scholarly journals A Novel Line Immunoassay Based on Recombinant Virulence Factors Enables Highly Specific and Sensitive Serologic Diagnosis of Helicobacter pylori Infection

2013 ◽  
Vol 20 (11) ◽  
pp. 1703-1710 ◽  
Author(s):  
Luca Formichella ◽  
Laura Romberg ◽  
Christian Bolz ◽  
Michael Vieth ◽  
Michael Geppert ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Responses ◽  
Virulence Factors ◽  
Gastrointestinal Disease ◽  
Individual Risk ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Serologic Test ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloricolonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome.H. pylorivirulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to importantH. pylorivirulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed inEscherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) wereH. pylorinegative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), therecomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, therecomLine assay provides a valuable tool for the diagnosis ofH. pyloriinfection.

scholarly journals Production of Autoantibodies by Murine B-1a Cells Stimulated with Helicobacter pylori Urease through Toll-Like Receptor 2 Signaling

2011 ◽  
Vol 79 (12) ◽  
pp. 4791-4801 ◽  
Author(s):  
Fumiko Kobayashi ◽  
Eri Watanabe ◽  
Yohko Nakagawa ◽  
Shingo Yamanishi ◽  
Yoshihiko Norose ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
B Cells ◽  
Specific Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Autoantibody Production ◽  
Content Type ◽  
Murine Spleen ◽  
Neutralizing Capacity ◽  
Toll Like Receptor 2 ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purifiedH. pyloriurease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purifiedH. pyloriurease orH. pyloricoated onto plates (referred to hereafter as plate-coatedH. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted fromH. pyloribut was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coatedH. pyloristimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matchedH. pyloridid not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pyloriurease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pyloriurease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coatedH. pyloriorH. pyloriurease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface ofH. pyloriwill directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders.

scholarly journals Chemokines and Antimicrobial Peptides Have acag-Dependent Early Response to Helicobacter pylori Infection in Primary Human Gastric Epithelial Cells

2014 ◽  
Vol 82 (7) ◽  
pp. 2881-2889 ◽  
Author(s):  
Pascale Mustapha ◽  
Isabelle Paris ◽  
Magali Garcia ◽  
Cong Tri Tran ◽  
Julie Cremniter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Inflammatory Response ◽  
Antimicrobial Peptides ◽  
Virulence Factors ◽  
Inflammatory Mediator ◽  
Host Cells ◽  
Gastric Epithelial Cells ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection systematically causes chronic gastric inflammation that can persist asymptomatically or evolve toward more severe gastroduodenal pathologies, such as ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Thecagpathogenicity island (cagPAI) ofH. pyloriallows translocation of the virulence protein CagA and fragments of peptidoglycan into host cells, thereby inducing production of chemokines, cytokines, and antimicrobial peptides. In order to characterize the inflammatory response toH. pylori, a new experimental protocol for isolating and culturing primary human gastric epithelial cells was established using pieces of stomach from patients who had undergone sleeve gastrectomy. Isolated cells expressed markers indicating that they were mucin-secreting epithelial cells. Challenge of primary epithelial cells withH. pyloriB128 underscored early dose-dependent induction of expression of mRNAs of the inflammatory mediators CXCL1 to -3, CXCL5, CXCL8, CCL20, BD2, and tumor necrosis factor alpha (TNF-α). In AGS cells, significant expression of only CXCL5 and CXCL8 was observed following infection, suggesting that these cells were less reactive than primary epithelial cells. Infection of both cellular models withH. pyloriB128ΔcagM, acagPAI mutant, resulted in weak inflammatory-mediator mRNA induction. At 24 h after infection of primary epithelial cells withH. pylori, inflammatory-mediator production was largely due tocagPAI substrate-independent virulence factors. Thus,H. pyloricagPAI substrate appears to be involved in eliciting an epithelial response during the early phases of infection. Afterwards, other virulence factors of the bacterium take over in development of the inflammatory response. Using a relevant cellular model, this study provides new information on the modulation of inflammation duringH. pyloriinfection.

