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Abstract
BACKGROUND Bile tolerant helicobacter species such as H hepaticus andH bilis have frequently been reported to cause hepatitis in mice and other rodents. AIMS To investigate the possible pathogenic role of these and other helicobacter species in chronic liver disease in humans. METHODS Serum samples from 144 patients with various chronic liver diseases, 30 patients with primary sclerosing cholangitis (PSC), and 48 healthy blood donors were analysed for antibodies against H hepaticus murine strain CCUG 33637 and H pylori strain CCUG 17874. Cell surface proteins of H hepaticuswere extracted by acid glycine buffer and used in an enzyme immunoassay (EIA) and immunoblot (IB). RESULTS 56 of 144 (39%) patients with chronic liver diseases and six of 30 (20%) with PSC showed increased antibody concentrations in the H hepaticus EIA; in the H pylori EIA the numbers were 58% and 13% respectively. Compared with the healthy blood donors the antibody reactivity against the two helicobacter species was not increased (46% and 48% respectively). Patient serum samples retested by the H hepaticus EIA after absorption with sonicated H pyloricells remained positive in 12 of 37 (33%) serum samples. Distinct antibody reactivity to 55–65 kDa proteins was observed byH hepaticus IB, after the absorption step, and was considered specific for H hepaticus. These 12 serum samples were from patients with chronic alcoholic liver disease. CONCLUSIONS Antibodies toH hepaticus, often cross reacting withH pylori, occur frequently in patients with chronic liver diseases, with no clear cut relation to specific diagnostic groups. The pathogenic significance of these findings is not known.
... Helicobacter hepaticus is a spirally shaped Gram-negative bacterium slightly smaller than H. pylori, has bipolar sheathed flagella and can live in both anaerobic and microaerophilic environments [183]. This pathogen has mainly been investigated in the mouse model, where it causes hepatic diseases such as chronic hepatis [184,185] but may also exhibit pathogenicity in humans as it is detected in patients with hepatobiliary disease [186,187]. Although closely related to H. pylori and sharing the ability to produce urease, H. hepaticus lacks other key virulence factors of H. pylori such as CagA and the three adhesins SabA, AlpA and BabA [183]. ...
... In fact, is has been demonstrated that CDT induces cell cycle arrest at G2/M checkpoint and may therefore also promote persistence of infection [188]. H. hepaticus infection causes liver cancer in A/Jct mouse models and correlates with the development of gallbladder polyps and gallbladder cancer in humans [183,184,186,187,189,190]. H. hepaticus as well as other Helicobacter species are frequently found in cholangiocarcinomas and it has been postulated that they may induce hepatobiliary malignancies [191,192]. ...
The intestinal epithelium serves as a barrier to discriminate the outside from the inside and is in constant exchange with the luminal contents, including nutrients and the microbiota. Pathogens have evolved mechanisms to overcome the multiple ways of defense in the mucosa, while several members of the microbiota can exhibit pathogenic features once the healthy barrier integrity of the epithelium is disrupted. This not only leads to symptoms accompanying the acute infection but may also contribute to long-term injuries such as genomic instability, which is linked to mutations and cancer. While for Helicobacter pylori a link between infection and cancer is well established, many other bacteria and their virulence factors have only recently been linked to gastrointestinal malignancies through epidemiological as well as mechanistic studies. This review will focus on those pathogens and members of the microbiota that have been linked to genotoxicity in the context of gastric or colorectal cancer. We will address the mechanisms by which such bacteria establish contact with the gastrointestinal epithelium-either via an existing breach in the barrier or via their own virulence factors as well as the mechanisms by which they interfere with host genomic integrity.
... and Helicobacter pylori in bile samples, gallbladder tissue, and gallstones were detected by several methods: using cultures [52,59,67,71], by immunohistochemistry [46], by urease test, Giemsa, and immunohistochemical stain [72], by 16S rRNA PCR-amplification, by nested and multiplex PCR [47,[54][55][56][59][60][61][62][63][64]67,71,[73][74][75][76][77], by PCR-DGGE [52], both by PCR and by an ELISA study of the specific antigen (H. pylori stool antigen) [47], by ELISA after a preabsorption step using H. pylori antigens [46,78], by a Western blot analysis of Helicobacter antibodies [67], or by WMS sequencing and 16S rRNA sequencing on bile samples [35]. ...
... A study from Greece indicated positive Helicobacter pylori serology in 51.3% of patients with calcular biliary and pancreatic disease [79]. However, there is cross-reactivity among H. bilis, H. hepaticus, and H. pylori [78]. ...
Gallstone disease (GSD) has, for many years, remained a high-cost, socially significant public health problem. Over the past decade, a number of studies have been carried out—both in humans and in animal models—confirming the role of the microbiota in various sections of the gastrointestinal tract as a new link in the etiopathogenesis of GSD. The microbiome of bile correlates with the bacterial composition of saliva, and the microbiome of the biliary tract has a high similarity with the microbiota of the duodenum. Pathogenic microflora of the oral cavity, through mechanisms of immunomodulation, can affect the motility of the gallbladder and the expression of mucin genes (MUC1, Muc3, MUC4), and represent one of the promoters of stone formation in the gallbladder. The presence of H. pylori infection contributes to the formation of gallstones and affects the occurrence of complications of GSD, including acute and chronic cholecystitis, cholangitis, pancreatitis. Intestinal bacteria (Clostridium, Bifidobacterium, Peptostreptococcus, Bacteroides, Eubacterium, and Escherichia coli) participating in the oxidation and epimerization of bile acids can disrupt enterohepatic circulation and lead to the formation of gallstones. At the same time, cholecystectomy due to GSD leads to the further transformation of the composition of the microbiota in various parts of the gastrointestinal tract, increasing the risk of developing stomach cancer and colorectal cancer. Further research is required to determine the possibility of using the evaluation of the composition of the microbiota of the gastrointestinal and biliary tracts as an early diagnostic marker of various gastroenterological diseases.
... Bile-tolerant helicobacter species such as H. hepaticus and H. bilis have frequently been reported to cause hepatitis in mice and other rodents [85] . The most comprehensively studied member of this group of enterohepatic Helicobacter species is H. hepaticus [86] . ...
... H. hepaticus was initially detected by immunofluorescence, electron microscopy, and culture in the livers of certain inbred mice and has been shown to cause multifocal necrotizing hepatitis, hepatic adenomas, and hepatocellular carcinomas [88,89] . The reason for further study of the possible role of these new helicobacter species in human liver disease has been to develop noninvasive serological assays for determining the seroprevalence of hepatic helicobacter infections [85] . Fox et al [90] stated that H. hepaticus infection increased the risk of liver cancer in hepatitis C virus and/or Hepatitis B virus infection. ...
The presence of concomitant diseases is an independent predictive factor for non-Helicobacter pylori (H. pylori) peptic ulcers. Patients contracting concomitant diseases have an increased risk of developing ulcer disease through pathogenic mechanisms distinct from those of H. pylori infections. Factors other than H. pylori seem critical in peptic ulcer recurrence in end stage renal disease (ESRD) and cirrhotic patients. However, early H. pylori eradication is associated with a reduced risk of recurrent complicated peptic ulcers in patients with ESRD and liver cirrhosis. Resistances to triple therapy are currently detected using culture-based and molecular methods. Culture susceptibility testing before first- or second-line therapy is unadvisable. Using highly effective empiric first-line and rescue regimens can yield acceptable results. Sequential therapy has been included in a recent consensus report as a valid first-line option for eradicating H. pylori in geographic regions with high clarithromycin resistance. Two novel eradication regimens, namely concomitant and hybrid therapy, have proven more effective in patients with dual- (clarithromycin- and metronidazole-) resistant H. pylori strains. We aim to review the prevalence of and eradication therapy for H. pylori infection in patients with ESRD and cirrhosis. Moreover, we summarized the updated H. pylori eradication regimens.
High-throughput multiplexed assays are needed to simplify detection of Helicobacter species in experimental infection and routine health monitoring of laboratory mice. Therefore, fluorescent bead-based hybridization assays for Helicobacter sp. DNA and serology were developed. Multiplex PCR amplicons (H. hepaticus, H. bilis, H. typhlonius, H. pylori, H. muridarum, H. pullorum, H. cinaedi, H. heilmanii, C. jejuni) and antibodies against H. pylori, H. hepaticus, H. bilis were assessed in naturally and experimentally infected mice, and results compared to conventional PCR. Species-specific and sensitive detection of seven Helicobacter spp. <100 copies/PCR, and of two species <1000 copies/PCR was successfully established in the Helicobacter multiplex DNA finder. The novel assay was highly comparable with conventional PCR (kappa = 0.98, 95%CI: 0.94–1.00). Antibody detection of H. hepaticus and H. bilis showed low sensitivity (71% and 62%, respectively) and cross-reactivity in H. typhlonius-infected mice. Infection experiments showed that antibodies develop earliest two weeks after DNA detection in feces. In conclusion, detection of Helicobacter antibodies showed low sensitivity depending on the timing relative to infection. However, Helicobacter multiplex DNA finder is a sensitive and specific high-throughput assay applicable in routine health monitoring for laboratory animals.
The gut microbiota has recently emerged as a unique mechanism of immunotherapeutic resistance or response within certain cancer patients. Certain adherent bacterial species that reside along the epithelial barrier within the gastrointestinal tract have been shown to be the most immunogenic and include several species within the Helicobacteraceae family. The role of these microbes in cancer remains controversial and varies according to species, immune status, and cancer type. Here, we hypothesize that the functional characteristics rather than the bacterial species of Helicobacteraceae dictate the type of immune response with either a benefit or a detriment to overall cancer progression.
Classic atherosclerosis risk factors do not explain all cases of chronic heart disease. There is significant evidence that gut microbiota may influence the development of atherosclerosis. The widespread prevalence of chronic Helicobacter pylori (H. pylori, HP) infections suggests that HP can be the source of components that stimulate local and systemic inflammatory responses. Elevated production of reactive oxygen species during HP infection leads to cholesterol oxidation, which drives atherogenesis. The aim of this study is to explore the link between persistent HP infection and a high-fat diet in the development of proinflammatory conditions that are potentially proatherogenic. An in vivo model of Caviae porcellus infected with HP and exposed to an experimental diet was investigated for the occurrence of a proinflammatory and proatherogenic endothelial environment. Vascular endothelial primary cells exposed to HP components were tested in vitro for oxidative stress, cell activation and apoptosis. The infiltration of inflammatory cells into the vascular endothelium of animals infected with HP and exposed to a high-fat diet was observed in conjunction with an increased level of inflammatory markers systemically. The arteries of such animals were the least elastic, suggesting the role of HP in arterial stiffness. Soluble HP components induced transformation of macrophages to foam cells in vitro and influenced the endothelial life span, which was correlated with Collagen I upregulation. These preliminary results support the hypothesis that HP antigens act synergistically with a high-fat diet in the development of proatherogenic conditions.
