Myo9B is associated with an increased risk of Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND: Reflux esophagitis (RE) and Barrett's esophagus (BE) are predisposing factors for development of esophageal adenocarcinoma (EAC), the solid tumor with the fastest rising incidence in the Western world. This RE-BE-EAC cascade involves multiple host factors and consequently multiple genes. Polymorphisms in the 3' region of myosin IXB (Myo9B) are associated with chronic inflammatory gastrointestinal disorders like celiac disease and ulcerative colitis, assuming that variation in Myo9B influences the intestinal permeability. AIM: To determine esophageal expression and the genetic variation of the Myo9B gene in the RE-BE-EAC cascade. METHODS: DNA from 886 Caucasian participants (198 non-reflux controls, 305 RE, 254 BE, 129 EAC) was collected for the determination of the Myo9B gene polymorphism (rs2305764). Esophageal Myo9B expression was determined on biopsies from normal, RE, BE and EAC epithelium. RESULTS: Genotype G/G was more common in BE (p = 0.032) and EAC (p = 0.046), but not in RE (p = 0.126) compared with the control group. Cytoplasmic Myo9B expression was determined in RE, BE and EAC, but most prominent in epithelial cells of BE and EAC. CONCLUSIONS: Genetic variation of Myo9B may play a role in the etiology of BE and EAC by increasing the permeability of the epithelial barrier.

NcoI TNF-ß gene polymorphism and TNF expression are associated with an increased risk of developing Barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVE: Esophageal cancer development is a sequence that starts with reflux esophagitis (RE), followed by Barrett's esophagitis (BE), dysplasia, and finally esophageal adenocarcinoma (EAC). Tumor necrosis factor (TNF) is a potent anti-neoplastic agent, hence DNA polymorphisms that reduce TNF levels potentially enhance the development of BE and EAC. The aim of the study was to determine the impact of TNF gene variation on the RE-BE-EAC cascade. METHODS: DNA from 887 Caucasian participants (197 controls, 305 RE, 257 BE, 128 EAC) was tested for the gene polymorphism TNF-ß NcoI, and TNF production was determined by TNF-α specific immunohistochemistry on esophageal biopsies from these BE (n = 31) and EAC (n = 4) patients. RESULTS: As compared with healthy controls, the TNF-ß NcoI A/A genotype was significantly more prevalent in BE (p = 0.04) and EAC patients (p = 0.02), but not in RE patients (p = 0.1). While TNF-α protein levels were invariably high in esophageal biopsies from EAC patients, most esophageal BE samples showed low to moderate TNF levels. CONCLUSIONS: Chronic inflammation, like in BE, markedly increase the risk of malignant transformation. In this study, the significantly higher frequency of the TNF-ß NcoI A/A genotype and the local TNF expression indicate that the pro-inflammatory cytokine TNF plays a role in the development of BE and EAC.

Functional single-nucleotide polymorphism of epidermal growth factor is associated with the development of Barrett's esophagus and esophageal adenocarcinoma.

Reflux esophagitis (RO) and Barrett's esophagus (BO) can cause esophageal adenocarcinoma (OAC). The esophageal mucosa in the RO-BO-OAC cascade is chronically exposed to gastro-esophageal reflux. Epidermal growth factor (EGF) has an important role in the protection and repair of mucosal damage, and non-physiologic levels are associated with gastrointestinal tumors. The aim is to determine the functional effect of EGF gene polymorphisms on RO, BO and OAC development. A cohort of 871 unrelated Dutch Caucasians consisted of 198 healthy controls, 298 RO patients, 246 BO patients and 129 OAC patients. The frequency of the EGF-production-associated 5'UTR A+61G polymorphism was determined in these four groups. EGF immunohistochemistry was performed on BO biopsies. EGF expression was significantly lower in the G/G genotype compared with the A/G (P=0.008) and A/A (P=0.002) group. The G/G genotype was significantly more prevalent in RO (odds ratios (OR)=2.6; 95% confidence intervals (95% CI): 1.3-5.2), BO (OR=3.0; 95% CI: 1.5-6.2) and OAC (OR=4.1; 95% CI: 1.8-9.7) than in controls. The G allele is associated with reduced EGF expression and increased risk for RO, BO and OAC development. This indicates that reduced mucosal protection resulting from genetically decreased EGF expression enhances esophageal tumor development.

Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma.

BACKGROUND: The continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma. METHODS: PXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender. RESULTS: PXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P<0.001), and RE patients (P=0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P=0.037). CONCLUSIONS: PXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease.

Inverse nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter mustelae.

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.

UreA2B2: a second urease system in the gastric pathogen Helicobacter felis.

Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism.

No association between tumor necrosis factor-alpha production and gene polymorphisms among inbred rat strains.

