Helicobacter pylori: a eubacterium lacking the stringent response

Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of Helicobacter pylori. All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA. This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype. The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms. However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism. These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches.     

T-cell receptor repertoires utilized in response to linear peptides representing an immunodominant MHC class II restricted T-cell epitope are far more diverse than that utilized in response to the same epitope in the nominal antigen

Previous analyses of the T-cell receptor (TCR) repertoire utilized in response to the 1-102 fragment of the lambda cI repressor protein and specific for the immunodominant amino acid 12-26 region in the context of I-Ek, have shown this repertoire to be extremely restricted. In contrast, here we show that the TCR repertoires utilized in two strains of I-Ek expressing mice in response to two linear peptides representing this immunodominant region are diverse. Despite their extensive diversity, these repertoires are somewhat overlapping. In addition, structural similarities were observed between the full lambda cI fragment (1-102) and peptide elicited TCR repertoires, including frequent use of the Valpha2 family of gene segments, particularly among peptide (12-26) elicited TCRs cross-reactive with 1-102/I-Ek. Nevertheless, these data indicate that it may be difficult to mimic the immune response to an immunodominant epitope of a protein antigen via immunization with linear peptides containing the amino acid sequence of that epitope. Possible explanations for differences in the levels of TCR diversity among T cells responding to an epitope present in a nominal antigen as compared to T cells responding to linear peptide antigens containing this same epitope are discussed.     

Identification of an immunodominant IgE epitope of the Parietaria judaica major allergen

Par j 1.0101 is one of the two major allergens of the Parietaria judaica (Pj) pollen, and its three-dimensional structure was built by three-dimensional structural homology modeling. The resultant model was used to identify putative IgE binding regions. Western blot analysis of gene fragmentation products showed that the 1 to 30 region was capable of binding specific IgE from a pool of sera (n = 30) of patients allergic to Pj pollen. Using the structural model as a guide, deletion and site-directed mutagenesis of the 1 to 30 region was performed, and the amino acids involved in IgE binding were identified. In addition, a synthetic peptide covering the 1 to 30 region was capable of binding human IgE without triggering histamine release from basophils of Pj allergic patients (n = 6) and thus represents a haptenic molecule with potential use as an immunotolerant agent. This epitope is also present on the Par j 2.0101 major allergen representing a common IgE epitope. It is an immunodominant epitope, since it was capable of inhibiting 30% of all specific IgE against the Pj major allergens, and therefore, it might be a candidate for the future development of immunotherapeutics.     

Helicobacter pylori does not migrate from the antrum to the corpus in response to omeprazole

BACKGROUND: Omeprazole is known to have an effect on Helicobacter pylori in vivo. One opinion is that H. pylori "migrates" from the antrum to the corpus in response to omeprazole therapy. METHODS: To determine whether H. pylori migrates in response to omeprazole, we assessed the presence of H. pylori in the antrum and corpus in duodenal ulcer patients receiving omeprazole for 4 wk. Culture and histological examination of antral biopsies (Genta stain) were performed before patients received omeprazole, at the end of therapy, and 4-6 wk later. The end points were presence or absence of H. pylori and the number of H. pylori colonies per biopsy. RESULTS: Seventy-two patients had H. pylori in both the antrum and corpus at entry and 4-6 wk after ending therapy. Three general patterns were prevalent at the end of omeprazole therapy: antrum- and corpus-positive (54%), antrum-negative and corpus-positive (24%), both antrum- and corpus-negative (21%), and one patient had antrum-positive with corpus-negative (1%). Evaluation of the number of colonies per biopsy in those who remained H. pylori-positive in both the antrum and corpus throughout showed that the number of H. pylori decreased in both the antrum and corpus during therapy (507 +/- 60 vs. 225 +/- 51, p < 0.01 and 415 +/- 58 vs. 290 +/- 46 0.1) for antrum and corpus, respectively, and tended to return to pre-therapy levels 4-6 wk later. The number of H. pylori in the corpus also decreased in the antrum-negative and corpus-positive group during therapy with omeprazole (433 +/- 87 vs. 185 +/- 61, p < 0.05). In most of the patients studied, the number of H. pylori in the corpus was less posttreatment than it was pretreatment. The decrease in H. pylori load was also reflected in the development of false-negative urea breath tests. CONCLUSIONS: Omeprazole is detrimental to H. pylori in both the antrum and the corpus; migration from the antrum to the corpus in response to omeprazole is a myth.     