C1q, as a LAIR-1 ligand, maintains monocytes quiescence and possess immunosuppressive properties. To understand the roles and molecular mechanisms, C1q mediated inflammation cytokines and several pivotal proteins in THP-1 cells after H. pylori infection were detected. The results showed that the expression of IL-8, IL-10, LAIR-1, phosphorylated/total JNK, phosphorylated/total p38-MAPK, phosphorylated/total AKT and phosphorylated/total NF-κB were up-regulated significantly in THP-1 cells after H. pylori infection. There was significant upregulation in IL-10 concentration, phosphorylated/total p38-MAPK and phosphorylated/total AKT, and downregulation in phosphorylated/total JNK in non-H. pylori infected THP-1 cells pretreated with C1q. C1q was also able to increase IL-8 and IL-10 production, and reduce LAIR-1 and phosphorylated/total p38-MAPK expression in pretreatment-C1q THP-1 cells after H. pylori infection. These results together indicated that H. pylori might induce IL-8 and IL-10 production through JNK, p38-MAPK, PI3K/AKT and NF-κB signaling pathway. C1q manipulate LAIR-1 to regulation IL-8 and IL-10 secretion in THP-1 cells after H. pylori infection through the p38-MAPK signaling pathway. This information is helpful to further understand the role and mechanisms of C1q on inflammation cytokines secretion in monocytes after H. pylori infection.
Background
Helicobacter pylori is an established cause of gastritis and has been implicated in extradigestive diseases.
Objective
To investigate the role of H. pylori in patients with unexplained refractory pruritus.
Methods
Ten patients with severe pruritus unresponsive to conventional therapy were evaluated for active H. pylori infection by H. pylori serology followed by either esophagogastroduodenoscopy (EGD) or urea breath test. Of the 10 patients, 8 were found to have active infection. All 10 received anti- H. pylori antibiotic therapy and were reevaluated for relief of pruritus.
Results
Of 8 patients with active H. pylori infection, 87.5% (7/8) had some type of pruritus relief after triple therapy. Of these, 62.5% (5/8) had complete relief and 25% (2/8) had temporary relief of pruritus. The remaining 12.5% (1/8) did not respond. Two control patients without active H. pylori infection had no relief of pruritus with therapy.
Conclusions
We have identified a population of patients with refractory pruritus and active H. pylori infection whose pruritus resolved after eradication of H. pylori.
Objective:
Helicobacter pylori is a Gram-negative, microaerophilic bacteria usually found in the stomach, which may evade its host's immune system and present long-term symptoms in affected individuals. This study aimed to evaluate the functional role of leukocyte-associated immunoglobulin (Ig)-like receptor-1 (LAIR-1) in the strategies and underlying molecular mechanisms by which H. pylori escapes the host's immune responses.
Methods:
LAIR-1 knockdown THP-1 cells were used to detect cell apoptosis, cell proliferation, interleukin-8 (IL-8), IL-10, and activation of intracellular signaling induced by H. pylori.
Results:
Cell apoptosis, cell proliferation, IL-8, and IL-10 were increased in THP-1 cells after 24 h of H. pylori infection. Functional analysis indicated LAIR-1 silencing obviously inhibited the phosphorylation of IκBα, eIF2α, JNK, and Smad2 in the THP-1 after H. pylori infection. In addition, there were no significant differences in proliferation rates between control siRNA group and LAIR-1 siRNA group regardless of whether THP-1 cells were infected by H. pylori.
Conclusion:
These results together indicated that LAIR-1 modulated cell apoptosis and inflammatory cytokines secretion in THP-1 cells, which might help sustain inflammation and prevent removal of the bacteria by the immune responses.
Primary biliary cirrhosis is a chronic progressive cholestatic liver disease that primarily targets middle-aged women. The pathogenesis is unknown, but several lines of evidence suggest genetic and environmental factors initiate an autoimmune process. Routine laboratory testing has led to the earlier detection of disease, so patients are often asymptomatic at the time of diagnosis. Initial symptoms are usually fatigue and pruritus. Signs of decompensated liver disease may eventually develop. Ursodeoxycholic acid is the only recommended treatment and may improve survival in selected patients. Prognostic models help predict natural history of the disease. Liver transplantation is the only definitive therapy once end-stage liver disease occurs.
The link between Helicobacter pylori and gastric infection and malignancy has been firmly established since the discovery of this organism 20 years ago, and, in addition, there are now several studies implicating H. pylori infection as a risk factor for cardiac1–3 and other extraintestinal diseases. Furthermore, among the more than 20 Helicobacter species currently described, several have been linked to hepatobiliary and intestinal diseases. The human liver is normally sterile and has effective systems to escape bacterial pathogens. The biliary tree is likewise normally sterile but, when bile stones are present various microbes can be cultured from or detected in bile or the gallbladder wall4,5. Salmonella spp and Enterococcus spp can grow in the presence of bile, having been regularly isolated from chronic carriers and patients with cholecystitis, respectively6. Various conditions such as common bile duct or cystic duct obstruction, or infection can decrease the pH of bile which in turn could enhance the survival of H. pylori in a bile-rich environment7.
Hepatocellular carcinoma (HCC) has a high incidence worldwide, with a low diagnostic rate in the early period and therefore a high death rate. Causes for some cases of liver cancer are not completely clear. The finding that the rate of liver cancer in male A/Jr mice is significantly higher makes researchers realize that Helicobacter hepati-cus may cause HCC in human beings. To get a new perspective for exploring causes of liver cancer, this paper introduces the biological nature of and detecting methods for Helicobacter hepaticus, as well as the relationship between Helicobacter hepaticus infection and HCC. Helicobacter hepaticus infection may play an important role in some HCC cases. © 2014 Baishideng Publishing Group Co., Limited. All rights reserved.
Primary biliary cirrhosis (PBC) is an immune mediated liver disease directed against the biliary epithelial cells of the small bile ducts. The disease is characterised by circulating antimitochondrial antibodies (AMA), as well as antinuclear antibodies (ANA). AMA is considered pathognomonic for PBC, with AMA positivity predicting disease development in asymptomatic individuals. Middle aged females are most commonly affected, with increased incidence in families. Sisters and daughters of PBC patients are especially at risk. Epidemiological and twin studies have demonstrated that genetic predisposition, combined with environmental factors likely act together in the disease initiation. Among the environmental risk factors, infectious agents have been implicated. The mechanism by which infectious agents contribute to the pathogenesis of PBC appears to be through molecular-mimicry, and cross reactivity to antigenic epitopes of mitochondrial antigens. Although several bacterial and viral pathogens have been identified with PBC, Escherichia coli, Novosphingobium aromaticivorans, and Lactobacillus delbrueckii subspecies bulgaricus have been considered the most significant infectious triggers, and have also been the most studied. The pathogenic significance of these bacteria may be reflective of their relationship with other identified risk factors, such as recurrent urinary tract infections and alterations in oestrogen metabolism. This review will examine the literature surrounding the epidemiological and molecular studies which have characterised the role of these bacteria in the pathogenesis of PBC.
Helicobacter pylori is a well-known causative organism of chronic gastric diseases and has been found in many hepatic carcinoma samples. To explore the expression of apoptosis-related proteins and carcinoma development in H. pylori-infected livers, we utilized BALB/cAnSlac mice to establish an H. pylori-infected model by oral inoculation and orthotopic grafts of hepatic tumors by H22 cells, respectively. We found that H. pylori colonies could not be cultured from all liver and tumor samples. However, its 16S rRNA was detectable in 85.3% of livers and 66.7% of tumors in the infected mice. Inflammatory cells were observed and thinly distributed in the lobule portions of the liver, and H. pylori mainly existed in the infected hepatic sinusoids and the necrotic areas of the infected tumors. No significant difference was found in liver to body weight ratio between the infected and uninfected. Moreover, the pathological tumor difference was unremarkable between the two. The proliferating cell nuclear antigen (PCNA) and Bcl-2-associated X protein (Bax) expression in the infected tumors was significantly higher and lower, respectively, than those of the uninfected tumors. However, no significant difference in Bcl-2 (B-cell lymphoma 2) expression existed. The results indicate that H. pylori found in the livers which were infected by H. pylori oral inoculation could contribute to the infiltration of inflammatory cells in livers. Although H. pylori has no significant impact on the liver to body weight ratio or tumor Bcl-2 expression, it may upregulate PCNA expression and downregulate Bax expression, respectively. All our findings show that H. pylori may promote proliferation and inhibit apoptosis of tumor cells.
Helicobacter hepaticus was discovered in 1992 as a cause of liver cancer in the A/JCr mouse model. In susceptible mice, infection by H. hepaticus causes chronic gastrointestinal inflammation leading to neoplasia. It can also cause morphological changes in breast-glands leading to neoplasm and adenocarcinoma in mouse models. Studies performed on humans have revealed that H. hepaticus may also be a human pathogen since infection by H. hepaticus can be associated with cholecystitis, cholelithiasis and gallbladder cancer. H. hepaticus is a close relative of H. pylori, but it lacks the major virulence factors of H. pylori including vacoulating cytotoxin A (VacA) and cytotoxin associated gene (cagA). Moreover, SabA, AlpA, and BabA, three important adhesin proteins of H. pylori, are absent in its genome. In contrast, the genome of H. hepaticus contains genes encoding some orthologus virulence factors of Campylobacter jejuni such as cytolethal distending toxin (CDT), and PebI adhesin factor. Other genes including 16S rRNA, 18 KDa immunogenic protein, and urease structural subunits are related to H. pylori. Its genome contains a small island consisting of 71 Kbp named HHGI1, which probably encodes a secretion system type IV (T4SS), and some other virulence factors. As far as the immunogenic antigens are concerned, the lipopolysaccharide (LPS) and flagellin of H. hepaticus are weak stimulants of the immune system, while pro-inflammatory responses are mainly induced by its lipoproteins and most likely by the peptidoglycan. Concerning the multidrug efflux pumps, a homologue of H. pylori TolC, HefA, has been observed in H. hepaticus which contributes to resistance to amoxicillin and bile acids.
Abstract:
Helicobacter bilis is a member of the murine enterohepatic Helicobacter spp which identified and isolated
from bile, liver, and intestine of aged, inbred mice and associated with liver and Hepatobiliary tract disease in human patients. The aim of the present study was to investigation of the presence of Helicobacter species, particularly Helicobacter bilis for the first time in liver of Mus musculus in Isfahan province (center of Iran) by the molecular method. DNA was extracted from 300 wild mice liver specimen using the DNA Extraction Kit and Nested PCR was performed on template DNA and amplified products were separated on 1.5% agarose gel. From 300 liver samples which assayed, 204 (68%) samples were positive for genus Helicobacter and 118 (39.33%) samples were positive for Helicobacter bilis (P≤0.05). In conclusion, PCR technique can detect H. bilis DNA in liver samples of Mus musculus in the center of Iran and wild mice may be carrying zoonosis infection such as H. bilis. According to the results of this study, wild mice may be important source and the potential of carrying Zoonosis infection such as H.bilis. These data suggest Mus musculus may have the potential of carrying H.bilis and zoonotic microorganisms that can be infectious in humans.
