Evaluation of frxA and rdxA gene mutations in clinical metronidazole resistance Helicobacter pylori isolates.

Metronidazole resistance is an important factor related to failure in the treatment of Helicobacter pylori. The mutation in the rdxA and frxA genes is the most important cause of resistance to metronidazole. Since the resistance rate of metronidazole is high in our region, we decided to assess the frequency of these mutations among H. pylori clinical isolates. Antral gastric biopsy specimens were cultured and minimal inhibitory concentrations (MICs) of metronidazole were determined by the E-test method. The rdxA and frxA genes were amplified in all isolates through the use of PCR with the specific primers. PCR products were purified for sequencing. The resultant sequences were compared with the wild type reference sequences to find any possible mutations. According to our findings, the rate of metronidazole resistance was 77%, with the MICs ranging from 0.25-1 µg/ml for metronidazolesensitive group and from 16-256 µg/ml for resistance group. H. pylori isolates containing a single mutation in rdxA or frxA genes demonstrated a low MIC (8-16 µg/ml), while those containing mutations in both genes showed a higher MIC (32-256 µg/ml). In this study, all resistant H. pylori isolates contained single or multiple nucleotide substitutions in the mentioned genes. Nevertheless, no nucleotide substitutions were found in the sensitive clinical isolates. The results of our study showed that the mutations in rdxA are mostly related to metronidazole resistance, and mutations in frxA are able to enhance H. pylori resistance.

Evaluation of frxA and rdxA gene mutations in clinical metronidazole resistance Helicobacter pylori isolates

Metronidazole resistance is an important factor related to failure in the treatment of Helicobacter pylori. The mutation in the rdxA and frxA genes is the most important cause of resistance to metronidazole. Since the resistance rate of metronidazole is high in our region, we decided to assess the frequency of these mutations among H. pylori clinical isolates. Antral gastric biopsy specimens were cultured and minimal inhibitory concentrations (MICs) of metronidazole were determined by the E-test method. The rdxA and frxA genes were amplified in all isolates through the use of PCR with the specific primers. PCR products were purified for sequencing. The resultant sequences were compared with the wild type reference sequences to find any possible mutations. According to our findings, the rate of metronidazole resistance was 77%, with the MICs ranging from 0.25-1 μg/ml for metronidazole- sensitive group and from 16-256 μg/ml for resistance group. H. pylori isolates containing a single mutation in rdxA or frxA genes demonstrated a low MIC (8-16 μg/ml), while those containing mutations in both genes showed a higher MIC (32-256 μg/ml). In this study, all resistant H. pylori isolates contained single or multiple nucleotide substitutions in the mentioned genes. Nevertheless, no nucleotide substitutions were found in the sensitive clinical isolates. The results of our study showed that the mutations in rdxA are mostly related to metronidazole resistance, and mutations in frxA are able to enhance H. pylori resistance.

The effect of ω-fatty acids on mRNA expression level of PPARγ in patients with gastric adenocarcinoma.

BACKGROUND: The antineoplastic role of peroxisome proliferator-activated receptor gamma (PPARγ) ligandshas previously been demonstrated in several gastric cancer cell lines. Activation of PPARγ by polyunsaturated fatty acids (PUFAs) inhibits growth and proliferationof tumor cells. In this double-blind clinical study, we evaluate the effect of PUFAs on PPARγ mRNA expression in patients with gastric adenocarcinoma. MATERIALS AND METHODS: A total of 34 chemotherapy-naive patients diagnosed with gastric adenocarcinoma were enrolled in the present study. According to treatment strategies, all subjects were divided into two groups, the first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFAs supplements for 3 weeks. The gastric biopsy samples were obtained from all participants before and after treatment, and PPARγ mRNA expression levels were evaluated by quantitative real-time polymerase chain reaction using validated reference genes. RESULTS: Our findings revealed that PPARγ mRNA expression is significantly upregulated in group II afterreceiving cisplatin plus orally administered PUFAs supplements for three weeks (p < 0.0001), whereas PPARγ mRNA expression did not show significant alteration in group I after receiving cisplatin alone. CONCLUSION: The results of the study evidence that PPARγ may act as a potential target for the therapy of human gastric adenocarcinoma.

Combination effect of melatonin and dexamethasone on liver ischemia/reperfusion injury.

