Inhibition of fumarase by bismuth(III): implications for the tricarboxylic acid cycle as a potential target of bismuth drugs in Helicobacter pylori

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations and gastric cancer. Triple therapy combining bismuth compounds with two antibiotics is the cornerstone of the treatment of H. pylori infections. Up to now, the molecular mechanisms by which bismuth inhibits the growth of H. pylori are far from clear. In the bacterial tricarboxylic acid (TCA) cycle, fumarase catalyses the reversible hydration of fumarate to malic acid. Our previous proteomic work indicated that fumarase was capable of bismuth-binding. The interactions as well as the inhibitory effects of bismuth to fumarase have been characterized in this study. The titration of bismuth showed that each fumarase monomer binds one mol equiv of Bi³⁺, with negligible secondary structural change. Bismuth-binding results in a near stoichiometric inactivation of the enzyme, leading to an apparent non-competitive mechanism as reflected by the Lineweaver–Burk plots. Our collective data indicate that the TCA cycle is a potential molecular target of bismuth drugs in H. pylori.     

Helicobacter pylori: new developments and treatments

The authors highlight new developments in research on Helicobacter pylori. There is now consensus that all patients with newly diagnosed or recurrent duodenal or gastric ulcers who have a positive test result for H. pylori should be treated for the infection. Patients presenting with complications of ulcers, such as bleeding, should also be treated. H. pylori has recently been classified as a definite human carcinogen by the International Agency for Research on Cancer. In treatment, new combination regimens, consisting of 3 or 4 different drugs, cure the infection in more than 80% of patients. Currently, the best combinations are: (1) omeprazole (or another proton-pump inhibitor), clarithromycin and metronidazole, (2) omeprazole (or another proton-pump inhibitor), clarithromycin and amoxicillin, (3) bismuth subsalicylate, tetracycline and metronidazole, and (4) omeprazole, bismuth subsalicylate, tetracycline and metronidazole.     

Solubility, absorption, and anti-Helicobacter pylori activity of bismuth subnitrate and colloidal bismuth subcitrate: In vitro data Do not predict In vivo efficacy

Objectives. The aim of this study was to compare the dissolution, bioavailability, and anti– Helicobacter pylori activity of bismuth subnitrate and colloidal bismuth subcitrate. This could, first, provide insights into the mechanism of action of bismuth and, second, help to develop optimal therapeutic strategies.
Methods. Solubility and aquated size of bismuth species were determined in human gastric juice, while absorption into blood and urinary excretion of bismuth was determined in volunteers. Activity against H. pylori was determined in vitro in the presence and absence of antibiotics, while H. pylori eradication was compared in vivo.
Results. Bismuth from colloidal bismuth subcitrate was at least 10% soluble and ultrafilterable and was absorbed in volunteers (>0.5%), whereas that from bismuth subnitrate was insoluble and not absorbed (<0.01%). Colloidal bismuth subcitrate was active against H. pylori (mean inhibitory concentration, ≤12.5 μg/ml), while bismuth subnitrate was inactive (>400 μg/ml); neither was synergistic with antibiotics. With in vivo triple therapy, bismuth subnitrate was as effective as colloidal bismuth subcitrate in eradicating H. pylori (74% and 70% eradicated, respectively).
Conclusions. Colloidal bismuth subcitrate, unlike bismuth subnitrate, is partially soluble, absorbed in humans, and directly toxic to H. pylori in vitro. Surprisingly, however, these preparations had similar efficacy in vivo against H. pylori within triple therapy, suggesting that bismuth compounds may also exhibit indirect antimicrobial effects. We propose that this is an effect on the gastric mucus layer. Nonabsorbable bismuth compounds should be preferentially considered in bismuth-based therapies against H. pylori , as they would minimize toxicity while maintaining efficacy.     

Eicosanoid synthesis and Helicobacter pylori associated gastritis: increase in leukotriene C4 generation associated with H. pylori colonization.