The Helicobacter genus includes Gram negative bacteria which were originally considered to belong to the Campylobacter genus. They have been classified in a separate genus since 1989 because they have different biochemical characteristics, with more than 24 species having been identified and more still being studied.H. pylori is the best known. It has an important etiopathogenic role in peptic ulcer disease and gastric cancer. Enterohepatic Helicobacters (EHH) other than H. pylori colonize the bowel, biliary tree and liver of animals and human beings with pathogenic potential. The difficulties existing to correctly isolate these microorganisms limit the description of their true prevalence and of the diseases they cause. Many studies have tried to discover the different clinical implications of EHH. Diseases like chronic liver disease, autoimmune hepatitis, hepatocarcinoma, autoimmune hepatobiliary disease, biliary lithiasis, cholangiocarcinoma and gallbladder cancer, Meckel´s diverticulum, acute appendicitis and inflammatory bowel disease have been related with different EHH species with different results, although their prevalence is greater than in healthy subjects. However, these data are currently not sufficient to draw definitive conclusions. Finally, the best known role of EHH in bowel disease is production of acute and chronic diarrhea pictures initially referred to as Campylobacter. H. pullorum has been identified in patients with acute gastroenteritis. The correct identification of EHH as producers of infectious gastroenteritis is found in its antibiotic susceptibility. It is generally macrolide-susceptible and quinolone-resistant.
Accumulating evidence has implicated Helicobacter pylori (H. pylori) infection in extragastrointestinal diseases, including obesity, type 2 diabetes mellitus, cardiovascular disease, and liver disease. Recently, there has been a special focus on H. pylori infection as a risk factor for the development of nonalcoholic fatty liver disease (NAFLD). NAFLD is currently considered to be the most common liver disorder in western countries, and is rapidly becoming a serious threat to public health. The mechanisms of pathogenesis underlying NAFLD remain unclear at present and therapeutic options are limited. The growing awareness of the role of H. pylori in NAFLD is thus important to aid the development of novel intervention and prevention strategies, because the eradication of H. pylori is easy and much less expensive than long-term treatment of the other risk factors. H. pylori infection is involved in the pathogenesis of insulin resistance (IR), which is closely linked with NAFLD. It provides a new insight into the pathogenesis of NAFLD. This review probes the possible relationship between H. pylori and NAFLD, from the perspective of the potential mechanism of how H. pylori infection brings about IR and other aspects concerning this correlation.
Primary biliary cirrhosis (PBC) is a progressive cholestatic liver disease characterized by the autoimmune destruction of the biliary epithelial cells of the small and medium-size bile ducts. The disease affects middle aged women and usually affects more than one member within a family. The pathognomonic serological hallmark of the disease is the presence of circulating anti-mitochondrial antibodies, and disease-specific anti-nuclear antibodies. Susceptibility genes and environmental risk factors such as infections and smoking have been reported as important for the development of the disease. Among the environmental agents, infectious triggers are the best studied. Most of the work published so far has investigated the role of infections caused by Novosphingobium aromaticivorans and Escherichia coli. This review will discuss the popular and unpopular infectious agents causatively linked to PBC. It will also examine reports investigating the epidemiological aspects of the disease and their direct or indirect implications to bacterial-induced PBC.
Helicobacter (H.) hepaticus infection causes chronic active hepatitis and induces hepatocellular tumours in A/JCr mice, but evidence of this in humans is scarce. This study aimed to demonstrate the correlation between H. hepaticus and human primary hepatocellular carcinoma (HCC).
The sera of 50 patients with primary HCC were tested for the presence of anti-H. pylori and anti-H. hepaticus immunoglobulin G (IgG) antibodies. The liver tissues of patients who tested positive for serum antibody were analysed for H. hepaticus-specific 16S rRNA, H. hepaticus cdtB, H. pylori cagA, H. pylori vacA and H. pylori ureC genes using polymerase chain reaction.
After the anti-H. pylori antibodies in the serum samples were absorbed by H. pylori antigen, the anti-H. hepaticus IgG serum antibody detection rate was 50.0% in patients with primary HCC. This was significantly higher (p < 0.001) than the detection rate in the benign liver tumour (7.7%) and normal liver tissue (6.3%) groups. Of the 25 primary HCC samples that tested positive for anti-H. hepaticus IgG serum antibody, the H. hepaticus-specific 16S rRNA gene was detected in nine (36.0%) samples. Sequencing showed that the polymerase chain reaction-amplified product exhibited 95.5%-100% homology to the H. hepaticus-specific 16S rRNA gene. Among these nine primary HCC tissue samples, the H. hepaticus cdtB gene was detected in four (44.4%) samples, while no such expression was observed in the benign liver tumour or normal liver tissue groups.
The present study identified the presence of H. hepaticus infection in patients with primary HCC using serological and molecular biological detection, suggesting that H. hepaticus infection may be involved in the progression of HCC.
Background. The association of gallstones with Helicobacter pylori has been investigated but not clearly demonstrated. In this study, the presence of H. pylori in the gallbladder mucosa of patients with symptomatic gallstones was investigated. Method. Ninety-four consecutive patients with symptomatic gallstone disease were enrolled for the study. Gastroscopy and gastric H. pylori urease test were done before cholecystectomy to all patients who accepted. After cholecystectomy, the gallbladder tissue was investigated in terms of H. pylori by urease test, Giemsa, and immunohistochemical stain. Results. Overall 35 patients (37%) gallbladder mucosa tested positive for H. pylori with any of the three tests. Correlation of the three tests Giemsa, IHC, and rapid urease test was significant (r s : 0590, P > 0.001). Rapid urease test was positive in the gastric mucosa in 47 (58.7%) patients, and it was positive in the gallbladder mucosa in 21 patients (22%). In 15 patients both gastric and gallbladder tested positive with the urease test. There was significant correlation of rapid urease test in both of gallbladder and gastric mucosa (P = 0.0001). Conclusion. Study demonstrates the presence of H. pylori in the gallbladders of 37% of patients with symptomatic gallstones.
Helicobacter hepaticus and Helicobacter pylori both belong to Helicobacter species. Strains of Lactobacillus acidophilus, including L4 and L6, have shown significant inhibitory effects on H. pylori. Based on this phenomenon, we aim to investigate the inhibitory effect of L. acidophilus on H. hepaticus. Both standard and isolated H. hepaticus strains were grown under microaerophilic conditions at 37 °C in the presence of L. acidophilus supernatant, or lactic acid. The diameters of the inhibition zones were measured on the solid culture media. In liquid culture, the cell concentrations were measured and the urease activity was determined by phenol red staining. Sixteen strains of L. acidophilus isolated from human feces (named as L1-L16) showed anti-H. hepaticus effects. Two of them (L4 and L6) exhibited the most apparent effects on H. hepaticus inhibition. The L. acidophilus supernatant of L4 and L6 significantly increased the diameters of the inhibition zones compared with that of the lactic acid control (P < 0.05). The inhibitory role of L. acidophilus supernatant was independent of the pH value of solution (P > 0.05). Moreover, in liquid culture, L. acidophilus supernatant significantly reduced the cell growth rate and the urease activity of H. hepaticus cells in a time-dependent pattern (P < 0.05 compared with lactic acid control). No obvious difference was observed between the standard and isolated strain of H. hepaticus (P > 0.05). Our results indicate that L. acidophilus can decrease the viability and urease activity of H. hepaticus in vitro and this inhibition is independent of pH levels. This provides evidence for developing novel approaches for the prevention and treatment of H. hepaticus infection.
Die Entdeckung der Rolle des Helicobacter (H.) pylori als Verursacher des Ulcus pepticum war ein Meilenstein in der Gastroenterologie. Neben der Ulkuskrankheit könnte H. pylori auch bei anderen internen Erkrankungen wie der koronaren Herzkrankheit, der rheumatoiden Arthritis, der hepatischen Enzephalopathie und beim Sjoegren-Syndrom eine Rolle spielen. Alle diese möglichen Assoziationen sind widersprüchlich und bedürfen weiterer Studien. Andere Helicobacterstämme (H. bilis, H. hepaticus, H. rappani, H. pullorum) werden als Erreger chronischer Leber- und Gallenwegserkrankungen sowie von Durchfallserkrankungen diskutiert. Besonders die mögliche Rolle dieser Keime für die Krebsentstehung ist hochinteressant und Gegenstand intensiver Forschungsarbeit.
The Importance of Helicobacter – Also Beyond the Stomach
The discovery of H. pylori as causative agent for peptic ulcers was a milestone in gastroenterology. Apart from peptic ulcer, H. pylori may play a role in the pathogenesis of coronary heart disease, rheumatoid arthritis, Sjoegren's syndrome and hepatic encephalopathy. These associations are still controversial and need further studies. Other strains (H. bilis, H. hepaticus, H. rappani, H. pullorum) may cause chronic hepatobiliary diseases or diarrhea. The possible role of such strains in carcinogenesis is very interesting and should be further explored.
Helicobacter species, which may colonize the biliary tract, have been implicated as a possible cause of hepatobiliary diseases ranging from chronic cholecystitis and primary sclerosing cholangitis to gall-bladder carcinoma and primary hepatic carcinomas.
Research in this area has been limited by the lack of a gold standard in the diagnosis of these organisms in bile. Most published data to date have been based on molecular techniques that detect the DNA of Helicobacter species in bile, rather than evidence of viable organisms in bile.
Helicobacter species have not been shown to induce histological injury to the biliary epithelium or liver parenchyma. The strongest association of the presence of these organisms in bile is with cholestatic conditions. This article reviews the literature on this newly developing field as it has evolved historically, taking pertinent methodological issues into account.
Background. Studies on spiral−sharped bacteria of the genus Helicobacter have focused mainly on Helicobacter pylori. However, in the last few years a great number of novel Helicobacter species have been isolated from ani− mals and humans. Objectives. The aim of the study was to determine the correlation between infection by Helicobacter spp. such as H. pylori, H. hepaticus, and H. bilis and pathological hepatic changes in patients with chronic liver diseases. Material and Methods. The study included 56 patients aged 20–60 years diagnosed for various chronic liver dis− eases, e.g. chronic viral hepatitis types B or C, co−infection of HBV and HCV, autoimmune hepatitis, hemochro− matosis, Wilson's disease, and non−alcoholic fatty liver disease (NAFLD). Thick−needle hepatic biopsy specimens and serum samples from each patient were analyzed. The presence of Helicobacter spp. in the biopsy specimens was determined by culture on solid media and polymerase chain reaction (PCR). The level of anti−H. pylori IgG antibodies in the patients' sera was detected by ELISA. Results. Examination of the hepatic biopsies for Helicobacter spp. infection by different culture methods was neg− ative for all samples. The Helicobacter ureB gene was identified by PCR in 7 of the 56 biopsies (12.5%). Among these patients, 5 were diagnosed for hepatitis C or B, 1 for hemochromatosis, and 1 for NAFLD with no identified viral infection. Anti−H. pylori IgG antibodies in the serum samples were detected in 52% of the examined subjects and in all 7 patients with positive PCR results. Conclusions. The coexistence of H. pylori infection and chronic liver disease, especially viral hepatitis, might be possible in humans. Further studies are needed to clarify the relationship between H. pylori, H. hepaticus, and H. bilis infection and pathological hepatic changes in humans. So far, a detrimental effect of Helicobacter species on the liver could not be confirmed or excluded. There was no correlation between the presence of DNA detected by PCR assay of liver samples and the presence of specific antibodies in the patients' sera (Adv Clin Exp Med 2007, 16, 4, 537–542).
