Gastric mucosal inflammatory responses toHelicobacter pylori lipopolysaccharide: Suppression of caspase-3 and nitric oxide synthase-2 by omeprazole and sucralfate

Aims:Helicobacter pylori lipopolysaccharide is a primary virulence factor responsible for eliciting acute mucosal inflammatory responses associated withH. pylori infection. In this study, we investigated the activity of a key apoptotic protease, caspase-3, and the expression of inducible nitric oxide synthase (NOS-2) duringH. pylori lipopolysaccharide-induced acute gastritis, and evaluated the effect of anti-ulcer agents, omeprazole and sucralfate, on this process.Methods: Rats, pretreated twice daily with omeprazole at 40 mg/kg, sucralfate at 100 mg/kg, or the vehicle, were subjected to intragastric application ofH. pylori lipopolysaccharide at 50 mug/animal, and after 4 additional days on the anti-ulcer drug or vehicle regimen their mucosal tissue was used for histologic assessment, assays of epithelial cells apoptosis, and the measurements of caspase-3 and NOS-2 activities.Results: In the absence of anti-ulcer agents,H. pylori lipopolysaccharide induced acute reaction characterized by the inflammatory infiltration of the lamina propria, hyperemia, and epithelial hemorrhage. This was accompanied by an 11.2-fold increase in epithelial cell apoptosis, a 6.5-fold induction in mucosal expression of NOS-2, and a 5.4-fold increase in caspase-3 activity. Treatment with proton pump inhibitor, omeprazole, produced a 39.6% reduction in the extent of mucosal inflammatory changes elicited byH. pylori lipopolysaccharide and a 75.5% decrease in the epithelial cells apoptosis, while the activity of caspase-3 decreased by 26.8% and that of NOS-2 showed a 46.7% decline. The gastroprotective agent, sucralfate, evoked a 62.3% reduction in the extent of mucosal inflammatory changes caused by the lipopolysaccharide, epithelial cell apoptosis decreased by 85.8%, and NOS-2 showed a 68.2% decline, while the activity of caspase-3 decreased by 39.7%.Conclusions: Our findings implicate caspase-3 involvement in gastric mucosal inflammatory responses toH. pylori lipopolysaccharide, and point towards participation of NOS-2 in the amplification of the cell death-signaling cascade. We also show that anti-ulcer agents, omeprazole and sucralfate, are capable of suppressing the H. pylori-induced mucosal inflammatory responses by interfering with the events propagated by NOS-2 and caspase-3.     

Helicobacter pylori infection: Epidemiology, pathophysiology, and therapy

Helicobacter pylori is one of the most commonly encountered human pathogens. It has been shown to be closely associated with peptic ulcer disease (PUD), gastric adenocarcinoma, and the gastric mucosa-associated lymphoid tissue (MALT) that may lead to gastric lymphoma. The current diagnostic methods include histology, microbiological culture, classic serology, urease activity detection, polymerase chain reaction (PCR) and stool antigen detection. Its treatment modality options are multiple; however, a triple regimen consisting of a proton pump inhibitor (PPI), and two antibiotics for 10 to 14 days is preferred. Drug resistance is a growing problem in this organism and new therapeutic options are currently limited.     

多西环素配伍阿莫西林四联方案根除幽门螺旋杆菌的疗效观察

目的 观察不同药物四联方案根除幽门螺旋杆菌（Helicobacter pylori,Hp）的疗效。方法 选取初次和补救治疗的Hp感染患者各180例,单盲随机分成4组,分别为初治试验组（阿莫西林胶囊+多西环素胶囊）、初治对照组（阿莫西林胶囊+克拉霉素片）、补救治疗试验组（阿莫西林胶囊+多西环素胶囊）、补救治疗对照组（阿莫西林胶囊+左氧氟沙星片）,各组均联合埃索美拉唑肠溶片、胶体果胶铋胶囊,每天2次,疗程14 d。疗程结束4周后复查¹³C呼气试验,观察Hp根除率、不良反应、成本-效果比（cost-effectiveness ratio,C/E）,并做6,12个月随访,观察Hp复发率。结果 在Hp初治组中,试验组和对照组Hp根除率和复发率比较无统计学意义,但试验组C/E较低。在Hp补救治疗组中,试验组Hp根除率较高,疗效较好,与补救治疗对照组相比,差异有统计学意义（P<0.05）,C/E较低,复发率较低,差异无统计学意义。各组不良反应比较,初治试验组、补救治疗试验组、补救治疗对照组的不良反应发生率较低,与初治对照组相比差异有统计学意义（P<0.01）。结论 多西环素配伍阿莫西林四联方案根除Hp近远期疗效较好,成本较低。     

