Helicobacter Bilis Research Articles

Helicobacter bilis Colonization Enhances Susceptibility to Typhlocolitis Following an Inflammatory Trigger

Aberrant mucosal immune responses to antigens of the resident microbiota are a significant cause of inflammatory bowel diseases (IBD), as are genetic and environmental factors. Previous work from our laboratory demonstrated that Helicobacter bilis colonization of immunocompetent, defined microbiota mice induced antigen-specific immune responses to the resident microbiota, yet these mice failed to develop colitis, suggesting that the immunological provocation induced by H. bilis alone was insufficient to induce disease. The purpose of this study was to test the hypothesis that the introduction of a bacterial provocateur such as H. bilis enhances the host's susceptibility to IBD following an inflammatory event. Defined microbiota (DM) mice colonized with H. bilis were administered low dose (1.5%) dextran sodium sulfate (DSS) in drinking water for 5days followed by a 4-day restitution period. Severity of lesions was assessed grossly and microscopically. Differential expression of select mucosal genes and histopathologic lesions was characterized. Helicobacter bilis colonization increased the severity of intestinal inflammation induced by an inflammatory trigger in the form of low-dose DSS. An analysis of the molecular and cellular mechanisms associated with H. bilis colonization revealed significant increases in expression of mucosal genes associated with lymphocyte activation and inflammatory cell chemotaxis as well as increased infiltration of mucosal macrophages and T cells in mice colonized with H. bilis prior to DSS treatment versus DSS treatment alone. These results indicate that prior colonization with H. bilis heightens the host's sensitivity to enteric inflammation by altering mucosal homeostasis and initiating immune cell activation and migration.

Selective colonization with Helicobacter bilis or Escherichia coli induces differential host immune responses following an inflammatory trigger.

Selective colonization with Helicobacter bilis or Escherichia coli induces differential host immune responses following an inflammatory trigger.

Effect of Heparin, Fucoidan and Other Polysaccharides on Adhesion of Enterohepatic Helicobacter Species to Murine Macrophages

Helicobacter species have been isolated and cultured from both the gastric and enterohepatic niches of the gastrointestinal tract and are associated with a wide spectrum of diseases. Some members of the enterohepatic Helicobacter species (EHS), which include Helicobacter bilis, Helicobacter hepaticus and Helicobacter pullorum, are associated with chronic inflammatory and proliferative bowel inflammation, hepatitis and in experimental murine studies with hepatic cancer. The present study aimed to explore if polysulphated polysaccharides can prevent adhesion of EHS to the murine macrophage cell line J774A.1. A competitive binding assay showed that heparin and heparan sulphate at a concentration of 1.25mg/ml reduced binding of H. hepaticus and H. pullorum to the host cells, but not H. bilis. Of the tested Helicobacter spp, the highest inhibition by heparin was demonstrated for H. pullorum (P < 0.01), the most hydrophilic strain. Partially or completely de-sulphated heparin derivatives lost the ability to inhibit adherence of EHS, indicating the importance of sulphated groups of heparin. The most efficient inhibitor of EHS binding to macrophages was fucoidan, which reduced bacterial adhesion of the three enterohepatic Helicobacter species to a greater extent than heparin, 60-90% inhibition vs 30-70% inhibition by heparin. Identification of receptors that EHS ligands bind to is important for understanding the development of infection and may provide a rational target to prevent infection and therapy.

Could Helicobacter organisms cause inflammatory bowel disease?

The discovery of Helicobacter pylori sparked a revolution in the understanding and management of peptic ulcer disease and gastric cancer. Other Helicobacter species are recognized as important pathogenic agents in colitic diseases of rodents and primates, in particular Helicobacter bilis, Helicobacter fennelliae, Helicobacter hepaticus and Helicobacter trogontum. Helicobacter bilis and H. hepaticus are now routinely used to initiate rodent models of inflammatory bowel disease (IBD), particularly in immunocompromised hosts. Molecular evidence exists linking various non-pylori Helicobacter spp. with human IBD; however, attempts to culture organisms in this disease cohort have proved unsuccessful to date. Attributing causation has therefore proved elusive. Seven enterohepatic, non-pylori Helicobacter organisms have been successfully cultured from humans, namely Helicobacter canadensis, Helicobacter canis, Helicobacter cinaedi, H. fennelliae, Helicobacter pullorum, Helicobacter winghamensis and Helicobacter sp. flexispira taxon 8 (now classified as H. bilis). Of these, H. cinaedi and H. fennelliae are the closest to fulfilling Koch's postulates as causative agents in homosexual proctitis. The possibility that novel Helicobacter organisms have a role in the initiation of human IBD warrants further consideration and targeted investigations.