The importance of pro-inflammatory leukotriene C4 in Helicobacter pylori (H. pylori) associated gastritis in man is unknown. Fresh gastric biopsy specimens from 28 dyspeptic patients were obtained: 10 showed normal antral histology with no evidence of H. pylori, the remaining 18 patients exhibited histological gastritis and were H. pylori positive as assessed by histology, culture and urease test. Twelve of these 18 patients received 240 mg twice daily colloidal bismuth subcitrate for four weeks before re-endoscopy. Gastric biopsies from H. pylori positive patients were incubated under basal and Ca(2+)-ionophore mediated conditions: Radioimmunoassay analysis of the supernatant showed basal release of prostaglandin E2 and leukotriene C4 was slightly but not significantly elevated in H. pylori positive mucosa. However in H. pylori positive mucosa there was an 85% increase in leukotriene C4 synthesis when biopsies were incubated with ionophore, compared to only 13% increase in H. pylori negative mucosa (p less than 0.02). After eradication of H. pylori by colloidal bismuth subcitrate, there was a clearance of inflammatory cell infiltrate as assessed by histology and a significant reduction in ionophore-mediated leukotriene C4 formation compared with before treatment (p less than 0.02). These results suggest that H. pylori gastritis is associated with increased capacity to generate leukotriene C4, which may amplify the damaging effects of the bacteria on gastric mucosa.     

Helicobacter pylori. One bacterium and a broad spectrum of human disease! An overview.

Since the historical rediscovery of gastric spiral Helicobacter pylori in the gastric mucosa of patients with chronic gastritis by Warren and Marshall in 1983, peptic ulcer disease has been largely viewed as being of infectious aetiology. Indeed, there is a strong association between the presence of H. pylori and chronic active gastritis in histology. The bacterium can be isolated in not less than 70% of gastric and in over 90% of duodenal ulcer patients. Eradication of the organism has been associated with histologic improvement of gastritis, lower relapse rate and less risk of bleeding from duodenal ulcer. The bacterium possesses several virulence factors enabling it to survive the strong acid milieu inside the stomach and possibly damaging host tissues. The sequence of events by which the bacterium might cause gastric or duodenal ulcer is still not fully elucidated and Koch's postulates have never been fulfilled. In the majority of individuals, H. pylori infection is largely or entirely asymptomatic and there is no convincing data to suggest an increase in the prevalence of peptic ulcer disease among these subjects. An increasingly growing body of literature suggests an association between colonization by H. pylori in the stomach and a risk for developing gastric mucosa-associated lymphoid tissue (MALT), MALT lymphoma, gastric adenocarcinoma and even pancreatic adenocarcinoma. The bacterium has been implicated also in a number of extra-gastrointestinal disorders such as ischaemic heart disease, ischaemic cerebrovascular disease, atherosclerosis, and skin diseases such as rosacea, but a causal role for the bacterium is missing. Eradication of H. pylori thus seems to be a beneficial impact on human health. Various drug regimens are in use to eradicate H. pylori involving the administration of three or four drugs including bismuth compounds, metronidazole, clarithromycin, tetracyclines, amoxycillin, ranitidine, omeprazole for 1-2 weeks. The financial burden, side effects and emergence of drug resistant strains due to an increase in the use in antibiotics for H. pylori eradication therapy need further reconsideration.     

幽门螺杆菌感染新型原代人胃上皮细胞模型的建立

目的 培养三维(3D)人胃黏膜上皮类器官,转为二维(2D)原代胃黏膜细胞培养,并建成人2D原代胃上皮细胞的幽门螺杆菌感染模型。 方法 (1)从正常人胃上皮组织中分离胃腺,在含有多种生长调节和凋亡抑制等混合因子的培养基中,依附于基质胶而培养成3D类器官;(2)利用免疫荧光技术鉴定胃上皮类器官的相关分子标记;(3)研究正常原代胃上皮细胞被幽门螺杆菌感染后的形态学变化,利用免疫印迹技术鉴定幽门螺杆菌感染相关蛋白的表达水平。 结果 成功培养出可长期传代的人胃上皮3D类器官,具有典型的人胃黏膜上皮分子标记。而且3D类器官转为2D平面培养的原代胃上皮细胞,可作为幽门螺杆菌的体外原代细胞感染模型。 结论 3D胃上皮类器官,可作为2D原代胃上皮细胞的持久来源,为研究幽门螺杆菌感染人体胃上皮的分子机制带来个体化的新模型。     

Subtractive hybridization analysis of gastric diseases-associated Helicobacter pylori identifies peptidyl-prolyl isomerase as a potential marker for gastric cancer

Helicobacter pylori, a microaerophilic Gram-negative bacterium, is known to cause chronic gastritis, peptic ulcer and gastric cancer. Genes that are present in certain isolates may determine strain-specific traits such as disease association and drug resistance. In order to understand the pathogenic mechanisms of gastric diseases, identify molecular markers of the diseases associated with H. pylori strains and provide clues for target treatment of H. pylori-related diseases, a subtracted DNA library was constructed from a gastric cancer-associated H. pylori strain and a superficial gastritis-associated H. pylori strain by suppression subtractive hybridization. The presence of gastric cancer-specific genes was identified by dot blot hybridization, DNA sequencing and PCR-based screening. Twelve gastric cancer-specific high-copy genes and nine low-copy genes were found in gastric cancer compared with the superficial gastritis strain. These genes were confirmed by PCR analysis of H. pylori isolates. Notably, peptidyl-prolyl cis-trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer.     