胃粘附片及粘附辅料的体外评价

用改良的粘附力测定法,测定了片剂中常用的添加剂对几种辅料粘附性的影响,并以阿莫西林为模型药物进行了体外粘附性及药物释放的研究。     

益生菌四联对比铋剂四联疗法补救根除幽门螺杆菌感染的Meta分析

目的 系统评价益生菌四联疗法（PQT）对比铋剂四联疗法（BQT）补救根除幽门螺杆菌（Helicobacter pylori,Hp）感染的疗效和安全性。方法 检索关于PQT对比BQT补救根除Hp感染的临床试验,检索时限均为建库至2016年10月。由2名评价员,根据纳入与排除标准筛选文献、提取资料,依据改良Jadad量表评价纳入研究的方法学质量,采用RevMan 5.3软件进行meta分析。结果 最终纳入9个临床试验,共875例患者,结果显示：补救治疗中PQT的Hp根除率高于BQT,但差异无统计学意义（RR=1.03,95% CI：0.97~1.10,P=0.36）;按PP分析,PQT与BQT的Hp根除率相当,但差异无统计学意义（RR=1.00,95% CI：0.94~1.07,P=0.90）。PQT的不良反应发生率明显低于BQT,且差异有统计学意义（RR=0.31,95% CI：0.23~0.41,P<0.000 01）。结论 与BQT相比,补救治疗中PQT的不良反应发生率更低,但Hp的根除率无差异,该结论有待大样本高质量的研究验证。     

复方果胶铋抗幽门螺旋杆菌作用

目的研究复方果胶铋(compound colloidal bismuth pectin,CCBP)的抗幽门螺旋杆菌及治疗实验动物胃溃疡作用。方法采用幽门螺旋杆菌诱导的BALB/c小鼠和沙土鼠胃溃疡模型,灌胃给予不同剂量的复方果胶铋。结果结果显示复方果胶铋720、2 160 mg.kg-1组对上述胃溃疡模型动物均具有降低溃疡指数的作用,抑制率呈现剂量效应相关性,随剂量增加效果增强。复方果胶铋530、1 600 mg.kg-1、复方枸橼酸铋甲硝唑四环素(bismuth subcitrate potassium,metronidazole,and tet-racycline hydrochloride,简称PYLERA)组沙土鼠胃组织尿素酶活性比模型组显著降低,其中1 600 mg.kg-1组动物的体内抑菌率为65.1%。结论复方果胶铋对各种胃溃疡模型实验动物胃黏膜损伤有明显保护作用。同剂量比较复方果胶铋降低溃疡指数效果优于各单方,与市售抗溃疡药PYLERA作用相当。     

Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains

Background and the purpose of the study

Current anti-H. pylori therapies are based on the use of two antibiotics with a proton pump inhibitor and/or a bismuth component. Metronidazole is a key component of such combination therapies in Iran. The aim of this study was to determine the role of rdxA gene in resistant strains of H. pylori isolated from Shahrekord Hajar hospital to metronidazole.

Methods

This study was a cross-sectional method, which was carried out on 263 patients who referred to endoscopy department of Hajar hospital, in 2007. Biopsy samples were cultured on selective Brucella agar containing 10% blood and incubated under microerophilic condition at 370C for 3–7 days. Suspected colonies were tested by Gram staining, urease, oxidase and catalase activities. Organisms were confirmed to be H. pylori on the basis of the presence of ureC(glmM) gene by PCR.Specific primers were used for detection of rdxA gene mutation.

Results

Eighty and four strains of H. pylori determined by PCR method. Of the isolated strains, 49 (58.33%) were resistant, 7 (8.33%) were semi-sensitive to metronidazole and 200bp deletion in rdxA gene was observed in 2 strains.

