Examination of the correlation between Helicobacter pylori and T cell response with the determination of PD-1 expression level in gastric pathologies

Helicobacter pylori (H. pylori) is a gram negative microaerobic bacterium that coccoid or sphiral shaped bacterium that colonizes in stomach. Bacteria is around 2.5-3 μm length. Each H. pylori has around 6 flagella. This flagellas enables the bacterium movement and survival under harsh environmental conditions by escaping. More than 50% of people all around the world are infected by this bacterium and world health organization accepts H. pylori as primary cancerous. Most of the infected people by H. pylori do not show any clinical disease evidence, it maintains its existence sub clinically. Generally, infection starts at childhood and maintain lifelong. However, if bacterium could not detect early and treated properly, it caused several gastric pathologies. These pathologies are ulcer, gastritis, gastric carcinoma and MALT. Gastric Cancer has two main subtypes; intestinal type gastric cancer and diffuse type gastric cancer. Intestinal type gastric cancer is more frequently diagnosed than diffuse type. The prevalence of intestinal type is around 60% and there is a stepwise progression of disease. When the healthy gastric epithelium infected with H. pylori, host immune system responds it. Inflammation starts and if bacterium is not treated or eliminated by immune system, inflammation becomes a chronic phase; chronic gastritis. If bacterium does not eradicated atrophic changes in gastric epithelium takes place. One of the most important atrophic changes is degeneracy of glandular structure of gastric epithelium. After the degradation of gastric epithelium structure, disease could be progressed to intestinal metaplasia stage. Intestinal metaplasia is a pathological discrimination of gastric disease, which is mainly the differentiation of gastric epithelial cells to small intestine or colon epithelial cells. After the formation of these epithelial changes, treatments against H. pylori could not efficient to preventation of gastric cancer. Therefore, bacterium detection must be take place in early stages and this detection must be accurate. Detection of H. pylori could be happened by invasive or non-invasive methods. Non-invasive and invasive methods could be used at the same time for more accurate diagnosis. All those H. pylori related diseases are directly related with the host immune responses, genetic susceptibility and virulence factors of bacterium. Especially the resistance of bacterium to the treatment and increasing the survival period in host stomach is directly associated with the virulence factors. Even the genetic strain of H. pylori shows geographical differences in between countries, most important and prevalent virulence factors are urease subunit A (ureA), urease subunit B (ureB) Cytotoxin-associated gene A (cagA), vacuolating cytotoxin gene A (vacA), outer inflammatory protein A (oipA), blood group antigen-binding adhesion (BabA), putative neuraminyllactose-binding hemaggluttinin homolog A (hpaA), neutrophil activating protein A (NapA), duodenal ulcer promoting gene A (dupA). Not every Helicobacter pylori strain has to carry all of those virulence factors but they have to carry one of the urease subunit; ureA or ureB. These virulence factors are importants for bacterial infection, adhesion, colonization and survival periods. When the H. pylori infected, host immune system starts an immediate response against it by innate or adaptive immunity. Innate immunity gives immediate but non-specific responds. However, adaptive immune response is highly specific to pathogenic antigens. Especially, as an adaptive immune response, T cells have an importance on determining the clinical outcome of the infection. One of the recently discovered subtypes of T helper cell is Th17. T helper 17 (Th17) cells are functional in defending the host against the extracellular microorganism infections and has role in induction and propagation of autoimmunity. The master regulatory transcription factor of Th17 is Retineic-acid-receptor-related orphan nuclear receptor gamma t (RORγT). RORγT mainly expresses in thymus and controls the lineage of Th17. Th17 has been secreted 8 cytokines known. IL-17 is main cytokine groups of Th17. PD-1 is programmed death protein that act as immune checkpoint protein. It is expressing by the effector T cells as a surface receptor. Normally, absent on resting T cells. This surface receptor protein is responsible in controlling the T cell response of the body. When it binds its ligand PD-L1, it turns active T cells to inactive T cells. This feature of receptor is important in progression of many diseases. The effect of H. pylori on T cells and gastric epithelium is known but, effect on PD-1 is not clearly explained yet. In this thesis, we worked with the gastric biopsy and resection samples. In total of 60 patients were selected from 5 different pathological conditions. With in this 5 pathological groups; there is 15 normal, 20 active chronic gastritis, 3 inactive chronic gastritis, 9 intestinal metaplasia and 14 gastric cancer patient exists. Normal group was used as uninfected and unaffected control group for gastric malignecies. Patients were selected from patients who admitted to Acıbadem Hospital Gastroenterology Department who undergo endoscopies and Acıbadem Hospital General Surgery Department who undergo resection in between 18 to 80 years old admitted in this study. Each patient informed about the study, and his or her permissions taken. Endoscopic biopsy samples were collected from antrum of patients and resection samples were from tumor sites. Tumor site was detected by macroscopic examination and verified by hematoxylin and eosin staining. First step of this study, we detected the each virulence factors by PCR assay. H. pylori G27 strain used as positive control. G27 strain expresses all of those 9 virulence factors. We correlated the virulence factors expression profiles in between active chronic gastritis and intestinal metaplasia patients. Then verification of datas were confirmed with multiplex PCR assay. Then, we compared our PCR results with pathology reports of patients, which were prepared, based on histological appearance of H. pylori. According to this result, our PCR assay shows almost perfect agreement. Secondly, we examined the gene expression profiles of IL-17, RORγT and PD-1 by RT-PCR. We tried to correlate expression of these three genes with H. pylori infection. We observed that only PD-1 showed significant increase expression profile in infected patiens. The expressions of IL-17 and RORγT did not show significant varience between infected and uninfected patients. Then, we examined and tried to correlate expression profiles of those genes in between normal (uninfected and unaffected), precancerous lesions (inactive chronic gastritis, active chronic gastritis and intestinal metaplasia) and gastric cancer. For, IL-17 and RORγT, highest expression found in normal (uninfected and unaffected) patients. PD-1 had highest expression in active chronic gastritis. Consequently, there was not any stepwise increase or decrease of IL-17 and RORγT expression in between progression steps of gastric cancer but PD-1 showed increasing expression profile.