Helicobacter spp. other than Helicobacter pylori

Over the last 12 months, new insights into the association of non-Helicobacter pylori Helicobacters with a range of human diseases in children and adults, including hepatobiliary disease, Crohn's disease, sepsis, and gastric disease were published. Studies investigating the presence of non-H. pylori Helicobacters in domestic animals reinforce previous findings that cats and dogs harbor gastric Helicobacter species and thus may be an important source of these organisms in humans. The confounding effect of enterohepatic Helicobacters on the outcome of biomedical research was investigated in several studies and led to recommendations that animals should be screened prior to performing experiments. A number of important and novel investigations regarding pathogenic mechanisms and immune responses to enterohepatic Helicobacters were conducted. Genomic advances in non-H. pylori Helicobacters included description of the complete genome of Helicobacter canadensis, delineation of two Helicobacter bilis genomospecies, and identification of a novel cis-regulatory RNA. New insights concerning growth conditions, biochemical characterization, and the effect of certain dietary compounds on Helicobacter spp. have also been reported.

Pyoderma Gangrenosum–Like Ulcer in a Patient With X-Linked Agammaglobulinemia

Pyoderma gangrenosum-like ulcers and cellulitis of the lower extremities associated with recurrent fevers in patients with X-linked (Bruton) agammaglobulinemia have been reported to be caused by Helicobacter bilis (formerly classified as Flexispira rappini and then Helicobacter strain flexispira taxon 8). Consistent themes in these reports are the difficulty in recovering this organism in blood and wound cultures and in maintaining isolates in vitro. We confirmed the presence of this organism in a patient's culture by using a novel application of gene amplification polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry. An adolescent boy with X-linked agammaglobulinemia presented with indurated plaques and a chronic leg ulcer whose origin was strongly suspected to be an H bilis organism. Histologic analysis demonstrated positive Warthin-Starry staining of curvilinear rods, which grew in culture but failed to grow when subcultured. They could not be identified by conventional techniques. A combination of gene amplification by polymerase chain reaction and electrospray ionization time-of-flight mass spectrometry confirmed the identity of this organism. This novel technology was useful in the identification of a difficult-to-grow Helicobacter organism, the cause of pyoderma gangrenosum-like leg ulcers in patients with X-linked agammaglobulinemia. Correct identification of this organism as the cause of pyoderma gangrenosum-like ulcers in patients with X-linked agammaglobulinemia is of great importance for the early initiation of appropriate and curative antibiotic therapy.

Helicobacter bilis colonization of the biliary system in patients with pancreaticobiliary maljunction

Helicobacter bilis is considered to be a causative factor in the pathogenesis of biliary cancer. This study investigated the prevalence of H. bilis colonization of the biliary system of patients with pancreaticobiliary maljunction (PBM). Bile juice and biliary tissue samples were collected from 17 patients with PBM and 27 controls who had benign biliary disease without PBM. DNA extracted from each biliary sample was subjected to polymerase chain reaction (PCR) analysis for H. bilis and Helicobacter pylori. PCR assays revealed that 12 of the 17 patients with PBM were positive for H. bilis DNA, compared with eight of 27 patients without PBM (P = 0.009). Among patients with PBM, H. bilis DNA was identified in six of eight children, including a 2-month-old infant, and in six of nine adults. The high prevalence of H. bilis DNA in the biliary system of patients with PBM was independent of age, sex, common bile duct dilatation, configuration of the pancreatic and bile ducts, and amylase activity in bile. H. bilis colonization of the biliary system is extremely common in patients with PBM. This may point to a role in the pathogenesis of biliary cancer.