Conclusion

Because of the high metronidazole resistance in patients under study it was necessary to replace it by other antibiotics in therapeutic regimens. On the basis of low frequency of resistance mutation in rdxA gene, sequence analysis for identification of other mechanisms is suggested.     

Selective adherence of a sucralfate—tetracycline complex to gastric ulcers: Implications for the treatment of Helicobacter pylori

The adherence of a sucralfate—tetracycline complex to gastric ulcers and to nearby nonulcer sites was determined in the rabbit antrum. Persistent gastric ulcers were produced by a previously described method. The presence of the complex was assessed 1 and 4 h after dosing. Drug adherence was determined by quantitation of aluminum in stomach wall biopsies. Significantly more aluminum adhered to ulcer sites than to nearby nonulcer sites. Adherence of the complex did not significantly decrease from 1 to 4 h. The complexation of tetracycline to sucralfate did not alter the selective adherence of sucralfte to gastric ulcers, providing a mechanism of ulcer site-selective drug delivery in the treatment of Helicobacter pylori gastric ulcer disease.     

Effect of CYP2C19*17 gene variant on Helicobacter pylori eradication in peptic ulcer patients

Objective Eradication of Helicobacter pylori is an important treatment strategy in peptic ulcer patients. Current regimens of eradication consist of proton pump inhibitor (PPI) and two antibiotics. The principal enzyme involved in PPIs metabolism is CYP2C19, which exhibits an interindividual variability of activity, mainly due to genetic polymorphism. Two alleles (CYP2C19*2 and CYP2C19*3), responsible for slow PPIs metabolism, were previously associated with higher efficacy of eradication. Recently, a novel CYP2C19 gene variant (CYP2C19*17), associated with faster metabolism of CYP2C19 substrates, was described. In the present study, a potential association between CYP2C19*17 allele and lower H. pylori eradication efficacy was tested in a group of peptic ulcer patients.Methods A total of 125 peptic ulcer patients, positive for H. pylori infection, were treated with triple therapy (pantoprazole+amoxicillin+metronidazole). Subsequently, the patients were divided into two groups (group 1 – success, and group 2 – failure of eradication after therapy) and genotyped for the presence of CYP2C19 variant alleles (*2, *3, and *17).Results Frequency of CYP2C19 alleles in two groups of patients were: 56.4 versus 65 (p=0.060) for *1, 15.4 versus 5.3 (p=0.015) for *2, and 28.2 versus 25.5 (p=0.663) for *17 allele, respectively. CYP2C19*3 was not detected in the evaluated population. No significant differences in frequency nor distribution of *17 allele were found between two groups of patients. CYP2C19*2 allele was associated with successful H. pylori eradication (p<0.02), *2 allele carriers were found to be over 4-times more prone to the treatment compared to *1/*1 homozygotes (OR=4.2, p=0.015).Conclusion Our results suggest that, contrary to CYP2C19*2, CYP2C19*17 allele has no impact on efficacy of H. pylori eradication in peptic ulcer patients treated with pantoprazole.     

Design and evaluation of gastroretentive levofloxacin floating mini-tablets-in-capsule system for eradication of Helicobacter pylori

Gastroretentive levofloxacin (LVF) floating mini-tablets for the eradication of Helicobacter pylori (H. pylori) were prepared using the matrix forming polymer hydroxypropyl methylcellulose (HPMC K100M), alone or with Carbopol 940P in different ratios by wet granulation technique. Buoyancy of mini-tablets was achieved by an addition of an effervescent mixture consisting of sodium bicarbonate and anhydrous citric acid to some formulations. The prepared mini-tablets were evaluated for weight variation, thickness, friability, hardness, drug content, in vitro buoyancy, water uptake and in vitro release. The optimized formula was subjected to further studies: FT-IR, DSC analysis and in vivo examination in healthy volunteers. The prepared mini-tablets exhibited satisfactory physicochemical characteristics. Incorporation of gas-generating agent improved the floating parameters. HPMC K100M mini-tablet formulation (F1) offered the best controlled drug release (>8 h) along with floating lag time <1 s and total floating time >24 h. The obtained DSC thermograms and FT-IR charts indicated that there is no positive evidence for the interaction between LVF and ingredients of the optimized formula. The in vivo test confirmed the success of the optimized formula F1 in being retained in the stomach of the volunteers for more than 4 h. LVF floating mini-tablets based on HPMC K100M is a promising formulation for eradication of H. pylori.Abbreviations: LVF, levofloxacin; H. pylori, Helicobacter pylori; HPMC, hydroxypropyl methylcellulose; FT-IR, Fourier transform infrared spectroscopy; DSC, differential scanning calorimetry; PVP, polyvinyl pyrrolidone; SI, swelling index     