scholarly journals A Double Mutant Heat-Labile Toxin from Escherichia coli, LT(R192G/L211A), Is an Effective Mucosal Adjuvant for Vaccination against Helicobacter pylori Infection

2013 ◽  
Vol 81 (5) ◽  
pp. 1532-1540 ◽  
Author(s):  
Louise Sjökvist Ottsjö ◽  
Carl-Fredrik Flach ◽  
John Clements ◽  
Jan Holmgren ◽  
Sukanya Raghavan
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Immune Responses ◽  
Double Mutant ◽  
Bacterial Load ◽  
Mucosal Vaccine ◽  
Mucosal Adjuvant ◽  
Content Type ◽  
Heat Labile ◽  
H Pylori

ABSTRACTHelicobacter pyloriinfection in the stomach is a common cause of peptic ulcer disease and is a strong risk factor for the development of gastric adenocarcinoma, yet no effective vaccine againstH. pyloriinfection is available to date. In mice, mucosal vaccination withH. pyloriantigens when given together with cholera toxin (CT) adjuvant, but not without adjuvant, can induce protective immune responses againstH. pyloriinfection. However, the toxicity of CT precludes its use as a mucosal adjuvant in humans. We evaluated a recently developed, essentially nontoxic double mutantEscherichia coliheat-labile toxin, LT(R192G/L211A) (dmLT), as a mucosal adjuvant in an experimentalH. pylorivaccine and compared it to CT in promoting immune responses and protection againstH. pyloriinfection in mice. Immunization via the sublingual or intragastric route withH. pylorilysate antigens and dmLT resulted in a significant decrease in bacterial load after challenge compared to that in unimmunized infection controls and to the same extent as when using CT as an adjuvant. Cellular immune responses in the sublingually immunized mice known to correlate with protection were also fully comparable when using dmLT and CT as adjuvants, resulting in enhancedin vitroproliferative and cytokine responses from spleen and mesenteric lymph node cells toH. pyloriantigens. Our results suggest that dmLT is an attractive adjuvant for inclusion in a mucosal vaccine againstH. pyloriinfection.

scholarly journals Helicobacter pylori-Specific Immune Responses of Children: Implications for Future Vaccination Strategy

2002 ◽  
Vol 9 (5) ◽  
pp. 1126-1128 ◽  
Author(s):  
Günter Bode ◽  
Isolde Piechotowski ◽  
Dietrich Rothenbacher ◽  
Hermann Brenner
Keyword(s):  
Helicobacter Pylori ◽  
Confidence Interval ◽  
Immune Responses ◽  
Western Blot ◽  
Vaccination Strategy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Significant Difference ◽  
Specific Igg ◽  
H Pylori

ABSTRACT We analyzed the specific anti-Helicobacter pylori immunoglobulin G (IgG) antibody profile for a sample of 824 asymptomatic schoolchildren in southern Germany (mean age, 10.7 ± 0.65 years) with an H. pylori-specific IgG enzyme-linked immunosorbent assay and Western blot analysis. The prevalence of infection was 19.8% (95% confidence interval, 17.1 to 22.7%). The immunoresponses were characterized predominantly by antibodies against low-molecular-mass antigens of 14 and 29 kDa, with a significant difference between children of German and Turkish nationalities (P = 0.0012 and P < 0.0001, respectively).

scholarly journals Helicobacter pylori Infection and Strain Types in Adult Dyspeptic Patients Undergoing Endoscopy in a Specialized Hospital of Dhaka City

2009 ◽  
Vol 3 (1) ◽  
pp. 4-9
Author(s):  
Sufi HZ Rahman ◽  
M Anisur Rahman ◽  
MS Arfin ◽  
M Mahbub Alam ◽  
TM Bhuiyan ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Helicobacter Pylori Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rapid Urease Test ◽  
Type I ◽  
Urease Test ◽  
H Pylori