OBJECTIVE: Liver failure following ischemia-reperfusion (I/R) injury is a major concern in liver surgery. The purpose of this study was to evaluate combination pretreatment with melatonin (MEL) and dexamethasone (DEX) on liver I/R model. Male Wistar rats (n = 60) were assigned to 5 groups of 12 animals each: (1) Sham: laparotomy without I/R; (2) I/R: hepatic I/R; (3) I/R+MEL: hepatic I/R+melatonin injected intraperitoneally (20 mg/kg); (4) I/R+DEX: hepatic I/R+ dexamethasone injected intravenously (10 mg/kg); (5) I/R+MEL+DEX: hepatic I/R+ melatonin injected intraperitoneally+dexamethasone injected intravenously. The liver was subjected to ischemia by clamping the portal triad for 30 minutes and then reperfused for 6 hours after ischemia by removing the clamps. RESULTS: The levels of glutathione peroxidase (GPx) and superoxide dismutase (SOD) decreased after hepatic I/R in all groups. Levels of GPx and SOD were higher in I/R+MEL+DEX group compared to I/R, I/R+MEL and I/R+DEX groups and they were significantly higher in I/R+MEL group compared to I/R and I/R+DEX groups (p < 0.05). Levels of ALT, AST, TNF-α, hepatic tissue malondialdehyde (MDA), liver injury index, and apoptotic index increased after hepatic I/R. Levels of ALT, AST, tissue MDA, tissue injury index and apoptotic index were lower in I/R+MEL+DEX group compared to those in I/R, I/R+MEL and I/R+DEX groups, and in I/R+MEL they were significantly lower than in I/R+DEX group (p < 0.05). TNF-α level was lower in I/R+MEL+DEX group compared to other groups and it was significantly lower in I/R+DEX group than in I/R+MEL and I/R groups (p < 0.05). CONCLUSION: Combination therapy with melatonin and dexamethasone had better results in decreasing the liver injury compared to when each of them was administered alone (Tab. 3, Ref. 58).

Effects of probiotic yogurt consumption on metabolic factors in individuals with nonalcoholic fatty liver disease.

The aim of this study was to investigate the effects of probiotic yogurt consumption on some metabolic factors in nonalcoholic fatty liver disease (NAFLD) patients. This double-blind, randomized, controlled clinical trial was conducted on 72 patients with NAFLD (33 males and 39 females) aged 23 to 63 yr. Subjects in the intervention group (n=36) consumed 300 g/d of probiotic yogurt containing Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 and those in the control group (n=36) consumed 300 g/d of conventional yogurt for 8 wk. Fasting blood samples, anthropometric measurements, and dietary records (24h/d for 3 d) were collected at baseline and at the end of the trial. Probiotic yogurt consumption resulted in reductions of 4.67, 5.42, 4.1, and 6.92% in serum levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, and low-density lipoprotein cholesterol, respectively, compared with control group. No significant changes were observed in levels of serum glucose, triglycerides, or high-density lipoprotein cholesterol in either group. Probiotic yogurt consumption improved hepatic enzymes, serum total cholesterol, and low-density lipoprotein cholesterol levels in studied subjects and might be useful in management of NAFLD risk factors.

Lipoic acid prevents hepatic and intestinal damage induced by obstruction of the common bile duct in rats.

INTRODUCTION: Cholestatic liver diseases are characterized by impaired hepatocellular secretion of bile, resulting in intracellular accumulation of bile acids which result in a shift in the oxidant/prooxidant balance in favor of increased free radical activity and injury of different tissues including liver and intestine. The aim of this research was to study protective effect of lipoic acid (LA) as a potent antioxidant in cholestsis induced hepatic and intestinal injury in rats. MATERIALS AND METHODS: Forty five adult male Wistar rats were randomly assigned to four groups each containing fifteen rats as follows: sham operation (SO) (control), bile duct ligating (BDL), and BDL+LA (25 mg/kg). After fourteen days hepatic and intestinal tissue sampled and blood serum sampled for pathologic and biochemical studies. RESULTS: Levels of SOD and GPx antioxidant enzymes were higher in BDL+LA group comparing to BDL group, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyltranspeptidase (GGT), and pathologic scores in liver and intestine were lower in BDL+LA group comparing to BDL group significantly, but there is no significant difference in concentrations of total bilirubin between groups. CONCLUSIONS: Our results showed the protective potential of LA with liver and intestine damage. Despite improvements in operative technique and the development of potent, broad-spectrum antibiotics, biliary tract surgery in patients with obstructive jaundice is still associated with high morbidity and mortality rates In summary, our results show that BDL induced hepatic and intestinal injury were significantly attenuated by LA administration and the administration of LA could effectively diminish this damage.