In vitro anti-Helicobacter pylori activity of panaxytriol isolated from ginseng

This study investigated the effect that some polyacetylenes and protopanaxatriol, which were isolated from heated ginseng (family Araliaceae), have on inhibiting Helicobacter pylori (HP) growth. Among the compounds tested, panaxytriol was quite effective in inhibiting HP growth with an MIC of 50 microg/ml. Ginsenoside Rh1 and protopanaxatriol weakly inhibited H+/K+-ATPase from a rat stomach.     

Relationship between mucosal levels of interleukin 8 and toxinogenicity of Helicobacter pylori

Aim of study: To investigate if an association exists between in-vivo mucosal levels of IL-8 and bacterial expression of cytotoxin and cagA gene of H. pylori. Methods: Seventy-two dyspeptic patients referred for endoscopy were studied, including 36 patients with peptic ulcer (PU) and 36 with non-ulcer dyspepsia (NUD). Biopsies were taken for histology, H. pylori culture and measurement of IL-8 by ELISA. To test the ability of H. pylori to produce cytotoxin (VacA), broth culture supernatants were assayed on Vero cells and vacuolation measured. PCR was used to detect the cagA gene of H. pylori. Results: H. pylori was isolated in 52 of 72 patients studied. Among the 52 strains, 25 (49%) were VacA+ve/cagA+ve; 12 (23%) were VacA−ve/cagA−ve; the remaining 15 strains (28%) were either VacA+ve/cagA−ve or VacA−ve/cagA+ve. IL-8 levels (median (interquartile) pg/mg) in patients infected with VacA+ve (1.5 (0.64, 2.84)) or cagA+ve strains (1.25 (0.72, 2.34) were significantly higher than in those with VacA−ve (0.76 (0.4, 1.0)) or cagA−ve strains (0.5 (0.4, 1.5); p<0.05). The neutrophil infiltration score was also higher in patients infected with VacA+ve or cagA+ve strains than in those infected with VacA−ve or cagA−ve strains (p<0.05). Conclusion: VacA+ve/cagA+ve strains were associated with an enhanced production of mucosal IL-8 in vivo and correlated with a stronger infiltration of neutrophils. Enhanced mucosal production of IL-8 and its role in neutrophil chemotaxis and activation could be important in H. pylori-induced gastroduodenal inflammation and in the development of peptic ulcer disease.     

Anti-Helicobacter pylori effect of costunolide isolated from the stem bark ofMagnolia sieboldii

Helicobacter pylori (H. pylori) infection is now established as the major pathogenic factor in chronic gastritis and peptic ulcer disease. In addition, there is accumulating evidence thatH. pylori plays an important role in the process of gastric carcinogenesis. On the other hand, oriental traditional medicines have been used for stomach disease for thousands of years. In the present study, methanol extract from the stem bark ofMagnolia sieboldii (M. sieboldii) and its components were investigated on their inhibitory effects against urease activity and growth ofH. pylori in vitro. The methanol extract ofM. sieboldii significantly inhibited the growth ofH. pylori ATCC 43504 at 5 mg/ml. From the further fractionation, the chloroform fraction inhibited the bacterial growth dose-dependently. Among four fractions separated from the chloroform fraction by silica gel column chromatography, MS-C-2 was the most potent. Costunolide was isolated from the MS-C-2 subfraction by preparative TLC and recrystallization using n-hexane. Anti-H. pylori effect of costunolide was investigated using one commercial strain (H. pylori ATCC 43504) and three clinical strains (H. pylori 4, 43, 82548). Costunolide exhibited potent anti-H. pylori activity, and the MIC was around 100–200 μg/ml. However, costunolide had no inhibitory effect ofH. pylori urease activity at the concentration used for the growth inhibition assay. From these results, we conclude that costunolide inhibits the growth ofH. pylori by the independent manner ofH. pylori urease inhibition.     