Helicobacter pylori infection occurs worldwide with a high prevalence in developing countries. Virulence of H. pylori strains varies in different geographic regions. The aim of this study was to see H. pylori infection and its strain types in adult dyspeptic patients in Bangladesh and to analyze association of H. pylori strain types with clinical disease and severity of histological gastritis. Ninety consecutive adult dyspeptic patients undergoing diagnostic endoscopy were tested for H. pylori infection by culture, rapid urease test (RUT), histology and anti H. pylori IgG ELISA (Enzyme linked immunosorbent assay). H. pylori strain types were determined by Western Blot analysis. Association of strain types with clinical gastro-duodenal diseases and grades of histological gastritis were analyzed by χ2 test. Among the selected patients, 53 (58.9%) were culture positive, 48 (53.3%) were RUT positive, 31 (34.4%) were histology positive and 82 (91.1%) were anti-H. pylori IgG ELISA positive. By Western Blot analysis of the 90 sera samples, 48 (53.3%) showed antibodies to Type I strain of H. pylori, 21 (23.3%) Intermediate strain and 3 (3.3%) Type II strain. Endoscopically, 20 (22.2%) patients were found normal, 27 (30.0%) had gastritis, 9 (10.0%) had duodenitis, 28 (31.1%) had peptic ulcer disease, 4 (4.4%) had gastric carcinoma, and 2 (2.2%) had reflux esophagitis. Histologically, 34.4% had H. pylori, 44.4% had polymorhonuclear neutrophil (PMN), 100% had mononuclear cell (MNC) infiltration of different grades, 1.1% had atrophic gastritis and 2.2% had intestinal metaplasia of moderate grade. H. pylori strain types was not associated with clinical gastro-duodenal diseases or grades of PMN or MNC infiltration (p > 0.05) in these patients. Key words: Helicobacter pylori infection, H. pylori strain types, gastro-duodenal diseases, grades of gastritis   doi: 10.3329/bjmm.v3i1.2963 Bangladesh J Med Microbiol 2009; 03 (01): 4-9

scholarly journals Western Blot Method Evaluation for Detection of Helicobacter pylori Infections against H. pylori Ag in Stool Enzyme-Linked Immunoassay in Adult Egyptian Patients

2016 ◽  
Vol 4 (3) ◽  
pp. 352-358
Author(s):  
Mohamed E. Rashed ◽  
Amer M.M. ◽  
Mostafa Elnakeeb ◽  
Waleed Saeed Omer
Keyword(s):  
Helicobacter Pylori ◽  
Western Blot ◽  
Virulence Factors ◽  
Diagnostic Value ◽  
Type I ◽  
Western Blot Method ◽  
Egyptian Patients ◽  
Cytotoxin Associated Gene A ◽  
H Pylori ◽  
Medical Burden

Helicobacter pylori infection is tremendous medical burden especially in developing countries. Various immunological tests are available for diagnosis of H. pylori infection. Western blot method is proven to be promising for Precise, easy reading, sensitive and specific detection of H. pylori infections, besides it also permits the detection for the different virulence factors of CagA / VacA positive strains (type I). The objective of this study is to evaluate the diagnostic value of commercial Western Blot (WB) method in the serological diagnosis of H. pylori infections against the H. pylori Ag in stool (HpSAg) using commercial enzyme-linked immunoassay (ELISA) in adult dyspeptic Egyptian patients. Also we investigated the prevalence of virulence factors, cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) in the infected patients. Samples from 46 adult dyspeptic Egyptian patients were tested by the two methods. WB test gave accurate confirmed result with (82.6% accuracy and 89.5% sensitivity) compared to HpSAg test. Also the results indicated a high seroprevalence of cagA- and vacA-positive virulent H. pylori type I strains in adult infected population indicate that such strains may be common in this population and responsible for the majority of H. pylori infection among adult Egyptians. We concluded that WB method could be useful for the confirmatory detection of antibody profiles to H. pylori antigens and virulence factors in adult Egyptian patients.Int J Appl Sci Biotechnol, Vol 4(3): 352-358

scholarly journals Simultaneous Detection and Differentiation of Anti-Helicobacter pylori Antibodies by Flow Microparticle Immunofluorescence Assay

