138 J Contemp Med Sci | Vol. 2, No. 8, Autumn 2016: 138–140 Research Interleukin-8 251- A/T polymorphism related to peptic ulcer disease in H. pylori infected patient Israa Saeed Abbas ISSN 2413-0516 Department of Clinical Laboratories, College of Applied Medical Sciences, Karbala University, Iraq. Correspondence to Israa Saeed Abbas (email: alsultanyisraa81@gmail.com). (Submitted: 17 June 2016 – Revised version received: 03 October 2016 – Accepted: 10 October 2016 – Published online: 26 December 2016) Objectives This study includes the investigation of antifungal activity of the local propolis against dermatophytes and yeast. Methods A total of 92 tissue samples were taken from patients infected with H. pylori and 102 from uninfected individuals has been included. Genetic method used for the detection of H. pylori infection by polymerase chain reaction (PCR) for glM gene identification. IL-8- 251 A/T polymorphism was detected by allele-specific oligonucleotide polymerase chain reaction (ASO- PCR). Results Among the studied samples, the results improve that genetic polymorphism of IL-8-251 A/T genotype was found in a higher frequency with confidence interval 95% in duodenal ulcer H. pylori infected patient than control. Conclusion IL-8-251 A allele notice that it has been associated with severe inflammation lead to increase severity of disease. Keywords Helicobacter pylori, molecular biology (PCR), allele-specific oligonucleotide. Introduction Helicobacter pylori, is a bacterium that infects the stomach of humans, and strongly associated with gastroduodenal diseases such as chronic atrophic gastritis, peptic ulcer,1,2 and gastric cancer.3,4 Although this bacterium has been classified as a car- cinogen in human, keys of pathophysiological events in H. pylori infections is the induction of the inflammatory response in the gastric mucosa, which is mediated and also regulated by inflam- matory cytokines produced by gastric epithelial cells.5,6 Inter- leukin-8 (IL-8) was a small peptide (chemokine) secreted by a variety of cell types, which serves as a potent inflammatory mediator recruiting and activating neutrophils. Interleukin-8 (IL-8), was a potent chemo attractant for neutrophils and lym- phocytes, has been reported as a strong stimulator of angio- genesis in gastric adenocarcinoma.7,8 Several studies have demonstrated that H. pylori strains are capable of inducing IL-8 secretion from gastric carcinoma cells in vitro.9,10 Others researcher noticed that IL-8 is typically secreted by gastrointes- tinal epithelial cells in response to pathogenic bacteria.11 Genetic polymorphisms that affect innate immune response genes have also been linked to an increased risk of gastric cancer, and the levels of IL-8 are directly related to the severity of gastritis.12 The genetic polymorphism of IL-8-251 A allele notice to be associated with higher IL-8 production, more severe inflamma- tion, mucosal atrophy, and also intestinal metaplasia when com- pared with the IL-8-251 TT genotype of H. pylori-infected patients. The recent studies suggested a possible association of the IL-8-251 A allele with angiogenesis and inflammation in gastric carcinogenesis in H. pylori-infected Koreans 13,14 Materials and Methods Patients and gastric biopsy samples 194 tissue samples included, 128 women and 66 men with age range from 15 to 75 years (mean age 36 years). They were pre- sented with dyspepsia and referred to the Esophago Gastrodu- odeno Scope. The endoscopic diagnosis was grouped into three categories: firstly was peptic ulcer disease (PUD) patients who had endoscopic lesions of ulcers, secondly the non-peptic ulcer disease patients (NPUD) were defined as patients who had endoscopic with no lesions of ulcers but they have disease like gastritis or gastric atrophy or gastropathy, and lastly, the control group was defined as they did not have any type of gastric or duodenal disease. Two tissue biopsies were obtained from antrum. Rapid urease test was performed on one of the antral biopsies at the time of endoscopy. The other biopsy specimens placed in 1 ml of normal saline. The biopsy specimen was preserved immedi- ately at −20°C for molecular analysis. DNA extraction and PCR amplification conditions Total DNA extracted directly from gastric biopsy samples using tissue protocol (Geneaid, Korea). The final volume of DNA extraction product was 200 μl, with final concentration 1.25 ng/μl. Identification of H. pylori glM gene To confirm the presence of H. pylori, DNA in biopsies amplifi- cation and melting conditions were optimized for the PCR assay by using specific primer sequences for gene ureC (glmM) 294 (bp) PCR selected were as follows: glmM – forward primer (5´- AAGCTTTTAGGGGTGTTAGGGGTTT -3´), and glmM–reverse primer (5´-AAGCTTACTTTCTAACACT- AACGC -3´). PCRpremix™ kit from (Bioneer, Korea) was used to amplify the mentioned gene. Total reaction volume of 20 μl containing, 3 μl of extracted DNA, 1 μl of 10 pmol/μl of each forward and reverse primers for glmM gene in addition to 14 μl of molecular biology grade water then the mixture was added to lyophilized PCRpremix™ formula. Genotyping of IL-8 251A/T polymorphism gene (ASO) PCR techniques used for the detection of IL-8 251 pol- ymorphism. It is a simple method to detect of mutation involving single base changes or small deletion. It depends on the sequence of specific primer (specific PCR primer) that Israa Saeed Abbas 139J Contemp Med Sci | Vol. 2, No. 8, Autumn 2016: 138–140 Research Interleukin-8 251- A/T polymorphism related to peptic ulcer disease in H. pylori infected patient allows the amplification of test DNA only when target allele is contained with samples following reaction in the presence or absence of target