Differences in spontaneous allograft acceptance after liver transplantation among inbred rat strains might be explained by variation in the local production of TNF-alpha as a potent mediator of the inflammatory response. In this study, we hypothesize that nucleotide differences in the rat Tnf gene influence TNF-alpha protein expression. As such, polymorphisms in the Tnf gene may also provide a possible explanation for differences in survival of allogeneic liver grafts among inbred rat strains. We therefore investigated the capacity of mononuclear cells to produce TNF-alpha in response to a mitogenic stimulus and the Tnf locus was sequenced in six different inbred rat strains. Among the six strains (AUG, BN, DA, LEW, PVG and WF), 44 nucleotide differences including 36 single nucleotide polymorphisms (SNPs), five simple sequence length polymorphisms, two deletions and one insertion, were found in the Tnf gene. Although, the inbred rat strains differed significantly in mean levels of maximum TNF-alpha production (P = 0.001), no associations were found with nucleotide differences within the Tnf gene. In conclusion, our results indicate that differential in vitro TNF-alpha responses among inbred rat strains are not associated with nucleotide differences within non-coding regulatory regions of the rat TNF-alpha gene. Without an established relationship between polymorphisms and expression of the TNF-alpha gene, it is preliminary to address a possible association of Tnf gene polymorphisms with rat liver allograft survival.

The role of Helicobacter spp. in the pathogenesis of primary biliary cirrhosis and primary sclerosing cholangitis.

Helicobacter species DNA has been detected in liver tissue of patients affected by primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). To investigate a potential causative relation between Helicobacter species and PBC/PSC, we compared the presence of Helicobacter species-specific DNA in liver tissue of patients with PBC/PSC (n=18/n=13) with those of a control group of patients with various liver diseases with known cause (n=29). A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from paraffin embedded liver tissue. Control patients had hepatitis-B (n=9), alcoholic cirrhosis (n=14), or non-cirrhotic metabolic liver disease (n=6). There was no significant difference between the incidence of Helicobacter spp.-specific DNA in PBC/PSC (9/31; 29%) and the control group (10/29; 34%). Sequence analysis confirmed Helicobacter spp. DNA. Because Helicobacter spp. DNA can be found in approximately one-third of all samples tested, it is unlikely that PSC and PBC are caused by Helicobacter infection.

The presence of the cag pathogenicity island is associated with increased superoxide anion radical scavenging activity by Helicobacter pylori.

Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.

Detection of Helicobacter pylori in bile of cats.

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir.

The functional status of the Helicobacter pylori sabB adhesin gene as a putative marker for disease outcome.

BACKGROUND: Helicobacter pylori factors that contribute to disease outcome are largely unknown, but intimate contact with host cells mediated by outer membrane proteins is thought to play an important role. Expression of the outer membrane proteins OipA, HopZ, SabA, and SabB is regulated by phase-variable dinucleotide repeats in the coding regions of the respective genes. We have evaluated the correlation between the expression status of these four genes and disease outcome of H. pylori infection in a Dutch patient population. MATERIALS AND METHODS: H. pylori strains, isolated from 96 Dutch patients with gastritis (n = 29), duodenal ulcer (n = 28), gastric ulcer (n = 21), gastric carcinoma (n = 9), and lymphoma (n = 9), were analyzed for the 'on/off' expression status of the H. pylori genes oipA, hopZ, sabA, and sabB by direct DNA sequence analysis of amplified fragments. RESULTS: The off-status of sabB was significantly associated with duodenal ulcer (p =.036), but not with gastric ulcer. In contrast, the expression status of oipA, hopZ, and sabA did not correlate with disease outcome. Furthermore, lymphoma strains appeared to express a significantly smaller amount of putative adhesins when compared to gastritis, gastric ulcer, duodenal ulcer and gastric carcinoma strains (p <.02 for all groups tested). CONCLUSIONS: The off-status of sabB was found to be associated with duodenal ulcer disease, and thus represents a putative marker for disease outcome. Assuming that SabB is involved in bacterial adhesion, this association suggests that adherent H. pylori are more prone to elimination by the host immune system.

Transcriptional phase variation of a type III restriction-modification system in Helicobacter pylori.

Phase variation is important in bacterial pathogenesis, since it generates antigenic variation for the evasion of immune responses and provides a strategy for quick adaptation to environmental changes. In this study, a Helicobacter pylori clone, designated MOD525, was identified that displayed phase-variable lacZ expression. The clone contained a transcriptional lacZ fusion in a putative type III DNA methyltransferase gene (mod, a homolog of the gene JHP1296 of strain J99), organized in an operon-like structure with a putative type III restriction endonuclease gene (res, a homolog of the gene JHP1297), located directly upstream of it. This putative type III restriction-modification system was common in H. pylori, as it was present in 15 out of 16 clinical isolates. Phase variation of the mod gene occurred at the transcriptional level both in clone MOD525 and in the parental H. pylori strain 1061. Further analysis showed that the res gene also displayed transcriptional phase variation and that it was cotranscribed with the mod gene. A homopolymeric cytosine tract (C tract) was present in the 5' coding region of the res gene. Length variation of this C tract caused the res open reading frame (ORF) to shift in and out of frame, switching the res gene on and off at the translational level. Surprisingly, the presence of an intact res ORF was positively correlated with active transcription of the downstream mod gene. Moreover, the C tract was required for the occurrence of transcriptional phase variation. Our finding that translation and transcription are linked during phase variation through slipped-strand mispairing is new for H. pylori.