2004 ◽  
Vol 11 (1) ◽  
pp. 131-136 ◽  
Author(s):  
F. Bühling ◽  
G. Koch ◽  
T. Wex ◽  
A. Heimburg ◽  
M. Vieth ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Western Blot ◽  
Virulence Factors ◽  
Simultaneous Detection ◽  
Immunoblot Analysis ◽  
Measurement Range ◽  
Immunofluorescence Assay ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
H Pylori

ABSTRACT Helicobacter pylori is the key pathogen for gastroduodenal diseases. The clinical outcome of H. pylori infection is influenced by the presence of strain-specific virulence factors that are usually detected by the presence of specific anti-H. pylori antibodies in serum. Apart from the detection of these antibodies by enzyme-linked immunosorbent assay (ELISA), it is desirable to obtain additional information concerning the presence of certain virulence factors of H. pylori that are currently detected by immunoblot analysis. At present, the immunodiagnosis of an H. pylori infection includes two separate methods: ELISA and immunoblot analysis. Here, we report the development and evaluation of a new rapid flow microparticle immunofluorescence assay (FMIA) for detection of anti-H. pylori antibodies in human serum. The assay allows rapid qualitative and quantitative detection of anti-H. pylori antibodies by using crude antigen preparations as well as single recombinant antigens (urease A, urease B, CagA, and alkylhydroxy peroxide reductase) in the same sample with one measurement, and thus it combines the advantages of enzyme immunoassay and Western blot analysis. Seventy-five patient samples were analyzed by FMIA, ELISA, and Western blotting with respect to their immunoreactivity against crude H. pylori extracts and individual H. pylori antigens. Statistical analyses revealed an overall similarity of more than 90% among the results for FMIA, ELISA, and Western blot. Therefore, we conclude that FMIA is a powerful and time- and cost-saving assay system for the detection of antimicrobial antibodies, with higher sensitivity and a larger measurement range than ELISA.

scholarly journals Major virulence factors, VacA and CagA, are commonly positive inHelicobacter pylori isolates in Japan

Gut ◽  
1998 ◽  
Vol 42 (3) ◽  
pp. 338-343 ◽  
Author(s):  
S Maeda ◽  
K Ogura ◽  
H Yoshida ◽  
F Kanai ◽  
T Ikenoue ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factors ◽  
Japanese Patients ◽  
Type I ◽  
Activity Type ◽  
Vacuolating Cytotoxin ◽  
Specific Antibodies ◽  
High Prevalence ◽  
H Pylori ◽  
Very High

Background—VacA and CagA proteins have been reported to be major virulence factors of Helicobacter pylori. However, antibodies against these proteins are frequently found in the sera of Japanese patients regardless of their gastroduodenal status.Aim—To evaluate the expression of VacA and CagA proteins by H pylori strains isolated in Japan.Methods—By using specific antibodies raised against recombinant VacA and CagA proteins, the expression of VacA and CagA was evaluated in 68 H pylori strains isolated from Japanese patients; a vacuolating assay and genotyping of thevacA gene were also used in the evaluation. The results were analysed in relation to the gastroduodenal diseases of the hosts.Results—VacA and CagA proteins were expressed in 59/68 (87%) and in 61/68 (90%) isolates respectively. The vacuolating assay was positive in 57/68 (84%) isolates, indicating that most immunologically VacA positive strains produced active cytotoxin. The prevalence of infection with strains expressing CagA and positive for vacuolating activity (Type I) was very high, 54/68 (79%), irrespective of the gastroduodenal status of the host.Conclusion—Most H pylori isolates in Japan are positive for vacuolating cytotoxin and CagA, and thus these virulence factors cannot be used as markers to discern the risk of developing serious gastroduodenal pathologies in the hosts. However, the high prevalence of infection with strains positive for vacuolating cytotoxin and CagA may contribute to the characteristics of H pylori infection in Japan.