A characteristic spectrum of systemic inflammation involving the joints, hver, skin, eyes, and hematologic organs is associated with diverse types of intestinal injury (Table 1) [1-12]. Extraintestinal manifestations occur in 20-30% of patients with ulcerative colitis (UC), Crohn's disease (CD), or jejunoileal bypass for obesity but are relatively infrequent or rare in the other listed conditions. Although each of these intestinal disorders has a diff'erent pathogenesis, they share common properties of: (1) increased mucosal permeability (UC, CD, and celiac disease); (2) infection (enteric pathogens, Whipple's disease, and diverticulitis); or (3) overgrowth of predominantly anaerobic bacteria (jejunoileal bypass, pouchitis, and bacterial overgrowth associated with gastric or biliary surgery). These insults lead to increased systemic uptake of commensal or pathogenic enteric bacterial products. These clinical associations are supported by experimental observations that jejunal bacterial overgrowth [12, 13], colitis [6, 14], or ileocolitis [15 16] lead to hepatobiliary, joint, and hematologic abnormalities in rodents. Intestinal and systemic inflammation are further linked by finding asymptomatic intestinal inflammation in the majority of patients with primary sclerosing cholangitis (PSC) [17] and spondyloarthropathy [18]. This chapter discusses potential mechanisms and develops a unifying hypothesis explaining the association between intestinal and systemic inflammation. Mechanisms of non-inflammatory and specialized complications of inflammatory bowel diseases (IBD), such as gallstones or renal calculi associated with ileal CD, are discussed in Chapter 15. Clinical and experimental data, which are most extensive for PSC, ankylosing spondylitis, and reactive arthritis, are cited to provide evidence for each potential mechanism. (Table presented).
Patients with primary biliary cirrhosis (PBC) often have concurrent limited systemic sclerosis (SSc). Conversely, up to one-fourth of SSc patients are positive for PBC-specific antimitochondrial antibodies (AMA). The mechanisms responsible for the co-occurrence of these diseases are largely unknown. Genetic, epigenetic, environmental, and infectious factors appear to be important for the pathogenesis of the disease, but the hierarchy of events are not well defined. Patients with SSc and PBC have an increased morbidity and mortality compared with the general population, but whether the presence of both diseases in an affected individual worsens the prognosis and/or outcome of either disease is not clear. Some case reports suggested that the presence of SSc in PBC patents is associated with a more favorable prognosis of the liver disease, whereas others report an increased mortality in patients with PBC and SSc compared to patients with PBC alone. This paper discusses the features of patients with PBC-associated SSc. Our aims are to clarify some of the pathogenetic, diagnostic, and clinical challenges that are currently faced in the routine management of these patients. We also intend to provide some practical hints for practitioners that will assist in the early identification of patients with PBC-associated SSc.
Infection with Helicobacter hepaticus has been associated with development of hepatocellular carcinoma and gallstones in animal models. In humans, however, the association of H. hepaticus infection with biliary and pancreatic diseases has not been elucidated. The aim of this study was to serologically examine the prevalence of H. hepaticus infection in patients with biliary and pancreatic diseases.
Serum samples obtained from 55 patients with cholelithiasis, 18 with bile duct or gallbladder cancer and 19 with pancreatic cancer were studied. Sera were obtained from 34 control subjects who underwent endoscopy and were diagnosed as not having peptic ulcers or cancers. Seropositivity of H. hepaticus was examined by western blot analysis using a H. hepaticus-specific antigen. To validate the specificity, positive sera were also tested after absorption with H. hepaticus whole-cell sonicate. Serum samples were also tested for the presence of anti-Helicobacter pylori antibody.
Prevalence of antibody to H. hepaticus-specific antigen in patients with bile tract cancer was 38.8% and was significantly higher than in control subjects (13.1%, P < 0.05). Prevalence of antibody to H. hepaticus-specific antigen was 18.2% and 10.5% in patients with cholelithiasis and pancreatic cancer, respectively. Seropositivity for H. pylori was similar in all groups. Detection of the H. hepaticus-specific band was significantly decreased after the sera were absorbed with H. hepaticus whole-cell sonicate.
Infection with H. hepaticus might be associated with bile duct cancer. Results obtained from absorbed sera suggested high specificity of the western blot analysis.
Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic disease characterized by the presence of antibodies directed predominantly against the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). What provokes tolerance breakdown in PBC remains to be established, though there is evidence to indicate that microbes may induce anti-mitochondrial antibodies (AMA) through a mechanism of molecular mimicry.
Having found that urease beta (UREB)(22-36) antigen of Helicobacter pylori (HELPY) shares extensive (87%) similarity with PDC-E2(212-226), the major mitochondrial autoepitope, it was hypothesized that this would also lead to cross-reactivity. The UREB/PDC-E2 mimics were thus constructed and tested by ELISA in 112 PBC patients and 114 controls.
Reactivity to PDC-E2(212-226) was found in 104 patients but to UREB(22-36) in only 2. In these two patients, the double reactivity was not cross-reactive. The lack of surface antibody accessibility to UREB(22-36), as demonstrated through three-dimensional model prediction analysis, may explain this unexpected finding. There was some speculation on whether HELPY UREB(22-36) might act as a cross-reactive CD4 T-cell epitope. All seven PBC patients, tested in a standard proliferation assay against PDC-E2(212-226), gave a positive response. All seven were unresponsive to HELPY UREB(22-36). The pattern of reactivity to HELPY antigens by immunoblot was similar between anti-PDC-E2-positive and negative PBC cases, as well as between PBC patients and controls.
Contrary to common belief, extensive sequence homology (molecular mimicry) between self and microbe does not necessarily result in cross-reactivity. It is therefore likely that, when present, cross-reactivity between self and microbes is of biological importance.
Helicobacter hepaticus infection might be associated with liver and biliary tract diseases. To investigate its pathogenic role, the properties of anti-H. hepaticus serum antibody in patients with liver and diseases were elucidated.
Serum samples were collected from 166 patients-69 with liver diseases, 38 with upper gastrointestinal diseases, 17 with lower gastrointestinal diseases, 26 with biliary tract diseases, and 16 with pancreas diseases; 30 control sera were obtained from 30 healthy blood donors. Serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) and western blot using the new monoclonal antibody HR II-51.
Anti-H. hepaticus serum antibody concentrations in patients with liver disease (n = 69) were significantly increased compared with those in other disease groups (p = 0.014 to <0.001). Particularly, liver cirrhosis (n = 19) showed a significantly higher antibody level compared with other liver diseases (n = 50, p = 0.005) and healthy donors (n = 30, p = 0.0005), as well as a higher seroprevalence (68.4%) compared with other liver diseases (p = 0.05) and healthy donors (p = 0.004). Furthermore, the ELISA value in liver cirrhosis (n = 19) was significantly higher than that in patients with hepatitis B virus (HBV)-and/or hepatitis C virus (HCV)-infected chronic hepatitis (n = 15) (0.389 ± 0.084 vs. 0.350 ± 0.084, p = 0.029). However, there was no relationship between the total immunoglobulin concentration and the anti-H. hepaticus antibody level in each liver disease (Spearman's rank correlation coefficient [rs] < 0.225).
H. hepaticus infection might play a role in the development of liver diseases; in particular, it might increase the risk of the development of HBV- and/or HCV-infected liver diseases.
Most of the members of the genus Helicobacter do not normally colonize the gastric mucosa, but instead thrive in the mucosal surfaces of the intestinal tract and/or the liver of humans, other mammals, and birds. These enterohepatic Helicobacter species have features of ultrastructure and physiology in common with Helicobacter pylori and the other gastric Helicobacter species, and have been the subject of several recent reviews (35, 41, 108). Because the enterohepatic Helicobacter species were first recognized in laboratory rodents, in which they are highly prevalent in most inbred strains and outbred stocks, they have been considered a component of the resident microbiota, or "normal flora." It is now clear that some of the enterohepatic Helicobacter species, and perhaps all, have the ability to cause disease in normal, immunocompetent rodents. A growing number of enterohepatic Helicobacter species have also been reported to be associated with gastroenteritis, hepatitis, and other disease states in humans and in other animal species. The significance of the enterohepatic Helicobacter species in human disease and the true prevalence of these organisms in human populations remain to be determined. What follows is a survey of this emerging group of organisms. Early studies characterizing the resident microbiota in the gut of laboratory rodents led to the discovery of a diverse population of spiral-shaped bacteria uniquely adapted to thrive in the mucosal surfaces of the intestine. These early studies, which used transmission electron microscopy rather than culture and isolation, described two morphologic types of organisms, both of which are now known to be enterohepatic Helicobacter species. Members of the first group superficially resemble Campylobacter species but are longer and have a single polar flagellum at each end. Representative organisms are shown in Fig. 1 and are listed in Table 1 as having no periplasmic fibers. Members of the second group have periplasmic fibers that wrap helically around the body of the bacterium as well as bipolar tufts of sheathed flagella. Representative organisms are shown in Fig. 2 and are listed in Table 1 as having periplasmic fibers. In studies that characterized the patterns of bacterial colonization of the large intestine of laboratory rodents, Davis et al. identified both morphologic types of organisms in the mucus of the cecum and colon (18, 19). The bacteria could be found during the first week of life, and they remained on the surface of the intestinal epithelium and packed deep in the crypts throughout the life of the animals. Perhaps because their ultrastructure is less remarkable, the early literature contains fewer reports of the simple spiral-shaped organisms than of the organisms with periplasmic fibers. Nonetheless, the simple spiral-shaped organisms have been isolated from a variety of mammals, including humans, pigs, dogs, cats, mice, rats, hamsters, gerbils, and several wild and domestic species of birds. The distinction between these organisms and the organisms with periplasmic fibers has a morphologic basis only. No comparable phylogenetic dichotomy has been recognized. On the other hand, the presence of periplasmic fibers has facilitated the recognition of members of the second group of enterohepatic Helicobacter species in a variety of locations. Spiral-shaped bacteria with periplasmic fibers were observed free in the cytoplasm of enterocytes as well as deeper in the lamina propria of mice following treatment with nitrogen mustard (53). Such treatment results in a generalized loss of epithelial integrity, but it is interesting to note that the enterohepatic Helicobacter species were the only organisms found to invade under these conditions. The abundance of these organisms in the mucus deep in the crypts of the ileum and their proximity to the apical surface of the epithelial cells lining the crypts may account, at least in part, for these observations. Erlandsen and Chase exploited the ultrastructural characteristics of these organisms to ascertain the fate of bacteria following phagocytosis from the crypts by differentiated enterocytes in the ileum of untreated rats (26). Davis et al. also noted the occasional penetration of enterohepatic Helicobacter species into the epithelium of the rat cecum (19). More recently, invasion into the lamina propria of the cecum by enterohepatic Helicobacter species in mice following challenge with the spirochete Serpulina hyodysenteriae has been reported (58). The significance of cell entry and/or tissue invasion by enterohepatic Helicobacter species and the conditions under which these events take place have not been fully elucidated. Tissue invasion may be a prerequisite for or a consequence of Helicobacter-associated disease in the gastrointestinal tract. It may also play a role in bacterial translocation to the liver and/or into systemic circulation, either as a primary event or secondary to other disease states. The fact that many investigators have encountered enterohepatic Helicobacter species with periplasmic fibers in the gastrointestinal tract of laboratory rodents no doubt reflects the frequency with which these animals are used in biomedical research. Bacteria with the same morphology have also been isolated from the gastrointestinal tract of many mammal species, including humans, monkeys, sheep, pigs, dogs, cats, mice, rats, hamsters, and gerbils. The complete range of host species from which these organisms can be isolated is not known. It may be that such bacteria can flourish wherever a mucus-rich interface between epithelial cells and the lumen of an alimentary tract is found. Certainly the observation of bacteria that appear morphologically indistinguishable from enterohepatic Helicobacter species in the hind-gut of Periplaneta americana, the American cockroach (4), suggests that the distribution of these microbes is wide indeed.