Hepatitis B virus surface protein mutations clustered mainly in CTL immune epitopes in chronic carriers: results of an Iranian nationwide study.

Mutations within the coding region of hepatitis B surface antigen (HBsAg) have been found naturally in chronic carriers. To characterize the mutations of HBsAg from Iranian chronic carriers who were vaccine and/or medication naive. The surface genes from 360 patients were amplified and directly sequenced. The distribution of amino acid substitutions was classified according to different immune epitopes of the surface protein. All isolates belonged to genotype D. 222 (61.6%) of 360 patients contained at least one amino acid substitution. 404 (74.5%) of 542 amino acid changes occurred in different immune epitopes of HBsAg, of which 112 (27.7%) in 32 residues of B-cell epitopes (62 in the 'a' determinant); 111 (27.4%) in 32 residues of T helper; and 197 (48.7%) in 32 residues inside cytotoxic T lymphocyte (CTL) epitopes. One Th (186-197) and two CTL (28-51 and 206-215) epitopes were found to be hotspot motifs for the occurrence of 213 (52.7%) substitutions. 20 stop codons were identified in different epitopes. There was a significant association between amino acid substitutions and anti-HBe seropositivity; however, the correlation between such changes with viral load and ALT levels was not significant. In chronic hepatitis B virus(HBV) carriers, positive selection in particular outside the 'a' determinant appeared to exert influence on the surface proteins. These changes could be immune escape mutations naturally occurring due to the host immune surveillance especially at the T-cell level.

Some early results related to electrical impedance of normal and abnormal gastric tissue.

Gastric cancer is the fourth most common cancer and most patients with gastric cancer are being diagnosed in advanced stages of the disease so they do not gain any survival chance from conventional surgical, chemotherapeutic or radiotherapeutic methods. These are relatively high cost procedures in terms of both time and money. This study considers the introduction of a novel minimally invasive diagnostic technique which shows the relationship between histopathology and the electrical impedance spectrum in the human stomach. In this study, 4 electrode technique was used to differentiate tissues from each other using Tabriz Mark 1 electrical impedance system (30 different frequencies in the range of 2 kHz to 1 MHz). A total of 97 points from 45 patients were studied in terms of their biopsy reports matching to the electrical impedance measurements (in vivo). After impedance measurements and applying calibration factors, a non-parametric statistical technique, the Kruskal-Wallis test was used to evaluate the difference among the groups. According to the calculation of respective data using this spectroscopy system, the resistivity of the normal group was higher than that of the benign group, and the resistivity of these groups were higher than that of the malignant group at frequencies between 470 kHz and 1 MHz (P < 0.05). In these frequencies, the impedivity of the dysplastic tissue was significantly lower than that of the other groups (P < 0.05). Also, Cole equation fitting procedure was used to generate a scatter plot of the malignant and benign points: it shows in general, benign points had higher values of R than the malignant points. Therefore, electrical impedance spectroscopy can be a useful technique to characterize the stomach tissue.

Rapid urease test, touch cytology and histopathologic assessment in determining infection by Helicobacter pylori in outpatient setting.

Helicobacterpylori (HP) is a common cause of gastric infection with serious consequences which is detected by different methods. This study aimed at comparing the diagnostic value of Rapid Urease Test (RUT), Touch Cytology (TC) and histopathologic assessment in outpatients setting. In this cross-sectional study, 51 candidates for upper gastrointestinal endoscopy were recruited in Tabriz Imam Khomeini Teaching Centre in a 24 month period of time. Three biopsy specimens were obtained from gastric antrum during endoscopic intervention. The RUT, TC and histopathologic assessment were performed on each biopsy specimen in each patient. Definite infection by HP was considered when at least 2 out of 3 tests indicated presence of infection. Fifty one patients, 29 females and 22 males with a mean age of 40.10 +/- 12.54 (range: 18-72) years enrolled in this study. Infection by HP was definite in 41 cases (80.4%). The infection rates by RUT, TC and histopathologic examination were 82.4, 82.4 and 76.5%, respectively. The sensitivity, specificity and accuracy of RUT, TC and histopathologic assessment were 92.7, 60 and 66.75%; 100, 90 and 98% and 95.1, 100 and 96.1%, respectively. There were significant agreements between outcomes of the three methods in diagnosis of infection by HP. In conclusion, TC was the most sensitive and histopathologic assessment was the most specific method in diagnosis of infection by HP in outpatient setting. The diagnostic value of RUT was rather low in this regard.