scholarly journals Riboregulation in the Major Gastric Pathogen Helicobacter pylori

2021 ◽  
Vol 12 ◽  
Author(s):  
Alejandro Tejada-Arranz ◽  
Hilde De Reuse
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factors ◽  
Antisense Rna ◽  
Small Rnas ◽  
Genetic Regulation ◽  
Type I ◽  
Control Of Gene Expression ◽  
Ulcer Disease ◽  
Interesting Study ◽  
H Pylori

Helicobacter pylori is a Gram-negative bacterial pathogen that colonizes the stomach of about half of the human population worldwide. Infection by H. pylori is generally acquired during childhood and this bacterium rapidly establishes a persistent colonization. H. pylori causes chronic gastritis that, in some cases, progresses into peptic ulcer disease or adenocarcinoma that is responsible for about 800,000 deaths in the world every year. H. pylori has evolved efficient adaptive strategies to colonize the stomach, a particularly hostile acidic environment. Few transcriptional regulators are encoded by the small H. pylori genome and post-transcriptional regulation has been proposed as a major level of control of gene expression in this pathogen. The transcriptome and transcription start sites (TSSs) of H. pylori strain 26695 have been defined at the genome level. This revealed the existence of a total of 1,907 TSSs among which more than 900 TSSs for non-coding RNAs (ncRNAs) including 60 validated small RNAs (sRNAs) and abundant anti-sense RNAs, few of which have been experimentally validated. An RNA degradosome was shown to play a central role in the control of mRNA and antisense RNA decay in H. pylori. Riboregulation, genetic regulation by RNA, has also been revealed and depends both on antisense RNAs and small RNAs. Known examples will be presented in this review. Antisense RNA regulation was reported for some virulence factors and for several type I toxin antitoxin systems, one of which controls the morphological transition of H. pylori spiral shape to round coccoids. Interestingly, the few documented cases of small RNA-based regulation suggest that their mechanisms do not follow the same rules that were well established in the model organism Escherichia coli. First, the genome of H. pylori encodes none of the two well-described RNA chaperones, Hfq and ProQ that are important for riboregulation in several organisms. Second, some of the reported small RNAs target, through “rheostat”-like mechanisms, repeat-rich stretches in the 5′-untranslated region of genes encoding important virulence factors. In conclusion, there are still many unanswered questions about the extent and underlying mechanisms of riboregulation in H. pylori but recent publications highlighted original mechanisms making this important pathogen an interesting study model.

scholarly journals Helicobacter pylori Virulence Factors—Mechanisms of Bacterial Pathogenicity in the Gastric Microenvironment

Cells ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 27
Author(s):  
Jacek Baj ◽  
Alicja Forma ◽  
Monika Sitarz ◽  
Piero Portincasa ◽  
Gabriella Garruti ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
Virulence Factors ◽  
Premalignant Lesions ◽  
Bacterial Pathogenicity ◽  
Current State ◽  
Genetic And Environmental Factors ◽  
H Pylori ◽  
Stimulation Of

Gastric cancer constitutes one of the most prevalent malignancies in both sexes; it is currently the fourth major cause of cancer-related deaths worldwide. The pathogenesis of gastric cancer is associated with the interaction between genetic and environmental factors, among which infection by Helicobacter pylori (H. pylori) is of major importance. The invasion, survival, colonization, and stimulation of further inflammation within the gastric mucosa are possible due to several evasive mechanisms induced by the virulence factors that are expressed by the bacterium. The knowledge concerning the mechanisms of H. pylori pathogenicity is crucial to ameliorate eradication strategies preventing the possible induction of carcinogenesis. This review highlights the current state of knowledge and the most recent findings regarding H. pylori virulence factors and their relationship with gastric premalignant lesions and further carcinogenesis.

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