Enterohepatic Helicobacter species (EHS) have previously been found in adults with hepatobiliary diseases. Here, we report the prevalence of Helicobacter pylori and EHS in liver and gastric tissue in children and adolescents with chronic liver disease (CLD).
Seventy-seven consecutive children and adolescents with CLD with or without ulcerative colitis or Crohn's disease (UC/CD) were investigated. Tissue samples were analysed using a Helicobacter genus-specific 16S rDNA polymerase chain reaction (PCR) assay and DNA-sequence analysis. Sera from 61 subjects were also analysed using enzyme immunoassay and immunoblotting.
The Helicobacter PCR was positive in 3/23 (13%) livers from patients with primary sclerosing cholangitis and UC, and in 1/2 livers from patients with autoimmune hepatitis (AIH) and UC. Sequenced PCR products matched the 16S rDNA of H. hepaticus, H. muridarum, H. canis, and H. pylori, respectively. H. ganmani and H. bilis were detected in gastric tissues from two AIH patients. H. hepaticus and H. pullorum were found in livers from two patients with acute liver failure and intrahepatic cholestasis. Antibody reactivity to Helicobacter cell-surface proteins was negative.
H. pylori and EHS can be detected in the livers of some patients with UC and concomitant liver disease, as well as in other children with liver diseases. Multicentre studies from different locations are needed to find out whether these bacteria play a pathogenetic role or whether their presence is an epiphenomenon.
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.
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.
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.
Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.
Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria.
The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate.
HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus-antigen-based ELISA.
Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.
Primary biliary cirrhosis and primary sclerosing cholangitis are well recognized chronic cholestatic liver diseases that are considered to have an autoimmune basis. Recent progress in the study of autoimmune liver diseases has improved the recognition and characterization of these conditions. An important component of this progress has been the identification of liver disease-associated autoantibodies and their respective target antigens, and the development of specific assays for these autoantibodies. In addition, some nonhumoral immunological findings imply an involvement of specific immunopathogenic mechanisms in the development of these conditions. Furthermore, immunogenetic factors associated with increased susceptibility to some of these diseases have been identified. This article reviews the most relevant information relating to the postulated autoimmune pathogenesis of these diseases, with special emphasis on their associated humoral and cellular immunological abnormalities and immunopathogenetic factors. Some of the remaining important unresolved issues relating to the pathogenesis of these diseases, that need to be addressed in further research, are highlighted.
The etiopathogenesis of primary biliary cirrhosis (PBC) remains speculative. Epithelioid granulomas are often found in the vicinity of damaged interlobular bile ducts in PBC, raising the possibility of a reaction to microbial materials. In this study, we tried to detect and identify bacterial DNA within granulomatous lesions in PBC. Using liver sections from 9 patients with PBC and 13 control livers, granuloma in portal tracts, portal tracts without granuloma, and adjacent hepatic parenchyma were selectively microdissected from sections, and then DNA was extracted from them. First, part of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from DNA samples extracted from 5 PBC and 6 control livers, and their amplicons were sequenced for the identification of bacterial species. Several indigenous bacteria were identified. Among them, Propionibacterium acnes (P. acnes) was detected as a major clone in 20% to 50% of sequenced clones from granuloma of PBC, but the detection rate of P. acnes was 0% to 20% in those cloned from adjacent hepatic parenchyma of PBC. Then, a P. acnes-specific PCR was performed using all microdissected samples. Distinct PCR products were identified in epithelioid granuloma in all 9 PBC cases. The result that P. acnes DNA is present as a major clone in granulomas of PBC, suggest that P. acnes is involved in the pathogenesis of granuloma in PBC.
Helicobacter hepaticus causes disease in the liver and lower intestinal tract of mice. It is strongly urease positive, although it does not live
in an acidic environment. The H. hepaticus urease gene cluster was expressed in Escherichia coli with and without coexpression of the Helicobacter pylori nickel transporter NixA. As for H. pylori, it was difficult to obtain enzymatic activity from recombinant H. hepaticus urease; special conditions including NiCl2supplementation were required. The H. hepaticus urease cluster contains a homolog of each gene in the H. pyloriurease cluster, including the urea transporter gene ureI. Downstream genes were homologs of the nik nickel transport operon of E. coli. Nongastric H. hepaticusproduces urease similar to that of H. pylori.
Hepatocellular carcinoma (HCC) is a long-term consequence of chronic liver disease, whose aetiology could result from viral, environmental and hereditary causes. Viral infection, by itself, could only partially explain the pathogenesis of cirrhosis and HCC. A new aetiologic agent capable of inducing chronic active hepatitis and hepatocellular tumours was discovered: it is a bacterium belonging to the genus Helicobacter, and named H. hepaticus. Presence of sequences belonging to the 16S rRNA of Helicobacter species (spp.) has been demonstrated in liver of most patients with cirrhosis and HCC. H. pylori and related bacteria, such as H. hepaticus, produce toxins that kill hepatocyte by a granulating effect on liver cell lines. In vivo, such toxins might reach the liver through the portal tract, thereby causing hepatocellular damage. The recognition of Helicobacter spp. as a possible risk factor for cirrhosis and HCC might have a practical impact on the general population: the treatment of this infection is easy and far less expensive than liver transplantation or any long term treatment for the other risk factors of HCC. Any confirmation of the involvement of Helicobacter in liver disease would eventually come from the success of culturing the bacterium from liver tissues. Future research is needed to clarify the importance of Helicobacter spp. in respect to the other pathogens already known as causative agents of chronic inflammation of the liver and its long term sequelae, namely cirrhosis and HCC.
The number of species in the genus Helicobacter has rapidly expanded over the past decade. The genus now includes at least 24 formally named species as well as numerous other helicobacters awaiting formal naming. This review highlights the expanding role that other helicobacters, although not as well known as H pylori, play in gastrointestinal and systemic disease in humans.
Bile-tolerant Helicobacter spp. are emerging human and animal pathogens. However, due to their fastidious nature, which requires nutrient-rich complex media to grow, infection with these bacteria may be underestimated. The accumulation of toxic metabolites in cultures may be one of the main obstacles for successful culture of these organisms. The present study examined various potential growth-enhancing substances for Helicobacter spp. and, furthermore, how they may affect spiral to coccoid conversion. Five Helicobacter spp. were cultured on agar and in broth media supplemented with activated charcoal, beta-cyclodextrin, or porcine gastric mucin. Growth was determined by estimating the numbers of colony-forming units and colony diameter, as well as bacterial cell mass. Coccoid transformation was estimated every 24 h by both Gram and acridine-orange staining. Activated charcoal was superior in supporting growth and increased cell mass on agar and in broth media. beta-Cyclodextrin delayed spiral to coccoid conversion by Helicobacter pylori and Helicobacter canis, whereas activated charcoal delayed the conversion to coccoid forms of Helicobacter hepaticus and Helicobacter bilis. The progression to coccoid forms by Helicobacter pullorum on agar media was not influenced by any growth supplement. The spiral to coccoid conversion was more rapid in broth media than on agar media. The growth enhancement observed is probably related to the capacity of activated charcoal to remove toxic compounds in culture media.
The biliary tract is normally sterile, but bile-tolerant bacteria are frequently isolated from patients with cholecystitis. Since the identification of about 25 Helicobacter species, some of which may grow in bile, studies have addressed the role of these organisms in primary biliary cirrhosis, primary sclerosing cholangitis, and cholelithiasis. Most of these bacteria show the presence of Helicobacter DNA or antigens in the bile tract and in liver samples. Altogether, data from studies on biliary and hepatic diseases, as well as pancreatic disorders, suggest that bile-tolerant Helicobacter species may induce a chronic infection with possible malignant transformation.
Helicobacter pylori is an established cause of gastritis and has been implicated in extradigestive diseases.
To investigate the role of H. pylori in patients with unexplained refractory pruritus.
Ten patients with severe pruritus unresponsive to conventional therapy were evaluated for active H. pylori infection by H. pylori serology followed by either esophagogastroduodenoscopy (EGD) or urea breath test. Of the 10 patients, 8 were found to have active infection. All 10 received anti-H. pylori antibiotic therapy and were reevaluated for relief of pruritus.
Of 8 patients with active H. pylori infection, 87.5% (7/8) had some type of pruritus relief after triple therapy. Of these, 62.5% (5/8) had complete relief and 25% (2/8) had temporary relief of pruritus. The remaining 12.5% (1/8) did not respond. Two control patients without active H. pylori infection had no relief of pruritus with therapy.
We have identified a population of patients with refractory pruritus and active H. pylori infection whose pruritus resolved after eradication of H. pylori.
The ecological niches occupied by various species of Helicobacter are not yet known and the full spectrum of diseases associated with Helicobacter infections are not yet defined. Since these fastidious microaerofilic bacteria require special growth conditions new and improved molecular and serologic diagnostic methods have been developed to increase our understanding of their pathogenesis and virulence characteristics. Immunogenic cell surface proteins of Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus were characterised by proteomic techniques using two-dimensional electrophoresis and immunoblotting with antisera from immunised rabbits. Cross-reactivity between the three Helicobacter species were analysed after a four-step cross-absorption experiment. For H. pullorum, H. bilis and H. hepaticus 21, 13 and 27 specific immunogenic proteins, respectively, were identified. These proteins could be of important sero-diagnostic value for analyses of sera from humans, laboratory animals and for the veterinarian field.