Delineation of two Helicobacter bilis genomospecies: implications for systematics and evolution

The evolution and taxonomy of Helicobacter bilis strains isolated in Italy and Finland were studied by phylogenetic analysis of different genes, comparative analysis of small rRNA gene intervening sequence (IVS), amplified fragment length polymorphism analysis and DNA-DNA hybridization. The results of this study divided the H. bilis strains into two distinct and divergent genomic groups. In the absence of a specific phenotype or pathotype to distinguish these groups, however, they may be referred to as two genomospecies: H. bilis sensu stricto and Helicobacter sp. FL56. The phylogenetic network of gyrB and ureB gene sequences, as well as the comparative analysis of small rRNA gene IVS, suggests independent evolution of the two genomospecies. In particular, Helicobacter sp. FL56 seems to be the result of adaptation of an ancestral H. bilis strain in a new host. The phenomenon of adaptation to different hosts, or different intestinal niches in the same host, associated with high mutation and recombination rates could explain the evolution and the complex taxonomy of the genus Helicobacter. A comprehensive phylogenomics study of this genus would be useful to properly investigate this hypothesis.

Chronic hepatitis, hepatic dysplasia, fibrosis, and biliary hyperplasia in hamsters naturally infected with a novel Helicobacter classified in the H. bilis cluster.

We recently described helicobacter-associated progressive, proliferative, and dysplastic typhlocolitis in aging (18- to 24-month-old) Syrian hamsters. Other pathogens associated with typhlocolitis in hamsters, Clostridium difficile, Lawsonia intracellularis, and Giardia spp., were not indentified. The presence of Helicobacter genus-specific DNA was noted by PCR in cecal and paraffin-embedded liver samples from aged hamsters by the use of Helicobacter-specific PCR primers. By 16S rRNA analysis, the Helicobacter sp. isolated from the liver tissue was identical to the cecal isolates from hamsters. The six hamster 16S rRNA sequences form a genotypic cluster most closely related to Helicobacter sp. Flexispira taxon 8, part of the Helicobacter bilis/H. cinaedi group. Livers from aged helicobacter-infected hamsters showed various stages of predominantly portocentric and, to a lesser extent, perivenular fibrosis. Within nodules, there was cellular atypia consistent with nodular dysplasia. The livers also exhibited a range of chronic active portal/interface and lobular inflammation, with significant portal hepatitis being present. The inflammation was composed of a mixture of lymphocytes, neutrophils, and macrophages, indicative of its chronic-active nature in these aged hamsters infected with Helicobacter spp. The isolation of novel Helicobacter spp., their identification by PCR from the diseased livers of aged hamsters, and their taxonomic classification as belonging to the Helicobacter bilis cluster strengthen the argument that H. bilis and closely related Helicobacter spp. play an etiological role in hepatobiliary disease in both animals and humans.

Effect of Helicobacter bilis Infection on Human Bile Duct Cancer Cells

Helicobacter pylori infection is known to be associated with chronic atrophic gastritis, peptic ulcers, and gastric malignancies. However, the effects of other Helicobacter species have not been investigated extensively. In mice, a close relationship is observed between Helicobacter hepaticus and hepatocellular carcinoma, and Helicobacter species can be found in humans, most commonly in extragastric organs. There have also been reports that H. bilis may be associated with biliary malignancies in humans. The effect of H. bilis infection on a human bile duct cancer cell line was investigated in this study. We prepared HuCCT-1, the human bile duct cancer cell line, which was cocultured with H. bilis and cultured alone as a control. HuCCT-1 with and without H. bilis were transfected with the NF-kappaB, E2 transcription factor (E2F), and cyclic AMP response element (CRE) luciferase vectors. The activity of NF-kappaB between H. bilis and the infected and noninfected HuCCT-1 cells was also measured by dual luciferase reporter assay. The concentration of vascular endothelial growth factor (VEGF) in the cocultured medium and control medium were measured by ELISA. To investigate the effect of H. bilis infection on HuCCT-1 with regard to human umbilical vein endothelial cell (HUVEC) tube formation, HUVECs and fibroblasts were cocultured in 24-well plates with and without the conditioned medium. NF-kappaB, E2F and CRE activity, production of VEGF, and angiogenesis in H. bilis-infected cell lines were enhanced compared with controls. H. bilis infection in a human bile duct cancer cell line activates transcript factors such as NF-kappaB that stimulate production of VEGF and lead to enhancement of angiogenesis. H. bilis infection may play an important role in malignancies in the biliary tract.

