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Indian Journal of Medical Microbiology, Vol. 28, No. 1, January-March, 2010, pp. 40-44 Original Article Molecular detection of host cytokine expression in Helicobacter pylori infected patients via semi-quantitative RT-PCR M Eshagh Hosseini, A Oghalaie, G Habibi, A Nahvijoo, ZM Hosseini, M Tashakoripoor, *M Mohammadi Biotechnology Research Center, Pasteur Institute of Iran, Tehran 13164, Iran Correspondence Address: *Biotechnology Research Center, Pasteur Institute of Iran, Tehran 13164, Iran, mmohammadi@yahoo.com Date of Submission: 31-Jan-2009 Code Number: mb10010 PMID: 20061762 DOI: 10.4103/0255-0857.58727 Abstract Background: Helicobacter pylori (Hp) is a bacterium recognised as a main causative agent for the development of chronic active gastritis, peptic ulcer disease, gastric adenocarcinoma and primary gastric lymphoma. Keywords: Cytokine, IL-4, IFN-γ, immune response Introduction H. pylori (Hp) causes chronic gastrointestinal complications infecting more than half of the world's adult population. Despite a vigorous inflammatory response, the immune system is usually unable to clear the infection. [1] The international agency for research on cancer has declared this bacterium as an independent class one carcinogen as it has an aetiological role in the development of gastric cancer. [2] In the face of the fact that Hp infects more than 50% of world's population, only a small proportion manifests clinical outcomes such as peptic ulcer or gastric cancer. [3] It can be postulated that such variability in the manifestations of Hp infection is due to differences in bacterial pathogenesis or host immune responses to the pathogen. [4] Infection with Hp induces infiltration of neutrophils and macrophages into the gastric mucosa. On the other hand, host inflammatory responses to Hp is an indicative factor for gastric mucosal injury. [5] Previous studies have shown that immune response, (specific T cell response) plays a critical role in inducing gastric mucosal inflammation. [6] Immune T helper cells are divided into two groups based on production of different cytokines: T helper one cells promote cell-mediated immunity and produce IFN-γ as a pro-inflammatory cytokine with anti-microbial activity; T helper two cells, on the other hand, induce humoral immunity and produce high levels of IL-4 as an anti-inflammatory cytokine. [7] The main goal of this study was to evaluate host cytokine expression in gastric cancer (GC) and peptic ulcer disease (PUD) patients via RT-PCR and determine the role of Th1 and Th2 cytokines as predisposing factors for Hp-associated gastrointestinal manifestations. Material and Methods Gastric biopsies Biopsy specimens were collected from three groups of non ulcer dyspepsia (NUD = 38), peptic ulcer patients (PUD = 20) and gastric cancer patients (GC = 18) undergoing endoscopy or gastric resection following provision of informed consent. RNA isolation Collected biopsy specimens were instantly frozen in liquid nitrogen, homogenised and total RNA was extracted from the gastric tissue under RNase free conditions, using high pure RNA tissue kit [Roche, Germany]. The concentration and quality of RNA was estimated by determining the absorbance at 260/280 nm and agarose gel electrophoresis respectively. Total RNA was extracted from peripheral blood lymphocytes and used as positive control. Complementary DNA synthesis Complementary DNA (cDNA) was synthesised from total RNA using a reverse transcription system (Invitrogen, Promega Co., WI, USA). Briefly, 0.5-1 μg of RNA was added to 20 U of M-MuLV reverse transcriptase, 10 U RNase Inhibitor, 500 mM of each dNTP, 160 pM of oligo (dT) 12 primer and 5 mM of MgCl 2 in a total volume of 20 μl. The reaction was incubated at 37ΊC for one hour, followed by 10 minutes at 70ΊC to inactivate the enzyme. PCR assays Oligonucleotide primers for HPRT, IFN-γ and IL-4 were selected from published sequences [8] [Table - 1] and synthesised at the Biotechnology Department of Pasteur Institute of Iran. The mentioned cytokines were PCR amplified and cDNA normalised against HPRT expression. Two microliters of the cDNA were separately amplified with each primer pair. Each reaction contained in a total of 25 μl, 2 μl of cDNA, 2.5 μl of 10X PCR buffer which contain 100 mM Tris-HCl (pH9.0), 500 mM KCl, 3 mM MgCl 2 , 1 mM spermidine), 0.5 μl of dNTP mix (10 mM), 0.7 μl of each primer (50 pmol/μl), 0.5U/reaction of Taq DNA polymerase. All reaction tubes were covered by 15 μl mineral oil. PCR amplification was performed under the following condition: 3 min at 95°C for initial cycle, followed by (HPRT: 30 cycles at 94°C for 35sec; IFN- γ: 33 cycles at 66°C for 35 sec and IL-4: 38 cycles at 72°C for 35 sec), followed by a final extension lasting for 4 min at 72°C in a DNA Eppendorf Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany). Analysis of DNA amplification Following PCR amplification, 10 μl of PCR products plus 3 μl of loading dye were run on 2% agarose gel containing 1 μg/ml ethidium bromide. The PCR products were visualised and scanned with Uvidoc, Gel Documentation system (Vilbert Lourmat, France) and the intensity of each band present in each lane was determined by the Molecular Analyst software (Bio-Rad Philadelphia, PA, USA, v. 1.4). Statistical analysis The one way ANOVA test was applied to assess correlation between independent and dependent studied variables (clinical groups, IL-4 and IFN-γ, respectively). For those cases for which such an assumption could not be established, its nonparametrical equivalent, Kruskal Wallis test was used for the analysis. For all tests, the first kind of error (Alpha) was accepted up to five per cent. Results The ratio of male to female for each mentioned group of patients was 9:10, 3:2, 7:2 respectively. The mean age and its standard deviation (SD) for the studied groups were 41.9 ± 16.32 in NUD group, 42.1 ± 18.9 for PUD group