Development and In vitro Evaluation of Mucoadhesive Buccal Films of Nebivolol

Nebivolol, a cardioselective β-blocker undergoes extensive metabolism in the liver after its oral administration resulting in low bioavailability. Oral administration of nebivolol also causes gastrointestinal disturbances characterised by stomach ache. To overcome these short comings, mucoadhesive buccal films of nebivolol were prepared using different concentrations of hydroxypropyl methylcellulose and hydroxyl ethylcellulose in the ratios of 2:1, 4:1 and 6:1 and hydroxypropyl methylcellulose and methylcellulose in the ratio of 2:2, 4:3 and 6:4 by solvent casting technique. All the prepared films were found to be smooth, elegant and uniform in thickness and weight. Among the three polymer combinations used, 6:4 (BFN₆) showed increased in vitro residence time, which appeared to be mainly due to mucoadhesive nature of hydroxylpropyl methylcellulose and methylcellulose. Evaluation of the films showed uniform dispersion of the drug throughout the formulation (96.21±0.71 to 97.02±0.12%). In vitro drug release studies showed better results at the end of 8 h. The release profile of all the formulations was subjected to kinetic analyses, which suggested that the drug was released by diffusion mechanism following super case-II transport.     

Trials of novel13C-urea-containing capsule for more economic and sensitive diagnosis ofHelicobacter pylori infection in human subjects

To develop a 13C-urea-containing capsule for more economic and sensitive diagnosis of Helicobacter pylori infection, the 13C-urea-containing capsules were prepared with various additives such as polyethylene glycol, microcrystalline cellulose, sodium lauryl sulfate and citric acid. Their dissolution test and 13C-urea Breath Test in human volunteers were then performed. Polyethylene glycol increased the initial dissolution rates of urea and difference delta 13C values from 13C-urea, while microcrystalline cellulose and sodium lauryl sulfate decreased them. Irrespective of addition of citric acid, the compositions with polyethylene glycol showed higher values from 13C-urea compared to a commercial 76 mg 13C-urea-containing capsule due to higher initial dissolution rate. The capsules with 38 mg 13C-urea and 1.9 mg polyethylene glycol, which showed higher Helicobacter pylori-positive value of about 8 per thousand at 10 min, improved the sensitivity of 13C-urea in human volunteers. Thus, the 13C-urea-containing capsule with polyethylene glycol would be a more economical and sensitive preparation for diagnosis of Helicobacter pylori infection.     

Synthesis and in vitro anti-Helicobacter pylori activity of 2-[(chlorobenzyl)thio]-5-(5-nitro-2-furyl)-1,3,4-thiadiazoles

A new series of 2-[(chlorobenzyl)thio]-5-(5-nitro-2-furyl)-1,3,4-thiadiazoles (6a–h) were synthesized and evaluated by the disc diffusion method against Helicobacter pylori. Four compounds which exhibited strong anti-H. pylori activity at concentration of 8–32 μg/disc (average of inhibition zone >20 mm) were further tested against 20 clinical isolates of H. pylori at lower concentrations. The averages of inhibition zone diameters indicated that all selected compounds exhibit better anti-H. pylori activity profile against clinical isolates of H. pylori with respect to standard drug metronidazole. Compound 6c, containing the 3-chlorobenzylthio moiety, was the most potent compound tested.     

The Versatility of the Helicobacter pylori Vacuolating Cytotoxin VacA in Signal Transduction and Molecular Crosstalk

By modulating important properties of eukaryotic cells, many bacterial protein toxins highjack host signalling pathways to create a suitable niche for the pathogen to colonize and persist. Helicobacter pylori VacA is paradigm of pore-forming toxins which contributes to the pathogenesis of peptic ulceration. Several cellular receptors have been described for VacA, which exert different effects on epithelial and immune cells. The crystal structure of VacA p55 subunit might be important for elucidating details of receptor interaction and pore formation. Here we discuss the multiple signalling activities of this important toxin and the molecular crosstalk between VacA and other virulence factors.     