allele. PCR working solution The master mix component for the detection of IL-8 251 poly- morphism gene adopted by ARMS PCR as shown in Table 1. PCR protocol PCR was performed in a thermo cycler under the following conditions adopted in Table 2. PCR amplification analysis Eskandar et al. 2006 336 CCA CCA TTT GGT GAA TTA TCA ATIL-8 - F 1 CCA CCA TTT GGT GAA TTA TCA AA IL-8 - F 2 TGC CCC TTC ACT CTG TTA ACIL-8 - R Table 1. The master mix components of PCR used for detection of IL-8 251 A/T polymorphism ComponentConcentrationAmount (μl) Deionized water7.0 PCR Buffer 2X2.0 PCR F-1 Primers10X1.5 PCR R Primers10X1.5 Taq DNA Polymerase5 U/μl1 dNTP 0.5 Mgcl 2 1.5 Specimen DNA or Control DNA from kit(200 ng/ μl) 10X5.0 Total volume20 Table 3. Distribution of disease group among positive and negative H. pylori infected samples Total H. pylori –veH. pylori +veGroups 37 829PUD 102 5646NPUD 2 20C.A 53 3617Control 194 10292Total Table 2. The PCR protocol for detection of gene No.Step Temperature (C°) Time No. of Cycles 1Initial denaturation941 min1 2 Initial annealing and extension5760 sec 35 Denaturation9360 sec Annealing and extension7260 sec 3Final extension725 min1 Fig.1 Representative results relating to the IL-8 genotyping. ASO PCR was used. By means of the allele-specific primers, the homozygote mutant (AA), and the heterozygote (AT ) and the homozygote TT variants. The image is from a representative gel electrophoresis of PCR amplification products of IL-8 gene (336 bp) were distinguishable. M: marker of the DNA. Fig. 2 Representative results relating to the IL-8 genotyping. ASO PCR was used. By means of the allele-specific primers, the homozygote mutant (AA), and the heterozygote (AT) and the homozygote TT variants. The image is from a representative gel electrophoresis of PCR amplification products of IL-8 gene (336 bp) were distinguishable. M: marker of the DNA. Table 5. The presence of the genotype polymorphisms of IL-8251 A/T gene in H. pylori-positive patients among disease groups Total H. pylori +ve N (%) Groups 3729 (78%)PUD 10246 (45%)NPUD 20 (0%)C.A 5317 (32%)control 19492 (47%)Total Statistical analysis The Chi-square test or Fisher’s exact test was used to com- pare between proportions. The values of P < 0.05 were con- sidered statistically significant. A prevalence ratio (PR) with a 95% confidence interval (CI) was calculated to evaluate the relationship between IL-8 251A/T genotyping polymor- phisms with gastroduodenal diseases and with H. pylori infection. Table 4. Prevalence of IL-8-251 A/T polymorphism among positive and negative H. pylori infected samples All H. pylori –ve % H. pylori +ve % Total (N = 194 ) (N = 102) (N = 92) AA 4 3.9% 0 0% 4 AT 86 84.3% 92 100% 178 TT 12 11.8% 0 0% 12 Total 102 100% 92 100% 194 140 J Contemp Med Sci | Vol. 2, No. 8, Autumn 2016: 138–140 Interleukin-8 251- A/T polymorphism related to peptic ulcer disease in H. pylori infected patient Research Israa Saeed Abbas Results In this study, a total of 141 patients who were affected with different forms of gastric diseases [37 of them had peptic ulcer diseases (PUD) and 102 had non-peptic ulcer disease (NPUD) and 2 patients had gastric cancer (C.A)]. were compared with 53 who had just epigastric pain while their endoscopic exami- nations revealed, they were regarded as a control group. The presence of the genotype polymorphisms of IL-8251 A/T gene in H. pylori-positive patients among disease groupe show in Table 5. In this table from total 92 IL-8251 A/T gene in H. pylori-positive samples all peptic ulcer disease grouped and all non-peptic ulcer disease grouped show IL-8251 A/T gene, improve that there was significant difference in contrast to the control grouped. Discussion Polymorphism in the IL-8 (–251) has been associated with the increased expression of IL-8 (15). Several papers have demon- strated an association between IL-8 (-251) polymorphism and an increased risk of developing gastroduodenal diseases.16 A significantly higher frequency of the IL-8 251AT genotype was observed among the H. pylori-positive DU patients than among the H. pylori-positive healthy subjects without gastrointes- tinal problems. This genotype reflects a higher IL-8-producing ability.17 The association with IL-8 was higher reconnoitered at the level of a single SNP (-251 A/T). The higher incidence of the -251 AT genotype with a concomitant higher IL-8-producing potential18,17 highlights the importance of the genetic determina- tion of IL-8 production in H. pylori-induced DU. We observed a greater frequency in the AA allele geno- types among patients with C.A stomach than in the control group or in other disease group, with the presence of allele A being associated with the risk of developing gastric cancer. Other researcher noted that presence of the A allele in the -251 position of the IL-8 gene was associated with an increase in the risk of stomach cancer in Japanese, Korean, Chinese and Iranian populations.13 Additionally, some studies have demon- strated that patients carrying the A allele present higher pro- duction of IL-8, leading to alteration in the quality and intensity of inflammatory responses produced by the host after exposure to H. pylori.13,19 Conversely, the frequency of the TT genotype (with a relatively low IL-8-producing potential) was significantly higher among the H. pylori negative, healthy individuals. This suggests the possibility that a relative protection from DU disease is observed in association with the TT geno- type. This observation is consistent with the results of,20 who concluded that H. pylori-positive healthy individuals with the IL-8-251 TT genotype might display a milder inflammatory reaction. 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