Pathophysiology and clinical relevance of Helicobacter pylori.

Considerable knowledge has recently accumulated on the mechanism by which Helicobacter pylori (H. pylori) induces chronic gastritis. Although H. pylori is not an invasive bacterium, soluble surface constituents can provoke pepsinogen release from gastric chief cells or trigger local inflammation in the underlying tissue. Urease appears to be one of the prime chemoattractants for recruitment and activation of inflammatory cells. Release of cytokines, such as tumor necrosis factor alpha, interleukin 1 and 6, and oxygen radicals, leads to a further tissue inflammation accompanied by a potent systemic IgA and IgG type of immune response. Chronic inflammation and antigens on glandular epithelial cells lead to a progressive destruction with loss of the epithelial barrier function. Within the gastric mucosa, patches of intestinal metaplasia develop, which may be a risk factor for subsequent development of gastric carcinoma. Hyperacidity in duodenal ulcer patients induces gastric metaplasia in the duodenal bulb, which represents a target for H. pylori colonization and ulcer formation. H. pylori can be detected in the majority of patients with peptic ulcers and, compared to age-matched healthy people, it is also found more often in patients with dyspepsia and gastric carcinoma. Although H. pylori can be detected in healthy people, the marked reduction of the ulcer recurrence rate by eradication of H. pylori (80 percent versus 20 percent relapse within one year) suggests that H. pylori is a major risk factor for duodenal ulcer formation. The potential role of H. pylori in non-ulcer dyspepsia and carcinogenesis is under investigation. Current regimens aimed at eradicating H. pylori use a combination of several drugs that are potentially toxic. Since the risk of complications may exceed the potential benefit in most patients, eradication treatment should be limited to clinical trials and to patients with aggressive ulcer disease. New drug regimens, e.g., the combination of proton pump inhibitors with one antibiotic, may provide less toxic alternatives. Beyond ulcer treatment, effective and well-tolerated eradication regimens may have a place in prophylaxis of gastric carcinoma.     

SagA of CagA in Helicobacter pylori pathogenesis

Much attention has recently been given to the role of the Helicobacter pylori CagA protein, the only as yet identified H. pylori protein that is delivered into the host gastric epithelial cells by a type IV secretion system, in the development of H. pylori-associated diseases, including gastric carcinoma. This review summarizes the latest advances in our understanding of pathogenic actions of H. pylori CagA, particularly focusing on the molecular mechanisms underlying CagA entry into the host cells as well as CagA-mediated perturbation of host cell signaling involved in proliferation, motility, differentiation, and polarity, which contributes malignant transformation of mammalian cells.     

Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.     

Randomised controlled trial of short term treatment to eradicate Helicobacter pylori in patients with duodenal ulcer.

OBJECTIVE--To determine whether one week''s drug treatment is sufficient to eradicate Helicobacter pylori in patients with duodenal ulcer. DESIGN--Single blind, randomised controlled trial. SETTING--Specialised ulcer clinic in a teaching hospital. PATIENTS--155 patients with H pylori and a duodenal ulcer verified endoscopically which had either bled within the previous 24 hours or was causing dyspepsia. INTERVENTIONS--Patients were allocated randomly to receive either omeprazole for four weeks plus bismuth 120 mg, tetracycline 500 mg, and metronidazole 400 mg (all four times a day) for the first week (n = 78), or omeprazole alone for four weeks (n = 77). Further endoscopy was performed four weeks after cessation of all drugs. MAIN OUTCOME MEASURES--Presence or absence of H pylori (by urease testing, microscopy, and culture of antral biopsy specimens), duodenal ulcer, and side effects. RESULTS--Eradication of H pylori occurred in 70 (95%) patients taking the four drugs (95% confidence interval 86% to 97%) compared with three (4%) patients taking omeprazole alone (1% to 11%). Duodenal ulcers were found in four (5%) patients taking the four drugs (2% to 12%) and in 16 (22%) patients taking omeprazole alone (14% to 32%). Mild dizziness was the only reported side effect (six patients in each group) and did not affect compliance. CONCLUSIONS--A one week regimen of bismuth, tetracycline, and metronidazole is safe and effective in eradicating H pylori and reduces the number of duodenal ulcers four weeks after completing treatment.     