Performance characteristics of an enzyme-linked immunosorbent assay for determining salivary immunoglobulin G response to Helicobacter pylori

We evaluated the salivary immunoglobulin G (IgG) immune response to Helicobacter pylori in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive H. pylori culture showed significantly higher titers of antibodies than subjects with no detectable H. pylori: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-H. pylori IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.     

A new urea broth-based test to detect Helicobacter pylori presence in upper gastrointestinal biopsies

PURPOSE: To evaluate the performance before and after sterilization of Enhance and Min-Identoflex finishing and polishing systems on TPH and Z100 composites. MATERIALS AND METHODS: Finishing and polishing instruments consisted of a light-cured resin cup impregnated with an abrasive and a silicon dioxide impregnated rubber cup. Sterilization methods included microwaving and autoclaving. RESULTS: Performance of the finishing and polishing instruments were affected more by the composite being finished and polished than by autoclaving or microwaving.     

Utilization of proteinase K-treated cells as lipopolysaccharide antigens for the serodiagnosis of Helicobacter pylori infections

We have evaluated the use of proteinase K (PK)-treated cells isolated from Helicobacter pylori as lipopolysaccharide (LPS) antigens in an immunoblot assay and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of H. pylori infection. The sera from patients with chronic gastritis, gastric ulcer, duodenal ulcer or gastric cancer, and from healthy adults with or without H. pylori infection were assayed with three commercial serodiagnostic kits (HM-CAP, Helico-G, and G.A.P. II) and novel methods relying on the use of PK-treated cells. The PK-treated cells used in these assays were selected on the basis of their possibility to possess a common epitope in the O-polysaccharides of H. pylori, which is known to be highly immunogenic in humans. Of the sera from these patients, 71-94% were positive with the commercial kits, 97% with immunoblot assay, and 90% with ELISA. On the other hand, of the healthy adults infected with H. pylori, 72-97% were positive with the commercial kits, 86% with immunoblot assay, and 72% with ELISA. PK-treated cells that did not contain the common epitope were unsuitable as an antigen for immunoblot assay or ELISA. Furthermore, the reactivity of these sera reacted specifically with H. pylori PK-treated cells but not with LPSs from other gram-negative bacteria, such as Campylobacter, Proteus, Bordetella, and Salmonella. These results demonstrate that the serological assays relying on the use of H. pylori PK-treated cells possessing a highly antigenic epitope are potentially useful as a serodiagnostic test for H. pylori infection.     

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen

We identified an expansion of the CAG trinucleotide repeat in the coding region of the Machado-Joseph disease gene in 7 of 24 American families diagnosed with autosomal dominant ataxia. All affected individuals were heterozygous for an expanded allele that ranged from 67 to more than 200 CAG repeats, whereas the normal allele had 14 to 33 repeats. In contrast to the Azorean-Portuguese origins of Machado-Joseph disease, the two largest American families were of German and Dutch-African descent. Clinical, pathologic, and genetic evaluations suggest that American families with spinocerebellar ataxia type 3 differ from those with Machado-Joseph disease by their ethnic origins, predominant spinopontine atrophy, lack of dystonic features, and larger CAG repeat expansion.     

