it is possible that H. pylori HSP60 and TLR interaction in host cells contributes to the development of gastric inflammation caused by H. pylori infection.