Independent predictors of in-hospital re-bleeding, need of operation and mortality in acute upper gastrointestinal bleeding.

Prediction of outcome is difficult in patients with acute upper gastrointestinal bleeding (AUGIB). Some factors have been proposed in this regard with varying accuracy. This study aimed to investigate probable predictors of in-hospital outcome in patients with AUGIB. One hundred sixty four patients with AUGIB were studied prospectively in Tabriz Imam Reza Teaching Centre. All these patients were evaluated endoscopically by an expert. Patients' age, gender, presenting complains, transfusion, clinical findings and previous medical history were compared between survived vs. expired, re-bled vs. non re-bled and operated vs. non operated patients. There were 117 males and 47 females with the mean age of 57.12 +/- 17.32 (range: 32-78) years in this study. Hematemesis was the sole independent predictor of in-hospital mortality (82.1 vs. 100%; p < 0.001). In univariate analysis, however, female gender, major hemorrhage and previous neurological disease were associated with higher rate of expiration. Comparing two re-bled and non re-bled groups, hematemesis (76.5 vs. 95.9%; p = 0.003) and need of transfusion > 2U (36.1 vs. 71.4%; p = 0.006) were independent predictors of re-bleeding. In univariate analysis, hematocrit < 30%, major hemorrhage and previous history of hepatic disease or hypertension were predictive of re-bleeding. In comparison between operated and non operated groups no significant predictor was detected. In conclusion, this study showed that presence of hematemesis at the time of admission and need of transfusion > 2U were independent predictors of poor outcome in patients with AUGIB.

Pioglitazone attenuates ischemia/reperfusion-induced liver injury in rats.

INTRODUCTION: Hepatic ischemia/reperfusion (I/R) injury leads to free radical generation and acute inflammatory responses that cause liver damage, an important problem for liver transplantation. Pioglitazone is known to protect I/R injury in various tissues; however, the mechanism of cytoprotection is not well understood. This study investigated the effects of pioglitazone administration in a warm hepatic I/R model on tumor necrosis factor (TNF)-alpha level, tissue injury, and antioxidant enzyme activity. MATERIALS AND METHODS: Eighty wistar strain rats were divided into 4 groups (n = 20): Group 1 sham hosts; Group 2 hepatic I/R; Group 3 hepatic I/R + pioglitazone (10 mg/kg); and Group 4 hepatic I/R + vehicle. Rat livers were subjected to 30 minutes of ischemia followed by 6 hours of reperfusion. After reperfusion rats were humanely killed to obtain liver tissue to study glutathione peroxidase (GPx), superoxide dysmutase (SOD), malondialdehyde (MDA) levels and for histopathologic assessment. TNF-alpha, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured in serum. RESULTS: Pioglitazone pretreatment significantly reduced liver enzyme content (ALT, 176.80 +/- 13.75 vs 235.28 +/- 31.92 and AST, 748.20 +/- 79.29 vs 944.85 +/- 101.87) and TNF-alpha level (9:8.60 +/- 8.67 vs 138.28 +/- 9.99) after I/R compared with the control group. MDA level (3.02 +/- 0.37 vs 4.36 +/- 0.38) and hepatocytic degeneration were reduced in the pioglitazone-treated group. GPx (2.40 +/- 0.25 vs 1.36 +/- 0.31) and SOD activity (2.22 +/- 0.30 vs 1.40 +/- 0.35) were significantly higher in the pioglitazone-treated group compared with the control group. CONCLUSION: The present study showed that pioglitazone administration improved hepatic I/R injury that was associated with enhanced antioxidant enzyme activities and suppression of TNF-alpha, ALT, and AST levels. Because peroxisome proliferator-activated receptor-gamma agonists are widely used to treat diabetic patients, it may be relatively easy to expand their clinical indication. However, further investigations will be required to delineate protective mechanisms by which pioglitazone attenuates hepatic tissue injury after I/R.