Mucosal gene expression profiles following the colonization of immunocompetent defined-flora C3H mice with Helicobacter bilis: a prelude to typhlocolitis

An aberrant immune response to the commensal microbiota is widely hypothesized to contribute to the development of inflammatory bowel disease. Helicobacter bilis colonization of defined-flora mice has been shown to trigger host immune responses to the commensal flora. However, the magnitude of the effects on mucosal homeostasis following colonization with H. bilis has not been determined. Using microarray analysis, differential gene expression within the cecal mucosa was assessed at 15, 30, or 45 days following H. bilis colonization using Affymetrix Genechips. H. bilis colonization induced marked upregulation of genes associated with protein metabolism, immune responses, and downregulation of genes associated with fatty acid metabolism and detoxification which peaked at 15 days postinfection. A set of genes associated with glycoprotein synthesis and detoxification including Fut2, B3galt5, Ceacam12, Cyp4b1, and Ugt8a were uniquely identified and found to be similarly expressed following the induction of typhlocolitis by dextran sodium sulfate or Brachyspira hyodysenteriae. This study provides preliminary evidence as to the types of factors or changes in the intestinal mucosa that potentially predispose the host to the development of typhlocolitis.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Differential susceptibility of P-glycoprotein deficient mice to colitis induction by environmental insults

P-glycoprotein (P-gp), the product of the multidrug resistance gene (MDR), is an ATP-dependent transmembrane pump, which is expressed in multiple cell lineages including epithelial and hematopoetic cells. The human MDR gene is located on chromosome 7 (7q21.1), a susceptibility loci for inflammatory bowel disease (IBD). A significant number of IBD patients carry mutations in this gene and P-gp-deficient FVB/N mice develop a severe spontaneous colitis, characterized by impaired intestinal barrier function and immune reactivity to intestinal bacterial antigens. In this work we explored the role of mouse strain, as well as environmental insults, on the development of colonic inflammation in the absence of P-gp. Among the induction methods utilized, dextran sodium sulfate (DSS) disrupts the intestinal epithelium, while piroxicam is a nonsteroidal antiinflammatory (NSAID) drug that inhibits prostaglandin production and initiates colitis in IL10-deficient animals. Helicobacter bilis is a known mediator of bacterial-induced colitis. We demonstrate that crossing this mutation onto the C57BL/6 strain confers protection from spontaneous colitis. C57BL/6.mdr1a-deficient animals demonstrated increased histological inflammation, colonic shortening, fecal blood, and reduced body weight after 7 days of treatment with 2.25% DSS. C57BL/6.mdr1a-deficient mice treated with piroxicam or infected with H. bilis showed no weight loss, or alterations in colonic histology. These data indicate that the effects of P-gp deficiency are significantly modulated by background strain influences, but that the epithelium continues to have increased susceptibility to chemical injury in the C57BL/6 model.

Helicobacter bilis colonization enhances susceptibility to DSS-induced colitis

Helicobacter bilis colonization enhances susceptibility to DSS-induced colitis

Helicobacter bilis colonization enhances susceptibility to DSS-induced colitis

Helicobacter bilis colonization enhances susceptibility to DSS-induced colitis

Cross-Reactivity between Immune Responses to Helicobacter bilis and Helicobacter pylori in a Population in Thailand at High Risk of Developing Cholangiocarcinoma

Helicobacter bilis DNA has been detected in human tissue and is a candidate for etiologic investigations on the causes of hepatic and biliary tract diseases, but reliable serologic tests need to be developed in order to pursue such investigations. The scope of this study was to assess the specificity of two assays for H. bilis immune response allowing for H. pylori, and their cross-reactivity in a population in Thailand at high risk for cholangiocarcinoma. Plasma samples from 92 Thai volunteers were independently tested in two laboratories (Massachusetts Institute of Technology [MIT] and Lund). MIT performed three analyses of H. pylori and H. bilis based either on (i) outer membrane protein (OMP) with no preabsorption or on antigens derived from whole-cell sonicate before (ii) or after (iii) preabsorption with H. pylori sonicate protein. Lund used cell surface proteins from H. pylori and H. bilis as antigens. Testing for H. bilis was preabsorbed with a whole-cell lysate of H. pylori. More than 80% of the samples were positive for H. pylori in both laboratories. As tested by MIT, 58.7% (95% confidence interval, 47.9 to 68.9%) were positive for H. bilis by OMP and 44.5% (34.1 to 55.3%) were positive for H. bilis sonicate protein, but only 15.2% (8.6 to 24.2%) remained positive after preabsorption with H. pylori sonicate protein. Lund found 34.5% of the samples positive for H. bilis (22.0 to 41.0%), which was statistically compatible with all three MIT results. Serologic responses to OMPs of the two bacteria coincided in 66 and 45% of the samples in the MIT and Lund assays, respectively. We found high cross-reactivity between the immune responses to H. pylori and H. bilis antigens. More-specific H. bilis antigens need to be isolated to develop serologic tests suitable for epidemiological studies.