The killing of Helicobacter pylori by low-power laser light in the presence of a photosensitiser

Activation and proliferation of human T lymphocytes in vitro can be obtained by various stimuli including specific antigens, mitogens, and cytokines. Here we describe the effect of interleukin-10, interleukin-12 and tumor necrosis factor-alpha on the interleukin-2 dependent proliferation and function of established human CD4+ and CD8+ alloreactive T-cell clones in the absence of antigen presenting cells. IL-12 and TNF-alpha both demonstrated an inhibitory effect on the proliferation of CD8+ cytotoxic T lymphocyte clones, whereas IL-10 enhanced the proliferation. IL-12-induced inhibition of CD8+ CTL clones was not mediated by the endogenous production of TNF-alpha by these clones. The strong inhibitory effect of IL-12 and TNF-alpha did not result in apoptosis. These cytokines did not alter the cytotoxicity of CD8+ CTL clones. When CD4+ T-cell clones were tested in the absence of APC, no significant change in IL-2-dependent proliferation due to IL-10, IL-12, and TNF-alpha could be measured. Since these effects on established CTL clones are in contrast to the effects of IL-10, IL-12, and TNF-alpha during the induction phase of immune responses, a dichotomy of immunomodulatory cytokines such as IL-10, IL-12, and TNF-alpha early and late in the immune response is suggested.     

CTL response to Mycobacterium tuberculosis: identification of an immunogenic epitope in the 19-kDa lipoprotein

The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88-97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88-97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.     

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.     

Eradication of Helicobacter pylori: an objective assessment of current therapies

The purpose of the present review was to determine objectively the optimal treatment for the eradication of H. pylori amongst the currently used regimens. A comprehensive literature search provided a data-base relating to the following treatments: dual therapy with an anti-secretory drug plus either amoxycillin or clarithromycin; standard triple therapy, with or without additional anti-secretory drugs; proton pump inhibitor triple therapy; and H2-receptor antagonist triple therapy. Emphasis was placed on intention-to-treat analyses of eradication rates using all of the available evidence. The criteria used to select the optimal treatment were efficacy (eradication rates), frequency of side-effects, simplicity of the regimen (number of tablets per day and duration of treatment) and cost. The analysis showed that proton pump inhibitor triple therapy (that is, a proton pump inhibitor plus any two of amoxycillin, clarithromycin or a nitroimidazole) was the preferred treatment for the eradication of H. pylori. In particular, the 1-week, low-dose regimen with omeprazole plus clarithromycin plus tinidazole produced the highest eradication rates (> 90%) with the lowest frequency of side-effects and at only modest cost.     

Resistance to polyoma virus-induced tumors correlates with CTL recognition of an immunodominant H-2Dk-restricted epitope in the middle T protein

The natural mouse pathogen polyoma virus is highly oncogenic in H-2k mice carrying the endogenous superantigen encoded by the mouse mammary tumor provirus Mtv-7. This superantigen results in deletion of Vbeta6 TCR-expressing polyoma-specific CD8+ CTL, which appear to be critical effectors against polyoma tumorigenesis. Here we have isolated cloned lines of CD8+ T cells from resistant (i.e., Mtv-7-) H-2k mice that specifically lyse syngeneic polyoma virus-infected cells and polyoma tumor cells. Nearly all these CTL clones express Vbeta6 and are restricted in their recognition of virus-infected cells by H-2Dk. Screening a panel of synthetic peptides predicted to bind to Dk, for which no consensus peptide binding motif is known, we identified a peptide corresponding to a nine-amino acid sequence in the carboxyl-terminus of the middle T (MT) protein (amino acids 389-397) that was recognized by all the Vbeta6+ CD8+ CTL clones. The inability of MT(389-397)-reactive CTL to recognize cells infected with a mutant polyoma virus encoding a MT truncated just proximal to this sequence indicates that MT(389-397) is a naturally processed peptide. The frequencies of precursor CTL specific for polyoma virus and MT(389-397) peptide were similar, indicating that MT(389-397) is the immunodominant epitope in H-2k mice. In addition, polyoma-infected resistant mice possess a 10- to 20-fold higher MT(389-397)-specific precursor CTL frequency than susceptible mice. This highly focused CTL response to polyoma virus provides a valuable animal model to investigate the in vivo activity of CTL against virus-induced neoplasia.     