A novel therapeutic approach for Helicobacter pylori infection: the bismuth-based triple therapy monocapsule

Helicobacter pylori infection causes peptic ulcer disease and must be regarded as a serious infectious disease. Over the past two decades treatment of the infection has been a controversial issue. Treatment is purely empirical and based on combinations of two, three or four existing drugs. Antimicrobial resistance is important and an observed increase in the prevalence of resistance may change the relative importance of certain antibiotics. Bismuth-based triple therapy with bismuth, tetracycline and metronidazole is a well investigated, cheap and FDA approved regimen to cure the infection. Adding a proton pump inhibitor (PPI) increases efficacy. A novel monocapsule (‘Helicide^®’) that contains bismuth, tetracycline and metronidazole simplifies the regimen. This new and patient-friendly drug has been investigated in clinical studies and is expected to be released in North America in 2002.     

Incidence of Helicobacter pylori infection and their clarithromycin-resistant strains in otitis media with effusion regarding phenotypic and genotypic studies

Helicobacter pylori (H. pylori) are pathogenic bacteria that infect a half of the human population, colonize gastric mucosa and can be found in gastric juice. Reflux of gastric juice has been suggested to be associated with glue ear in children. It has been suggested that tonsil and adenoid tissues are potential reservoirs of H. pylori infection. These observations raise the question as to whether H. pylori infection might have a role in otitis media with effusion (OME) in children. The objectives of this research were to evaluate the incidence and possible role of H. pylori in the pathogenesis of OME in children and to evaluate the clarithromycin-resistant strains. Molecular assessment was done to evaluate the culture results vs. molecular study. A total of 60 children, who were prone to ventilation tube insertion, adenoidectomy and/or tonsillectomy were included in the study. The control group consisted of 40 children who underwent adenoidectomy and/or tonsillectomy without the history of OME. Samples of the middle ear fluid and mucosa, adenoid tissue, tonsillar tissue and gastric lavage were cultured and underwent polymerase chain reaction (PCR) analysis then were assembled by using QIAxcel System as capillary electrophoresis for H. pylori detection. There was significant difference between the results of cultures and PCR (P < 0.05). Middle ear fluid culture was positive for H. pylori in 40% of the patients vs. 56.7% PCR results while middle ear mucosa culture was positive in 20% vs. 26.7% PCR results. Gastric lavage culture was positive in 46.6% of the patients and PCR was positive in 63.3% of the patients. Adenoid culture and PCR were positive in 56.3% for each, while tonsil culture was positive in 70% and PCR was positive in 90%. H. pylori presence in the gastric lavage, the tonsillar and adenoid tissues by culture and PCR was significantly more frequent in the study group compared to the control group. The minimum inhibitory concentration (MIC) values of clarithromycin-resistant isolates ranged from 1.5 to 8 μg/ml. This study showed the presence of H. pylori in around 50% of the patients with OME. PCR revealed its sensitivity than culture techniques. The incidence of clarithromycin resistance was found to be high among the isolates (39.6%).     

Effectiveness of three times daily lansoprazole/amoxicillin dual therapy for Helicobacter pylori infection in Korea

AIM

We compared three times daily dual therapy with standard triple therapy for effectiveness and safety in H. pylori infection.

METHODS

Two hundred and four H. pylori positive patients with peptic ulcer were randomly assigned to one of two regimens: (i) triple therapy with amoxicillin, clarithromycin and lansoprazole twice daily for 2 weeks or (ii) dual therapy with amoxicillin and lansoprazole three times daily for 2 weeks. The success of eradication was evaluated 4 to 5 weeks after completing treatment.

RESULTS

The eradication rate was 82.8% in the triple therapy group and 78.4% in the dual therapy group by per protocol analysis. This difference was not significant (P= 0.573). Adverse events were more frequent in the triple therapy group than in the dual therapy group (P= 0.002).

CONCLUSIONS

Because dual therapy had fewer side effects than triple therapy and a similar eradication rate, dual therapy may provide an acceptable alternative first line therapy for H. pylori eradication in Korea.