Serum biomarkers in a mouse model of bacterial-induced inflammatory bowel disease

The diagnosis and classification of inflammatory bowel disease (IBD) require both clinical and histopathologic data. Serum biomarkers would be of considerable benefit to noninvasively monitor the progression of disease, assess effectiveness of therapies, and assist in understanding disease pathogenesis. Currently, there are limited noninvasive biomarkers for monitoring disease progression in animal IBD models, which are used extensively to develop new therapies and to understand IBD pathogenesis. Serum biomarkers of early and late IBD were identified using multianalyte profiling in mdr1a(-/-) mice with IBD triggered by infection with Helicobacter bilis. The correlation of changes in these biomarkers with histopathology scores and clinical signs in the presence and in the absence of antibiotic treatment was determined. Serum levels of interleukin (IL)-11, IL-17, 10-kDa interferon-gamma-inducible protein (IP-10), lymphotactin, monocyte chemoattractant protein (MCP)-1, and vascular cell adhesion molecule (VCAM)-1 were elevated early in IBD. In late, more severe IBD, serum levels of IL-11, IP-10, haptoglobin, matrix metalloproteinase-9, macrophage inflammatory protein (MIP)-1gamma, fibrinogen, immunoglobulin A, MIP-3 beta (beta), VCAM-1, apolipoprotein (Apo) A1, and IL-18 were elevated. All late serum biomarkers except Apo A1 correlated with histopathology scores. Antibiotic treatment improved clinical signs of IBD and decreased mean serum values of many of the biomarkers. For all biomarkers, the individual pathology scores correlated significantly with individual serum analyte levels after treatment. Serum analyte measurement is a useful, noninvasive method for monitoring disease in a mouse model of bacterial-induced IBD.

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.

Nature of Bacterial Provocation Determines Susceptibility To Colitis (B117)

Abstract Development of inflammatory bowel disease (IBD) involves predisposing and triggering events including aberrant immune responses against microbial antigens. Colonization of defined flora (DF) mice with Helicobacter bilis has been shown to induce immune responses to the resident flora. Aim: To determine the mechanisms underpinning susceptibility to colitis after colonizing DF mice with new bacterial species. Methods: Following colonization of DF mice with H. bilis or Escherichia coli, mice were treated with a low dose (1.5 %) of dextran sulfate sodium (DSS) (i.e., inflammatory trigger). Following euthanization, severity of colonic lesions were assessed as well as flora-specific antibody and T cell responses. Results: Colonization of DF mice with H. bilis, unlike E. coli, significantly (p&amp;lt;0.05) increased the severity of DSS-induced colitis. T cells from mice colonized with H. bilis and E. coli displayed in vitro ASF-specific recall responses but only the responses from the H. bilis-infected mice correlated with severity of lesions. T cell responses from E. coli-colonized mice produced more ASF-specific IL-10 than those from H. bilis colonized mice. Implications: These studies indicate that bacterial colonization can perturb mucosal homeostasis by impairing mucosal regulatory function(s) and exacerbate host susceptibility to subsequent colitic insults resulting in IBD. Supported by F31DK070563-01A2.

Specific and quantitative detection of PCR products from Clostridium piliforme, Helicobacter bilis, H. hepaticus, and mouse hepatitis virus infected mouse samples using a newly developed electrochemical DNA chip

We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9 ± 7.2, 31.9 ± 8.1, 29.3 ± 10.1, and 27.6 ± 3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3 ± 2.9, 3.7 ± 2.4, − 1.0 ± 1.7, and − 2.3 ± 2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.