and 49 ± 12.7 among GC cases [Table - 2]. Total RNA was extracted from biopsy specimens and run on 0.8% agarose gel to be estimated for its concentration and purity [Figure - 1]. RT-PCR products for each cytokine were run on 2% agarose gel and visualized by ethidium bromide staining [Figure - 2]. Cytokine gene expression analysis Following PCR amplification of each cytokine, the intensity of bands was measured by densitometry and normalised against HPRT expression [Figure - 3]. Under ideal conditions, the amount of PCR products will be doubled during each cycle of PCR reaction which allows for determination of the relative initial amounts of the target sequence and the endogenous standard. Different numbers of amplification cycles for target and standard sequences are often required. In an assumption of a linear range of amplification, the following equation was applied to calculate the number of cycles as well as the amount of template molecules in amplification process. X 0t /X 0s = βX t /X s Where: β = constant amount for each PCR reaction X 0t = initial number of goal molecules X 0s = initial number of standard molecules X t = number of amplified target molecules X s = number of amplified standard molecules The constant number for target sequence depends on the PCR conditions for target sequence. [9] Each graph indicates the densitometry analysis of PCR products. The surface area for each peak [Figure - 3] represents the intensity of each band which is normalized against HPRT expression as a house keeping gene. Thus, the intensity of each cytokine gene to intensity of housekeeping gene indicates the rate of cytokine expression. Expression of cytokine mRNA in gastric specimens The cDNA samples were normalised by PCR using HPRT specific primers due to its presence in all cell types. Normalisation allowed the use of equivalent amounts of cDNA for each assay. The intensity of bands which represent the expression of cytokine genes are shown in [Figure - 3]. Levels of cytokine expression were compared between different clinical groups. The mean IFN-γ gene expression of NUD and PUD groups (non GC individuals) was similar (3.4 ± 0.57 and 3.43 ± 0.41, respectively) but in GC cases, it was significantly higher (5.51 ± 0.32). On the other hand, there were no considerable differences for IL-4 gene expression between NUD and GC patients (2.8 ± 0.43 and 2.3 ± 0.12), but PUD patients showed significantly higher amount of expression for this cytokine (3.7 ± 0.44) [Figure - 4]. Statistical analysis indicated that the differences between IFN-γ and IL-4 expression levels which were observed in different strata of diagnostic variable are significant (P < 0.01). Discussion We selected two cytokines (IFN-γ and IL-4) specifically as the predominant indicators of Th1 or Th2 types of immune responses which are believed to lead to more severe as opposed to milder inflammatory outcomes, respectively. Previous studies have shown that specific T cell responses have critical roles in inducing gastric mucosal inflammation [10],[11],[12],[13] and suggested a pathogenic role for IFN-γ exacerbating gastric inflammation potentially leading to gastric cancer. This may be associated with reduced IL-4 production, driving the immune response toward a Th1 pathway. [14],[15],[16],[17] Other investigators have introduced Hp as a major stimulating pathogen for Th1 responses leading to peptic ulcer disease. [3] As indicated in [Table - 2], our patient population constituted of Hp positive and negative patients but due to small number of samples in each clinical group, H. pylori positive and negative specimens were collectively studied and the resulting conclusion was based on the clinical categories and not H. pylori infection status. Despite the negative association between IL-4 and gastric cancer development following H. pylori infection, [18] our data showed no considerable difference in IL-4 gene expression between GC and NUD patients. Our findings, however, indicated that the gastric immune responses in PUD patients is Th2 predominant. The enhanced expression of the predominant Th1 cytokine, IFN-γ, which promotes macrophage activation and local tissue destruction, in gastric mucosa of cancer patients was in agreement with previous reports. [19],[20] RT-PCR allowed us to measure in situ cytokine gene expression. Previous studies have investigated the host immune responses during Hp infection most of which used biopsy samples as the source of cytokines mRNA molecules. Systemic (blood) PBMCs cytokine expression were not used as specific indicators of in situ host immune responses during Hp infection since it may represent host responses to any other pathogen or instability whereas the response measured in the gastric tissue is due to the infiltration of lymphocytes into the epithelial tissue as a specific result of Hp infection. Such responses can be measured in different categories of Hp infected patients including those with gastritis, peptic ulcer and gastric cancer. This study presents an easy, precise method for isolation of cytokine mRNA molecule from gastric biopsy specimens followed by gene-specific RT-PCR with the goal of analysis of cytokine expression levels in various groups of patients following Hp infection. Despite the fact that one single cytokine cannot fully represent the pro or anti inflammatory cytokine status, this study only provides additional support to previous studies indicating host proinflammatory background as a promoting factor for gastric carcinogenesis. Acknowledgment The authors would like to thank Dr. Ramin Ghadimi and Dr. Amir Mirbagheri from Gastroenterology Department, Amir Alam Hospital, for providing biopsy specimens and Mr. Ramin Sarrami for statistical analysis. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10010f1.jpg] [mb10010f2.jpg] [mb10010f3.jpg] [mb10010t1.jpg] [mb10010f4.jpg] [mb10010t2.jpg] |
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