Indications for treatment of Helicobacter pylori infection: a systematic overview.

OBJECTIVE: To determine (a) the advantages and disadvantages of treatment options for the eradication of Helicobacter pylori and (b) whether eradication of H. pylori is indicated in patients with duodenal ulcer, nonucler dyspepsia and gastric cancer. DATA SOURCES: A MEDLINE search for articles published in English between January 1983 and December 1992 with the use of MeSH terms Helicobacter pylori (called Campylobacter pylori before 1990) and duodenal ulcer, gastric cancer, dyspepsia and clinical trial. Six journals and Current Contents were searched manually for pertinent articles published in that time frame. STUDY SELECTION: For duodenal ulcer the search was limited to studies involving adults, studies of H. pylori eradication and randomized clinical trials comparing anti-H. pylori therapy with conventional ulcer treatment. For nonulcer dyspepsia with H. pylori infection the search was limited to placebo-controlled randomized clinical trials. DATA EXTRACTION: The quality of each study was rated independently on a four-point scale by each author. For the studies of duodenal ulcer the outcome measures assessed were acute ulcer healing and time required for healing, H. pylori eradication and ulcer relapse. For the studies of nonulcer dyspepsia with H. pylori infection the authors assessed H. pylori eradication, the symptoms used as outcome measures and whether validated outcome measures had been used. DATA SYNTHESIS: Eight trials involving duodenal ulcer met our inclusion criteria: five were considered high quality, two were of reasonable quality, and one was weak. Six trials involving nonulcer dyspepsia met the criteria, but all were rated as weak. Among treatment options triple therapy with a bismuth compound, metronidazole and either amoxicillin or tetracycline achieved the highest eradication rates (73% to 94%). Results concerning treatment indications for duodenal ulcer were consistent among all of the studies: when anti-H. pylori therapy was added to conventional ulcer treatment acute ulcers healed more rapidly. Ulcer relapse rates were dramatically reduced after H. pylori eradication. All of the studies involving nonulcer dyspepsia assessed clearance rather than eradication of H. pylori. No study used validated outcome measures. A consistent decrease in symptom severity was no more prevalent in patients in whom the organism had been cleared than in those taking a placebo. Of the studies concerning gastric cancer none investigated the effect of eradication of H. pylori on subsequent risk of gastric cancer. CONCLUSIONS: There is sufficient evidence to support the use of anti-H. pylori therapy in patients with duodenal ulcers who have H. pylori infection, triple therapy achieving the best results. There is no current evidence to support such therapy for nonulcer dyspepsia in patients with H. pylori infection. Much more attention must be paid to the design of nonulcer dyspepsia studies. Also, studies are needed to determine whether H. pylori eradication in patients with gastritis will prevent gastric cancer.     

幽门螺杆菌感染的分子机制

幽门螺杆菌(Helicobacterpylori)是居留于人胃上皮组织并引起胃炎、消化性胃溃疡和胃癌的病原菌。近年来,随着幽门螺杆菌全基因组序列的报道和功能基因的研究深入,对幽门螺杆菌的感染的分子、免疫等机制逐渐阐明。现对幽门螺杆菌基因组特点和幽门螺杆菌黏附、毒性因子等对人体感染的分子机制等方面的研究进展做一综述。     

幽门螺杆菌抗生素耐药机制研究进展

幽门螺杆菌(Helicobacter pylori,H.pylori)感染可引起消化性溃疡、胃粘膜相关淋巴组织淋巴瘤和胃癌。随着抗生素耐药性的问题越来越严重,耐药机制的研究也不断深入。分子检测方法,尤其是核酸检测技术,可高效、快速、准确地检测幽门螺杆菌抗生素耐药基因及突变,对幽门螺杆菌感染的临床治疗发挥重要的指导作用,同时也可对幽门螺杆菌抗生素耐药性进行大规模及时有效监控。本文讨论了关于幽门螺杆菌抗生素耐药机制并着重总结了相关耐药基因及突变。     

益生菌在幽门螺杆菌根除治疗中的应用

幽门螺杆菌(Helicobacter pylori,H.pylori)是导致活动性胃炎、消化性溃疡、胃癌、胃黏膜相关淋巴组织淋巴瘤等消化系统疾病的重要病因之一,已被世界卫生组织确认为Ⅰ类致癌因子,根除H.pylori对防治上述疾病有重要意义。目前临床上主要采用含抗生素的三联或四联药物进行H.pylori的根除,虽然取得一定的疗效,但随着抗生素耐药率逐年增加,根除率持续下降,限制了其广泛应用。此外,初次或多次治疗失败后再治疗可选择的药物很少。近年来人们开始尝试将益生菌应用在H.pylori根除治疗中,并取得一定疗效。本文就益生菌在辅助根除幽门螺杆菌方面的研究进展作一简单综述。     

Therapy and prevention of gastric ulcer.