MEFV mutations in Iranian Azeri Turkish patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder with more than 60 disease-associated mutations in the responsible gene, MEFV. In the present study, we determined 15 MEFV mutations in Iranian Azeri Turkish FMF patients. Five hundred and twenty-four unrelated patients were tested for 15 known mutations in the MEFV gene using amplification refractory mutation system-polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism methods. Thirty-five different genotypes were characterized among the studied patients. Of the alleles investigated, the most common mutation was p.M694V (42.4%), followed by p.V726A (17%), p.E148Q (16.2%), and p.M680I (c.2040G>C) (15.2%). The p.R761H mutation (4.7%) was found to be the most frequent among the rare mutations. The mutations p.M680I (c.2040G>A), p.I692del, p.M694del and p.K695R were not found in this cohort. The remaining mutations account for 7.7% of the identifiable mutations. Five different types of complex alleles were also identified. The results show the diversity and the frequency of the mutations in the Iranian Azeri Turkish FMF patients. The p.R761H mutation is rather prevalent in Azeri Turks; therefore, it should be included in the routine molecular diagnosis of FMF patients from this ethnic group.

Simvastatin attenuates intestinal ischemia/reperfusion induced injury in rat.

Ischaemia/reperfusion (I/R) injury is commonly seen in the field of intestine surgical interventions, shock, trauma, and many other clinical conditions. Simvastatin is known to have antioxidant and anti-inflammatory properties. This study investigated the effect of simvastatin administration in a warm intestinal I/R model on TNF-alpha, antioxidant enzymes and intestinal tissue morphology. Thirty-six male wistar rats underwent laparotomy under general anaesthesia. Simvastatin was administered from four days before ischaemia induction. The rats were divided in to three groups (n = 12): the sham group, the I/R group, and the I/R + simvastatin group. Intestinal ischaemia was induced by superior mesenteric artery ligation with microvascular clamps for 60 minutes, and after ischaemia, blood perfusion was released into the tissue and a reperfusion phase was started, which lasted for 3 hours. After 3 hours, the animals were sacrificed and serum and tissue obtained for biochemical and histological study. In the simvastatin treated group, intestinal tissue injury, TNF-alpha level, and tissue malondealdehyde levels were significantly lower than in the I/R group (p < 0.05). Glutathion peroxidase and superoxide dismutase levels were significantly higher in the simvastatin treated group than in the I/R group (p < 0.05). Simvastatin pretreatment reduced intestinal I/R injury and was associated with down- -regulation of serum TNF-alpha and tissue malondealdehyde level, and simvastatin administration maintained cellular antioxidant enzyme contents compared to the I/R group after 3 hours reperfusion time.

Upper gastrointestinal bleeding during the first month after renal transplantation in the mycophenolate mofetil era.

Upper gastrointestinal (GI) bleeding remains a significant cause of mortality and morbidity among renal transplant recipients. We retrospectively analyzed the records of patients who received renal transplantations between January 2001 and July 2007 using mycophenolate mofetil (MMF) in their immunosuppressive regimens. The following data were recorded for those subjects with upper GI bleeding during the first month after transplantation (group B, cases): age, sex, acute rejection episodes, pretransplant upper GI endoscopic findings, Helicobacter positivity, and cytomegalovirus (CMV) seropositivity. The same parameters were studied among a group of patients, who did not have a history of upper GI bleeding (group A, controls). A statistical analysis was performed to ascertain potential risk factors. Among 523 patients (311 females, 212 males) of age range 7 to 58 years, 27 (5.2%) had upper GI bleeding: 13 males and 14 females of mean age 44 +/- 12 years. The most frequent endoscopic finding was erosive gastritis (n = 13; 48.1%) followed by duodenal ulcers. Binary logistic regression analysis comparing the 2 groups showed that acute rejection episodes (P = .015) and active CMV infection (P = .046) were the most prominent risk factors for upper GI bleeding during the first month after renal transplantation.

Tumor necrosis factor-alpha gene promoter polymorphism in Iranian patients with chronic hepatitis B.

BACKGROUND: Clearance of hepatitis B virus (HBV) infection requires a good host immune response. Cytokines like tumor necrosis factor-alpha (TNF- alpha) may play a role in such immune response. Genetic changes in TNF-a gene promoter region are known to influence TNF- alpha expression. We therefore studied the role of one such mutation in chronic HBV infection. METHODS: Presence of -308 G/A polymorphism in the promoter region of TNF- alpha gene was looked for in 100 patients with chronic HBV infection, 91 subjects with spontaneously recovered HBV infection and 89 healthy controls, using a PCR-RFLP method. RESULTS: Variant alleles (A/A or A/G) were found in 22 of 100 (22%) patients with chronic HBV infection, 21 of 91 (23%) subjects with spontaneous HBV clearance and 14 of 89 (15.7%) control subjects (p=ns for inter-group comparisons). CONCLUSION: TNF- alpha promoter polymorphism -308A is common in Iranian population, but has no association with development of chronic HBV infection.