Bile-tolerant Helicobacter species such as Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD)
in humans. Antibody responses to cell surface proteins of H. pullorum, H. bilis, and H. hepaticus in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using
enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to Helicobacter pylori. For analysis of a possible cross-reactivity between bile-tolerant Helicobacter species and H. pylori, sera from a subpopulation of each group were absorbed with a whole-cell extract of H. pylori and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for H. pullorum was significantly higher in patients with CLD than in healthy blood donors (P = 0.01). Antibody reactivity to cell surface protein of H. hepaticus was also significantly higher in the CLD patients than in the healthy blood donors and the population group (P = 0.005 and P = 0.002, respectively). Following the absorption, antibody responses to H. pullorum decreased significantly in all three groups (P = 0.0001 for CLD patients, P = 0.0005 for the population group, and P < 0.0001 for the blood donors), indicating that cross-reactivity between H. pylori and other Helicobacter spp. occurs. The antibody responses to H. hepaticus and H. bilis in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance
of this finding requires further investigations.
In this study stool samples from dyspeptic patients and healthy subjects were used for detection of specific Helicobacter pylori antigens and DNA by immunoenzymatic test (PPHpSA) and semi-nested PCR (ureA-PCR), respectively. The H. pylori status was estimated by invasive endoscopy-based rapid urease test and histology or noninvasive urea breath test (UBT), and by serology (ELISA, Western blot). The coincidence of H. pylori-negative invasive tests or UBT and negative antigen or DNA stool tests was very high (mean 95%). The PPHpSA results were found positive for 56% and ureA-PCR for 26% of individuals with H. pylori infection confirmed by invasive tests or UBT. The detection of specific H. pylori antigens and especially DNA in feces is not sufficient as a one-step diagnosis of H. pylori infection.
Helicobacter has been identified in isolated cases of hepato-biliary diseases, but its role in the pathogenesis of these conditions remains unclear.
To determine whether Helicobacter could be detected in bile obtained at endoscopic retrograde cholangiopancreatography, and to evaluate the prevalence of this infection in patients with hepato-biliary diseases.
Bile was collected from 125 patients with various hepato-biliary diseases undergoing endoscopic retrograde cholangiopancreatography. Among them, 75 were diagnosed with biliary stones, 15 with pancreatico-biliary malignancies and four with primary sclerosing cholangitis. The detection of Helicobacter in DNA extracted from these bile samples was performed using Helicobacter genus-specific primers (capable of detecting 100-1000 organisms/mL).
Helicobacter was detected in all positive controls. Only three samples had polymerase chain reaction inhibitors. All remaining bile samples (122 patients with hepato-biliary diseases) were negative for Helicobacter DNA.
Helicobacter can be detected in bile samples using polymerase chain reaction. This infection, however, was not present in any of our patients diagnosed with gallstones or hepato-biliary malignancies, raising doubt as to the possible association between Helicobacter and these entities. Given the low sample size of patients with primary sclerosing cholangitis, more studies are required to determine whether an association exists with this condition.
Infection with Helicobacter pylori is a major risk factor for the development of atrophic gastritis and gastric cancer. H. pylori strains can differ with respect to the presence of cagA (cytotoxin-associated gene A), a gene encoding a high-molecular-weight immunodominant antigen. H. pylori strains possessing cagA have been associated with enhanced induction of acute gastric inflammation.
We investigated the relationship between cagA status and the development of atrophic gastritis in a cohort of subjects infected with H. pylori.
Gastrointestinal endoscopy with biopsy sampling was used to study the natural history of gastritis in 58 subjects infected with H. pylori. Biopsy specimens were obtained before and after a mean follow-up period of 11.5 years (range, 10-13 years). The cagA status of each individual was determined at the follow-up visit with the use of an enzyme-linked immunosorbent assay designed to detect the presence of serum immunoglobulin G directed against the CagA protein. Two-sided Fisher's exact tests, McNemar's tests, Student's t tests, and Wilcoxon sum rank tests were used to analyze the data.
Twenty-four (41%) of the 58 evaluated subjects had serum antibodies against CagA (i.e., they were cagA positive), and 34 subjects were cagA negative. At the initial visit, moderate to severe atrophic gastritis was observed in eight (33%) of the cagA-positive subjects and in six (18%) of the cagA-negative subjects. At that time, positive cagA status and gastric atrophy were not significantly related (P = .22; Fisher's exact test; odds ratio [OR] 2.33; 95% confidence interval [CI] = 0.58-9.65). During follow-up, 16 (36%) of the 44 initially atrophy-negative subjects developed atrophic gastritis (eight [50%] of 16 cagA-positive subjects versus eight [29%] of 28 cagA-negative subjects; P = .20, Fisher's exact test; relative risk [RR] = 1.75; 95% CI = 0.82-3.76). In six of these 16 subjects (five cagA positive versus one cagA negative), atrophic gastritis was accompanied by the development of intestinal metaplasia (i.e., a change in the type of specialized cells present) (P = .02; Fisher's exact test; RR = 9.06; 95% CI = 1.16-71.0). One of the initially atrophy-negative, cagA-positive subjects developed early gastric cancer. Four (29%) of the 14 subjects initially diagnosed with atrophic gastritis showed regression of atrophy during follow-up (one cagA positive and three cagA negative). Therefore, at the end of follow-up, 15 (62%) of the 24 cagA-positive subjects had atrophic gastritis compared with 11 (32%) of the 34 cagA-negative subjects (P = .02; Fisher's exact test; OR = 3.48; 95% CI = 1.02-12.18).
Infection with cagA-positive H. pylori strains is associated with an increased risk for the eventual development of atrophic gastritis and intestinal metaplasia.
Several inbred strains of mice in closed breeding colonies were found to have spiral-shaped bacteria associated with active, chronic hepatitis. A new species of Helicobacter, H. hepaticus, was isolated from the infected livers of some strains of mice. Other strains of mice were colonised with H. hepaticus in the caecum and colon, but not the liver. Filtersterilised supernatant fluid from five strains of H. hepaticus was tested in a mouse liver cell line (ATCC no. CCL 9.1) for cytotoxic activity. All strains produced a toxic factor causing morphological changes in the cells at dilutions up to 1 in 1000. Toxicity was observed after exposure to the supernatant fluid for 48-72 h. Other Helicobacter spp. that also produced the cytopathic effect (CPE) in the liver cell line were H. felis, H. acinonyx, H. pylori and one strain of H. mustelae. "Helicobacter rappini" and H. muridarum did not cause CPE in the liver cells. The soluble factor was stable at 4 degrees C for up to 3 months. It was also stable at 56 degrees C for 30 min, but was inactivated by boiling for 15 min. It was inactivated by incubation with trypsin. A partially purified preparation of the cytotoxin had a mol. wt of c. 100,000 and did not have urease activity. The cytotoxin produced by H. hepaticus did not cause vacuole formation in HeLa cells.
To determine whether infection with a Helicobacter pylori strain possessing cagA is associated with an increased risk of development of adenocarcinoma of the stomach, we used a nested case-control study based on a cohort of 5443 Japanese-American men in Oahu, Hawaii, who had a physical examination and a phlebotomy during 1967 to 1970. We matched 103 H. pylori-infected men who developed gastric cancer during a 21-year surveillence period with 103 H. pylori-infected men who did not develop gastric cancer and tested stored serum specimens from patients and controls for the presence of serum IgG to the cagA product of H. pylori using an ELISA. The serum IgG assay using a recombinant CagA fragment had a sensitivity of 94.4% and a specificity of 92.5% when used in a clinically defined population; serological results were stable for more than 7 years. For men with antibodies to CagA, the odds ratio of developing gastric cancer was 1.9 (95% confidence interval, 0.9-4.0); for intestinal type cancer of the distal stomach, the odds ratio was 2.3 (95% confidence interval, 1.0-5.2). Age < 72 years and advanced tumor stage at diagnosis were significantly associated with CagA seropositivity. We conclude that infection with a cagA-positive H. pylori strain in comparison with a cagA-negative strain somewhat increases the risk for development of gastric cancer, especially intestinal type affecting the distal stomach.
Male A/JCr mice with naturally occurring Helicobacter hepaticus infection develop a progressive chronic active hepatitis and liver tumors, despite the presence of serum antibodies to Helicobacter proteins. A rabbit antiserum prepared against the bacterial proteins immunoreacted with hepatocytes present in liver sections from infected mice with progressive lesions. We found that sera from these mice contained IgG antibodies that reacted in immunoblots with recombinant heat shock protein 70 (DmaK from Escherichia coli) but not with heat shock protein 60 (GroEL) or heat shock protein 10 (GroES). A rabbit antibody to heat shock protein 70 reacted with H. hepaticus in tissue sections and to a H. hepaticus protein (70 kd) in Western blots. Immunohistochemistry and in situ hybridization for heat shock protein 70 revealed that individual hepatocytes and other cells expressed the protein in livers with hepatitis but not usually in normal livers. Liver tumors and preneoplastic lesions in infected mice did not usually express heat shock protein 70 except focally in a few tumors. In situ hybridization for H. hepaticus 16S rRNA showed that the bacteria was found throughout the liver associated with hepatitis but not within tumors. CD3+ T lymphocytes were found in close association with hepatic lesions. These data suggest a role for autoimmunity in progressive hepatitis and carcinogenesis in livers infected with H. hepaticus.
Helicobacter hepaticus causes hepatitis in selected strains of mice and in A/JCr mice is linked to liver cancer. To analyze whether H. hepaticus persists in specified ecological niches, to determine whether biomarkers of infection exist, and to analyze the influence of H. hepaticus on hepatocyte proliferation, a longitudinal study of H. hepaticus-infected A/JCr mice was undertaken. A/JCr mice were serially euthanatized from 3 through 18 months and surveyed by enzyme-linked immunosorbent assay; bacterial culture of liver, colon, and cecum; histology; electron microscopy; hepatocyte proliferation indices determined by using 5-bromo-2'-deoxyuridine; and measurement of the liver enzyme alanine aminotransferase. In infected animals throughout the 18-month study, H. hepaticus was consistently isolated from the lower bowel but only sporadically from the liver. By electron microscopy, H. hepaticus was noted infrequently and only in bile canaliculi. Infected mice, particularly males, showed chronic inflammation; oval cell, Kupffer cell, and Ito cell hyperplasia; hepatocytomegaly; and bile duct proliferation. The inflammatory and necrotizing lesion was progressive and involved the hepatic parenchyma, portal triads, and intralobular venules. Hepatic adenomas were noted only in male mice, whereas 5-bromo-2'-deoxyuridine proliferation indices were markedly increased in both sexes, but especially in males, compared to control A/J mice. Infected mice also developed sustained anti-H. hepaticus serum immunoglobulin G antibody responses and elevated alanine aminotransferase levels. H. hepaticus, which persists in the lower bowels and livers of A/JCr mice, is associated with a chronic proliferative hepatitis, and hepatomas in selected male mice indicate that this novel bacterium may cause an increased risk of hepatic cancer induction in susceptible strains of mice. This murine model should prove useful in dissecting the molecular events operable in the development of neoplasms induced by bacteria belonging to this expanding genera of pathogenic Helicobacter species.