After establishing the benign nature of a gastric ulcer, the treatment is primarily medical. This medical therapy is aimed to alleviate symptoms, to heal the ulcer and to prevent relapses. Based on the history of non-steroidal anti-inflammatory drugs (NSAIDs) and the Helicobacter pylori-status, gastric ulcer patients can be divided into four categories (1) H. pylori positive plus NSAID-use, (2) H. pylori positive without NSAID use, (3) NSAID use with negative H. pylori-status, (4) Negative H. pylori-status and no NSAID use. Patients taking NSAIDs should stop this therapy if possible. Patients with gastric H. pylori infection should be treated by a regimen of a proton pump inhibitor with at least two appropriate antibiotics. This treatment will result in early alleviation of symptoms, rapid healing of the ulcer and prophylaxis of ulcer relapse. In patients with gastric ulcer who cannot stop NSAIDs, maintenance therapy with prostaglandins or potent antisecretory drugs should be considered. The few patients with gastric ulcer who do not take NSAIDs and do not have gastric H. pylori infection should be treated by antisecretory drugs, and they should be carefully followed endoscopically to exclude malignant (carcinoma, lymphoma) or non-peptic (Crohn''s disease) disease. All patients with gastric ulcer should be re-endoscoped to verify complete ulcer healing. Surgery may be considered in gastric ulcer patients with complications, in those with severe dysplasia of the gastric mucosa, and in those who are not able or willing to take the medication.     

Molecular assessment of c-H-ras p21 expression in Helicobacter pylori-mediated gastric carcinogenesis

Helicobacter pylori (H. pylori) infection plays a significant role in causing gastric cancer; the exact molecular mechanisms of gastric carcinogenesis have not yet been fully determined. Therefore, this study was planned to examine the role of c-H-ras p21 expression in H. pylori infection at different stages of disease progression from precursor lesions to gastric carcinoma. This study was carried out in 200 patients, consisting of normal gastric mucosa (n = 20), mucosa with chronic gastritis (n = 63), intestinal metaplasia (n = 20), dysplasia (n = 11), and gastric adenocarcinoma (n = 86), in which the H. pylori status have been analysed. The expression of c-H-ras p21 was studied at mRNA as well as protein level using RT-PCR and western blotting, respectively. The localization of c-H-ras p21 was also studied semiquantitatively by immunohistochemistry. The RT-PCR and western blotting results of c-H-ras p21 mRNA and protein expressions were significantly increased in chronic gastritis, intestinal metaplasia, dysplasia, and gastric adenocarcinoma patients, respectively. Immunohistochemical study also showed the increased expression of c-H-ras p21 in the similar way. Overexpression of c-H-ras p21 might be due to H-ras mutation at codon 12 of ras gene family in H. pylori infection. The rate of expression of ras p21 was higher in the H. pylori-infected precursor lesions, chronic gastritis 49/56 (87.5%), intestinal metaplasia 16/17 (94%), and dysplasia 9/11(82%) whereas in the case of H. pylori negative cases these groups, show 12.5, 5.9, and 18.2%, respectively. The data suggested that H. pylori infection may increase the expression of c-H-ras p21 early in the process of gastric carcinogenesis.     

胃黏膜菌群与幽门螺杆菌的相关性

除幽门螺杆菌之外,胃黏膜内还定居着大量细菌,占主导地位的是厚壁菌门、变形菌门、拟杆菌门、放线菌门和梭杆菌门。幽门螺杆菌和胃黏膜菌群之间可通过竞争营养和空间、扰乱抑菌肽的分泌以及改变宿主胃生理环境等直接或间接相互影响。本研究总结了胃内正常菌群的组成特征,分析了胃黏膜菌群与幽门螺杆菌之间的相互关系及其潜在机制,并进一步探讨了胃黏膜菌群对幽门螺杆菌相关胃部疾病的影响,有利于深入理解慢性胃病的发病机制,为疾病预防及治疗提供理论依据。