Evidence against colonic mucosa colonisation by Helicobacter pylori. Lack of a specific antibody response in homogenates of rectal endoscopic biopsies

Aim of this study is to provide indirect evidence that human colonic mucosa harbour Helicobacter pylori. The antibody response of IgG and IgA class against Helicobacter pylori was examined in autologous homogenate of gastric and rectal endoscopic biopsies from 26 patients and in rectal samples of a further 36. All had a documented (histology and/or serology) Helicobacter pylori status. Helicobacter pylori specific IgG and IgA were measured by an in-house ELISA. In Helicobacter pylori positive patients having both gastric and rectal homogenate, mean level of Helicobacter pylori IgG and IgA was higher in gastric than in rectal samples (0.810 +/- 0.668 optical density vs 0.329 +/- 0.509 optical density for IgG, p = 0.007 and 0.660 +/- 0.477 vs 0.116 +/- 0.229 for IgA, p < 0.001, respectively). In each patient, level of the two isotypes was clearly higher in gastric than in autologous rectal sample. In the overall study population, mean level of Helicobacter pylori IgG in rectal homogenate was not significantly (p = 0.16) different between Helicobacter pylori positive (48/62, 77%, 0.243 +/- 0.388 optical density) and negative (14/62, 23%; 0.095 +/- 0.088) patients. In same material, levels of Helicobacter pylori IgA were very low and undetectable either in Helicobacter pylori positive or negative patients. Although Helicobacter pylori IgG are detectable in rectal homogenates of Helicobacter pylori positive patients, present data suggest that these antibodies may not be local in origin but rather reflect circulating response. These observations do not support the view that large bowel mucosa is colonised by Helicobacter pylori.     

Helicobacter pylori in peptic ulcer disease: report of an NIH consensus conference

Preoperative electromyographic examinations of superior levator muscle were done in 36 ptotic patients using the Neurematic 2000 electromyograph. Shortening of the levator palpebrae superior was done through a combined internal and external route. In 12 patients of mild ptosis the spike voltage was all above 30 mv during contraction. When the spike voltage was above 100 mv, better operative results were obtained. In 24 patients whose ptosis was moderate or severe, the graphs of contraction generally showed simple phases with low amplitude. In 9 patients whose spike voltage was below 30 mv, their palpebral fissure did not reach the anticipated width after the operation. On the contrary, in 15 patients whose spike voltage was above 30 mv the operative results were satisfactory. It is concluded that in patients whose ptosis is moderate or severe, the operative procedure should be based on electromyographic examinations. If the spike voltage of the superior levator muscls is higher than 30 mv on contraction, simple shortening of the levator will be successful. If the spike voltage is lower than 30 mv and the graph presents simple phase or electro-tranquilization, then suspension of the frontalis muscle is preferable.     

Anticarrier immunity suppresses the antibody response to polysaccharide antigens after intranasal immunization with the polysaccharide-protein conjugate

We have conjugated cholera toxin (CT) B subunit (CTB) to dextran and studied the effect in mice of previous immunization with CT and CTB on the response to dextran after intranasal immunizations with conjugate. Preexisting immunity to CTB was found to inhibit both the lung mucosal response and serum antibody response to dextran, but this effect could be overcome by using a higher dose of conjugate and delaying the conjugate immunization until the CTB antibody titers had declined. The role of anti-CTB antibodies on the mucosal surface was probably to prevent uptake of the conjugate through a mechanism of immune exclusion. Passively transferred serum antibodies against CTB, on the other hand, suppressed both the serum response and the local antibody response against CTB but did not affect the response to dextran after intranasal immunization with conjugate.     

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.