BACKGROUND/AIMS--The course of primary sclerosing cholangitis (PSC) is highly variable and unpredictable. This study describes the natural history and outcome of PSC. These data were used to construct a prognostic model for patients with PSC. METHODS--A total of 305 Swedish patients with PSC were studied. The median follow up time was 63 (1-194) months and all patients could be traced for follow up. Some 79 patients died or had a liver transplant. The prognostic significance of clinical, biochemical, and histological findings at the time of diagnosis were evaluated using multivariate analysis. RESULTS--The estimated median survival from time of diagnosis to death or liver transplantation was 12 years. Cholangiocarcinoma was found in 24 (8%) of the patients and 134 (44%) of the patients were asymptomatic at the time of diagnosis. The estimated survival rate was significantly higher in the asymptomatic group (p < 0.001). However, 29 (22%) of the asymptomatic patients became symptomatic during the study period. It was found that age, serum bilirubin concentration, and histological stage at the time of diagnosis were independent predictors of a bad prognosis. These variables were used to construct a prognostic model. CONCLUSIONS--This prognostic model developed from a large homogeneous population of PSC patients should be of value for the timing of transplantation and patient counselling in PSC.
On the basis of biochemical, phenotypic, and 16S rRNA analysis, a novel gram-negative bacterium, isolated from normal and diarrheic dogs as well as humans with gastroenteritis, has been recently named Helicobacter canis. A 2-month-old female crossbred puppy was submitted to necropsy with a history of weakness and vomiting for several hours prior to death. The liver had multiple and slightly irregular yellowish foci up to 1.5 cm in diameter. Histologically, the liver parenchyma contained randomly distributed, occasionally coalescing hepatocellular necrosis, often accompanied by large numbers of mononuclear cells and neutrophils. Sections of liver stained by the Warthin-Starry silver impregnation technique revealed spiral- to curve-shaped bacteria predominantly located in bile canaliculi and occasionally in bile ducts. Aerobic culture of liver was negative, whereas small colonies were noted on Campylobacter selective media after 5 days of microaerobic incubation. The bacteria were gram negative and oxidase positive but catalase, urease, and indoxyl acetate negative; nitrate was not reduced to nitrite, and the organism did not hydrolyze hippurate. The bacteria were also resistant to 1.5% bile. Electron microscopy revealed spiral-shaped bacteria with bipolar sheathed flagella. By 16S rRNA analysis, the organism was determined to be H. canis. This is the first observation of H. canis in active hepatitis in a dog and correlates with recent findings of Helicobacter hepaticus- and Helicobacter bilis-related hepatic disease in mice. Further studies are clearly warranted to ascertain whether H. canis-associated hepatitis is more widespread in canines as well as a cause of previously classified idiopathic liver disease in humans.
A filamentous, gram-negative, motile bacterium with a single polar sheathed flagellum was isolated from gallbladders of hamsters with cholangiofibrosis and centrilobular pancreatitis. Bacteria grew under microaerophilic conditions at 37 and 42 degrees C, were oxidase, catalase, arginine aminopeptidase, and L-arginine arylamidase positive, reduced nitrate to nitrite, were resistant to cephalothin, and exhibited intermediate susceptibility to nalidixic acid. Sequence analysis of the 16S rRNA gene indicated that the bacterium was a novel member of the Helicobacter genus, most closely related to Helicobacter pametensis. We propose to name this bacterium Helicobacter cholecystus. In epidemiologic studies, isolation of H. cholecystus correlated strongly with the presence of cholangiofibrosis and centrilobular pancreatitis; however, further studies are needed to define the role of this bacterium in pathogenesis.
During a study of the prevalence and distribution of gastric helicobacters in domestic pets, a novel group of Helicobacter-like organisms were identified. These "Helicobacter group 2" strains were initially distinguished from the species Helicobacter felis and Helicobacter bizzozeronii by their cellular morphology and the type of motility exhibited. Bacterial cells were only slightly spiral, 5 to 7 microns long, and 0.8 to 1.2 microns wide and showed an unusual slow wavelike motion. Each cell had tufts of sheathed flagella at one or both ends. Phylogenetic analysis by 16S ribosomal DNA sequence comparison revealed that H. felis, H. bizzozeronii, "Gastrospirillum hominis" 2, and the new group of helicobacters formed a distinct cluster with intraspecies similarity values of more than 98%. These taxa were clearly separated from all other known Helicobacter species. Dot blot DNA-DNA hybridization studies indicated that the Helicobacter group 2 strains are genetically homogeneous and distinct from other canine and feline gastric helicobacters. Quantitative DNA-DNA hybridization experiments showed that Helicobacter group 2 strains exhibit > 90% DNA homology to each other, but < 39% homology to the phylogenetically related taxa H. felis and H. bizzozeronii. We propose the name Helicobacter salomonis for the novel Helicobacter group 2 strains. The type strain is H. salomonis Inkinen (= CCUG 37845).
Whilst the mechanism by which Helicobacter pylori causes different gastroduodenal diseases is uncertain, strains producing the cytotoxin-associated protein (CagA) have greater pathogenicity. Hsps are immunogenic molecules induced by inflammatory mediators. The aim of this study was to assess pathogenicity of hsp antibodies in H. pylori-infected patients. ELISA techniques were used to assay sera of H. pylori-positive patients with gastritis, gastric atrophy, duodenal or gastric ulcer, and H. pylori-negative controls, for antibodies to CagA and to human, mycobacterial, and in 20 sera, H. pylori (hspB) 60-kD hsp. IgA antibodies to mycobacterial hsp60 in atrophy patients were elevated compared with patients with gastritis (P < 0.05) and with H. pylori-negative controls (P < 0.0005). IgA antibodies to human hsp60 in gastric atrophy patients were elevated compared with H. pylori-negative controls (P < 0.05). Patients with atrophy (P < 0.0005) and gastritis (P < 0.05) who were CagA-positive had raised titres of anti-mycobacterial hsp60 IgA antibodies compared with controls. IgA antibody levels to hspB were positively correlated with those to mycobacterial hsp60 (mhsp60) (P < 0.05) and human hsp60 (hhsp60) (P < 0.005). IgA antibodies to hsp60 are associated with gastroduodenal disease, particularly gastric atrophy, in H. pylori-infected patients. Increased humoral responses to hsp60 could either contribute to gastric atrophy or result from greater gastric mucosal damage induced by CagA-positive strains of H. pylori.
Cancer of the gallbladder is the number one cause of cancer mortality in Chilean women. Incidence rates for this tumor vary widely on a worldwide basis, being approximately 30 times higher in high-risk than in low-risk populations, suggesting that environmental factors such as infectious microorganisms, carcinogens, and nutrition play a role in its pathogenesis. Because several Helicobacter sp. colonize the livers of animals and induce hepatitis, the aim of this study was to determine whether Helicobacter infection was associated with cholecystitis in humans.
Bile or resected gallbladder tissue from 46 Chileans with chronic cholecystitis undergoing cholecystectomy were cultured for Helicobacter sp. and subjected to polymerase chain reaction (PCR) analysis using Helicobacter-specific 16S ribosomal RNA primers.
Recovery of Helicobacter sp. from frozen specimens was unsuccessful. However, by PCR analysis, 13 of 23 bile samples and 9 of 23 gallbladder tissues were positive for Helicobacter. Eight of the Helicobacter-specific PCR amplicons were sequenced and subjected to phylogenetic analysis. Five sequences represented strains of H. bilis, two strains of "Flexispira rappini" (ATCC 49317), and one strain of H. pullorum.
These data support an association of bile-resistant Helicobacter sp. with gallbladder disease. Further studies are needed to ascertain whether similar Helicobacter sp. play a causative role in the development of gallbladder cancer.
Background:
In the autumn of 1992, a novel form of chronic, active hepatitis of unknown etiology was discovered in mice at the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, Md. A high incidence of hepatocellular tumors occurred in affected animals. The disease entity was originally identified in A/JCr mice that were untreated controls in a long-term toxicologic study.
Purpose:
Our original purpose was to determine the origin and etiology of the chronic hepatitis and to quantify its association with hepatocellular tumors in mice of low liver tumor incidence strains. After a helical microorganism was discovered in hepatic parenchyma of diseased mice, we undertook characterization of the organism and investigation of its relationship to the disease process.
Methods:
Hepatic histopathology of many strains of mice and rats, as well as guinea pigs and Syrian hamsters, in our research and animal production facilities was reviewed. Steiner's modification of the Warthin-Starry stain and transmission electron microscopy were used to identify bacteria in the liver. We transmitted the hepatitis with liver suspensions from affected mice and by inoculation with bacterial cultures. Bacteria were cultivated on blood agar plates maintained under anaerobic or microaerophilic conditions and characterized morphologically, biochemically, and by 16S rRNA sequence.
Results:
We report here the isolation of a new species of Helicobacter (provisionally designated Helicobacter hepaticus sp. nov.) that selectively and persistently colonizes the hepatic bile canaliculi of mice (and possibly the intrahepatic biliary system and large bowel), causing a morphologically distinctive pattern of chronic, active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals.
Conclusions:
The novel Helicobacter is a likely candidate for the etiology of hepatocellular tumors in our mice. The Helicobacter-associated chronic active hepatitis represents a new model to study mechanisms of carcinogenesis by this genus of bacteria.
Implications:
Adenocarcinoma of the stomach, the second most prevalent of all human malignancies world-wide, is associated with infection at an early age with Helicobacter pylori. Infection leads to several distinctive forms of gastritis, including chronic atrophic gastritis, which is a precursor of adenocarcinoma. H. hepaticus infection in mice constitutes the only other parallel association between a persistent bacterial infection and tumor development known to exist naturally. Study of the H. hepaticus syndrome of chronic active hepatitis and liver tumors in mice may yield insights into the role of H. pylori in human stomach cancer and gastric lymphoma.
Cell surface proteins of Helicobacter pylori were solubilized by extraction with acidic glycine buffer, N-octyl-glucoside, lithium chloride, and distilled water, and by sonication. The preparations were evaluated as antigens in ELISA to detect serum IgG responses in patients and healthy subjects. SDS-PAGE analyses of the preparations from a type strain (NCTC 11637) and of acidic glycine extracts of 4 clinical isolates showed multiple protein bands. The sera were classified as HP+ve and HP-ve by culture of biopsy and immunoblotting. Sera were considered positive for H. pylori if they detected the specific 120kD antigen or 4-5 other bands. 49 sera were HP+ve; the 51 HP-ve sera did not react in immunoblotting. 35/44 sera (80%) that reacted with the 120kD antigen demonstrated high titers in ELISA with all antigen preparations, and the remaining 9(20%) sera gave discordant results. 4/5 HP+ve sera that did not react with the 120kD antigen, demonstrated high ELISA titers with all 5 antigen preparations. Glycine extracts of 3 isolates did not exhibit the 120kD protein, but were equally sensitive in ELISA. The role of 120kD antigen in our ELISA was not clear. Immunoblotting demonstrated that the 5 antigen preparations share similar antigenic components. All preparations were similarly high in sensitivity and specificity, indicating that surface antigens could be satisfactorily used in our ELISA. Our ELISA using the glycine extract was compared with commercial H. pylori ELISAs developed by Bio-Rad Laboratories, USA (GAP ELISA), Roche, Switzerland (EIA 2G), and Whittaker Bioproducts, USA (Pyloristat).(ABSTRACT TRUNCATED AT 250 WORDS)
Serum IgG antibodies of Helicobacter pylori were detected in single-dilution ELISA using glycine extracted material. Among 148 endoscopy patients 59% displayed antibodies; as expected, a higher occurrence (90%) was found in patients with positive gastric culture for H. pylori than in culture negative patients (37%). Among 68 blood donors the frequency of H. pylori antibodies was 28%. In 73 children less than 15 years of age examined for unrelated disorders the occurrence was 4%. By immunoblotting using the same extract, 3 prominent bands, 29K, 54K and 60K and several weak bands were identified. These were formed by 57%, 92%, and 65%, respectively, of the ELISA positive patient sera. Comparing culture positive and negative patients, the 3 bands occurred more often among the culture positive subjects though between 18 and 61% of the sera from culture negative patients gave either of the bands. When comparing the glycine extracts of 4 different H. pylori strains with separate haemagglutinating patterns no differences in the position of the major bands emerged. By absorption experiments no immunological cross-reactivity with components of Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni or C. fetus was found. Thus, the glycine extract seemed specific for the detection of antibodies to H. pylori.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The existence of Helicobacter pylori in the biliary tract was investigated. Seven bile samples were included in this study. Among them, six bile samples were collected by percutaneous transhepatic cholangiodrainage and the other by needle aspiration during cholecystectomy. Using nested PCR with two sets of primers homologous to the urease A gene, Helicobacter pylori DNA was detected. Three samples, one from a patient with advanced gastric cancer involving the pancreatic head and two from patients with pancreatic head tumor, were found to be positive for Helicobacter pylori DNA. On the other hand, three samples from patients with cholangiocarcinoma and one from a patient with chronic cholecystitis were all negative. To further verify the specificity of our PCR analysis, partial sequences of the PCR products from the three positive samples were analyzed by direct sequencing. Several silent mutations and a missense mutation (AAA to AGA; Lys-164 to Arg-164) were identified in the urease A gene. We conclude that Helicobacter pylori DNA can be easily detected in the bile samples. The possibility of asymptomatic cholangitis caused by this organism requires further investigation.
An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.
Enhanced serum IgA concentrations are common in alcoholic liver cirrhosis, but functional differences between IgA subclasses and their relation with interleukin-6 (IL-6) have not been described. Distinct immunoregulatory mechanisms may exist that selectively affect one subclass. This possibility prompted us to investigate the distribution of IgA1 and IgA2 subclasses in the serum of 25 heavy alcohol drinkers (alcohol: 80 to 200 g per day) without clinical disorders, in comparison with 35 patients affected by alcoholic liver cirrhosis, 29 viral hepatitis patients and 33 social drinkers as a control group. Mean (+/- SD) IgA2 concentration (0.56 +/- 0.31 g/l) was significantly increased (p < 0.01) in heavy alcohol drinkers, with an IgA2/IgA1 ratio of 0.33 +/- 0.12, while the mean total IgA concentration was similar to the control group. Mean IgA1 and IgA2 concentrations were significantly increased (p < 0.001) in alcoholic liver cirrhosis patients (6.13 +/- 4.52 g/l and 1.83 +/- 1.93 g/l respectively, with an IgA2/IgA1 ratio of 0.32 +/- 0.19) and viral hepatitis patients (3.66 +/- 2.59 g/l and 0.69 +/- 0.67 g/l respectively, with an IgA2/IgA1 ratio of 0.21 +/- 0.14) High serum IL-6 concentrations (34 +/- 33 ng/l) were correlated with elevated IgA1 and IgA2 concentrations only in patients with alcoholic liver cirrhosis. IgA2 subclass and IgA2/IgA1 ratio could therefore be used as markers of chronic alcohol abuse directly related to the extent and duration of the alcohol abuse and the effectiveness of alcohol withdrawal.
As a result of phylogenic studies using new molecular biology techniques and fundamental experimental studies, we now know more about helicobacteria in domestic carnivores, their morphologic characteristics, their taxonomia and more important we know more about their ecological niche. Few clinical studies have been carried out, but the ones that have been undertaken are interesting in that they confirm the extensive prevalence of Helicobacter infections in domestic carnivores and underline their role in the genesis of the inflammatory gastropathies observed in these species. Finally, recent observations have demonstrated the ubiquitous character of these helicobacteria by showing their presence in the stomach of man, dog and cat. This ubiquitous character has led some scientists to consider the potential zoonotic risk of the human infection by Helicobacter heilmannii, felis or pylori.
In 153 consecutive patients with cirrhosis we assessed: (1) the prevalence of IgG to Helicobacter pylori and compared it with that found in 1010 blood donors resident in the same area; and (2) the relationships of IgG to Helicobacter pylori with clinical and endoscopic features and with the risk of peptic ulcer. The IgG to Helicobacter pylori prevalence of cirrhotics was significantly higher than in blood donors (76.5% vs 41.8%; P < 0.0005) and was not associated with sex, cirrhosis etiology, Child class, gammaglobulins and hypertensive gastropathy. In both groups, the prevalence of IgG to Helicobacter pylori was significantly higher in subjects over 40. Among patients with cirrhosis a significantly higher prevalence of Helicobacter pylori was found in patients with previous hospital admission (P = 0.02) and/or upper gastrointestinal endoscopy (P = 0.01) and patients with peptic ulcer (P = 0.0004). Multivariate analysis identified increasing age and male sex as risk factors for a positive Helicobacter pylori serology and no independent risk factors for peptic ulcer. The high prevalence of Helicobacter pylori-positive serology found in the present series is related to age and sex and might also be explained by previous hospital admissions and/or upper gastrointestinal endoscopy. Our results do not confirm the role of Helicobacter pylori as risk factor for peptic ulcer in patients with liver cirrhosis.
Laboratory mice, rats, and rabbits may harbor a variety of viral, bacterial, parasitic, and fungal agents. Frequently, these organisms cause no overt signs of disease. However, many of the natural pathogens of these laboratory animals may alter host physiology, rendering the host unsuitable for many experimental uses. While the number and prevalence of these pathogens have declined considerably, many still turn up in laboratory animals and represent unwanted variables in research. Investigators using mice, rats, and rabbits in biomedical experimentation should be aware of the profound effects that many of these agents can have on research.
An increased frequency of peptic ulcer disease is noted in patients with cirrhosis, but the role of H. pylori in this disorder remains to be determined. The diagnosis of cirrhosis was confirmed by a combination of clinical, biochemical, radiological, and histological methods. The severity of cirrhosis was assessed by Pugh's modification of Child's criteria. Upper gastrointestinal endoscopy was performed consecutively to evaluate the presence of varices and gastroduodenal mucosa. H. pylori status was assessed by histology, urease test, and serology. In all, 130 patients with cirrhosis were recruited into the study; there were 86 males and 44 females with a mean (SD) age of 54.4 (12.7) years. The H. pylori prevalence was 76.2%. There was no difference in age between the H. pylori-positive and -negative cirrhotics (P = 0.29). The H. pylori prevalence revealed no difference among cirrhotics with Child A (77.8%), Child B (72.9%), and Child C (78.6%) (P = 0.8), and neither was there a difference in H. pylori prevalence in cirrhotics with and without congestive gastropathy (77% vs 73.7%, P = 0.84). The prevalence of H. pylori in cirrhotics with and without varices did not show a statistical difference (75% vs 81.8%, P = 0.68). There also was no difference in the H. pylori prevalence between cirrhotic patients with and without peptic ulcers (84.4% vs 69.7%, P = 0.09). In conclusion, the prevalence of H. pylori or peptic ulcer is independent of the severity of cirrhotic liver disease. The association between H. pylori infection and peptic ulcer disease is weak in cirrhosis.
Whilst the mechanism by which Helicobacter pylori causes different gastroduodenal diseases is uncertain, strains producing the cytotoxin-associated protein (CagA) have greater pathogenicity. Hsps are immunogenic molecules induced by inflammatory mediators. The aim of this study was to assess pathogenicity of hsp antibodies in H. pylori-infected patients. ELISA techniques were used to assay sera of H. pylori-positive patients with gastritis, gastric atrophy, duodenal or gastric ulcer, and H. pylori-negative controls, for antibodies to CagA and to human, mycobacterial, and in 20 sera, H. pylori (hspB) 60-kD hsp. IgA antibodies to mycobacterial hsp60 in atrophy patients were elevated compared with patients with gastritis (P < 0.05) and with H. pylori-negative controls (P < 0.0005). IgA antibodies to human hsp60 in gastric atrophy patients were elevated compared with H. pylori-negative controls (P < 0.05). Patients with atrophy (P < 0.0005) and gastritis (P < 0.05) who were CagA-positive had raised titres of anti-mycobacterial hsp60 IgA antibodies compared with controls. IgA antibody levels to hspB were positively correlated with those to mycobacterial hsp60 (mhsp60) (P < 0.05) and human hsp60 (hhsp60) (P < 0.005). IgA antibodies to hsp60 are associated with gastroduodenal disease, particularly gastric atrophy, in H. pylori-infected patients. Increased humoral responses to hsp60 could either contribute to gastric atrophy or result from greater gastric mucosal damage induced by CagA-positive strains of H. pylori.
Although hepatitis C virus (HCV) infection is recognized as an important causative factor in the development of liver cirrhosis and hepatocellular cancer (HCC), the strength of this correlation has been difficult to confirm in low-prevalence areas.
Stored serum samples from 987 consecutive (1978-88) patients with chronic liver disease were tested with an enzyme-linked immunosorbent assay for anti-HCV and further confirmed by immunoblot. To evaluate the long-term outcome, the cohort was followed up until 1995, for a median observation time of 10 years.
Anti-HCV, confirmed by immunoblot, was found in 9.5% (94 of 987) of the patients, and at inclusion most patients were asymptomatic irrespective of anti-HCV status. Of the 445 patients who died during the study period, 44 were HCV-positive. A liver-related cause of death was far commoner and the age-adjusted survival shorter among HCV-positive patients than among HCV-negative ones. At death 68% (30 of 44) of the HCV-positive subgroup had developed cirrhosis, and 30% (13 of 44) had concurrent HCC, as compared with 36% (142 of 393) (P = 0.001) and 8% (31 of 393) (P = 0.001), respectively, of the HCV-negative subgroup. HCV infection (P < 0.001), alcohol abuse (P < 0.001), and immigrant status (P = 0.045) were independent factors with regard to the development of cirrhosis, whereas HCV infection (P = 0.040) and immigrant status (P = 0.012) were independent factors with regard to HCC.
HCV infection is common among patients with chronic liver disease, even when clinical evidence of viral infection is sparse, and constitutes a significant cause of death even in a low-prevalence area.
identified in bile gallbladder tissue from Chileans with chronic cholecystitis
identified in bile gallbladder tissue from Chileans
with chronic cholecystitis. Gastroenterology 1998;114:755–
63.
of the head of bacteriophage T4
of the head of bacteriophage T4. Nature
1970;227:680–5.