LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

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http://hdl.handle.net/11527/19401

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Konu "Allergy and Immunology" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma
  • Öge
    Investigation of helicobacter pylori virulence genes and t-cell responses in pediatric gastritis patients
    (Graduate School, 2023) Özdemir, Niran ; Yazgan Sayı, Ayça ; 823936 ; Molecular Biology-Genetics and Biotechnology Programme
    Helicobacter pylori is a gram-negative, spiral-shaped and, microaerophilic gastric pathogen that infects more than half of the world. H. pylori is about 2.5-5.0 μm long and about 0.5-1.0 μm wide. H. pylori has 30 μm long and 2.5 nm thick flagella with four to six unipolar sheaths for motility. H. pylori, infects the individual through oral-oral, fecal-oral, and iatrogenic transmission. H. pylori infection is acquired in childhood and remains lifelong if left untreated. Numerous investigations have shown that if this childhood-acquired gastric pathogen is left untreated, it can cause gastritis, peptic ulcers, MALT, and gastritis gastric cancer. Additionally, since 1994, the World Health Organization and the International Agency for Research on Cancer have classified H. pylori, as a class I carcinogen related to the development of gastric cancer. Developed countries have a lower incidence of infection than underdeveloped countries, even though more than 50% of the world's population is infected with H. pylori. This percentage for adults in our country is more than 70%. The incidence of infection in children is lower than in adults. Furthermore, environmental hygiene, dietary conditions, income level, and water resources are some of the main risk factors that affect children's H. pylori infection. The incidence of H. pylori infection is also significantly influenced by these major risk factors. For the diagnosis of H. pylori, invasive, non-invasive, and molecular methods are frequently used nowadays. The diagnosis of H. pylori in children is more difficult than in adults. Non-invasive and molecular methods are preferred because invasive methods need an endoscopy. H. pylori-infected children have been treated with proton pump inhibitors (PPI) and triple antibiotic therapy, the same as adults. For the treatment of H. pylori infection, novel drug combinations or new treatment approaches have been needed due to the increasing antibiotic resistance in the population. Studies have shown that the interactions of virulence genes, host immune response, stomach microenvironment, and other environmental factors have an impact on clinical outcomes such as peptic ulcer, MALT, gastritis, and gastric cancer resulting from H. pylori infection. H. pylori-specific virulence genes are not only involved in inducing inflammatory responses but also maintain chronic inflammation by controlling and regulating immune responses. H. pylori-specific virulence genes allow the bacteria to colonize and survive in the gastric mucosa, causing more immune escape and induction of premalignant changes. Outer membrane proteins, of the virulence genes of H. pylori, ensure permanent colonization of H. pylori through specific interactions with host receptors. Although the genetic strain of H. pylori differs between geographies, there are the most important and common virulence genes. These are; babA2 (blood-group antigen-binding adhesion 2), oipA (outer inflammatory protein A), and sabA (sialic acid-binding adhesin gene A). Virulence genes that cause tissue damage by producing toxins; are cagA (cytotoxin-associated gene A), and vacA (vacuolating cytotoxin gene A). Other virulence genes are ureA (encoding urease enzyme geneA), ureB (encoding urease enzyme gene B), hpaA (putative neuraminyllactose-binding hemagglutinin homolog A), napA (neutrophile- activating gene A), GTT (γ-glutamyl-transpeptidase), iceA (induced by contact with epithelial gene A) and dupA (duodenal ulcer protein gene). The immune response induced by H. pylori infection is also important in determining clinical outcomes. Adult studies have demonstrated that cytokines and the immune response have a role in maintaining the development of chronic inflammation and regulating infection. In particular, T cells as an adaptive immune response are essential for the clinical consequences of H. pylori infection. T helper cells are mediators of the host's immune response. T helper 1 (Th1) cells are mainly involved in protecting the organism from intracellular pathogens. Th1 cells secrete interferon- ɣ (IFN-ɣ). Th2 cells mainly involve defending the organism against extracellular pathogens. Th9 cells play an important role in defense against helminth infections, tumor suppression, allergic responses, and autoimmunity. Th9 cells secrete IL-9. Th17 is very crucial in maintaining mucosal barriers. Th17 cells secrete IL-17. Th22 cells, on the other hand, play a role in autoimmunity, wound healing, and protective mechanisms against pathogens. Th22 cells secrete IL-22. In addition to helper T cells, T regulatory (Treg) cells are part of the adaptive immune system. Treg cells are immunosuppressive and suppress or reduce the proliferation of effector T cells. The main regulatory transcription factor of Treg is FOXP3. PD-1 is a programmed cell death protein, an immune checkpoint inhibitor. It is expressed in gastric tissue and epithelial cell. When PD-1 binds to PDL-1, active T cells convert to the inactive T cell. PDL-1 inhibits T cell activity on PD-1. According to studies, when H. pylori infection occurs, PD-1 and PDL-1 are associated with tumor development and gastric cancer. Expression of PD-1 and PD-L1 in pediatric gastric biopsies with gastritis infected with H. pylori is not well defined. The results obtained in previous scientific studies have demonstrated the relationship between T cells and H. pylori infection. Numerous studies have demonstrated that the T cell response can also be affected by H. pylori-specific virulence genes. However, there is no comprehensive study examining H. pylori-specific virulence genes and immune response in pediatric patients in the Turkish population. In addition, there is no study in the literature examining the cytokine expression levels of IL-9 produced by Th9 and IL-22 produced by Th22 in H. pylori-infected pediatric patients. One of the aims of our study is to characterize H. pylori-specific virulence genes in H. pylori- infected Turkish pediatric patients. Another aim of the study is to investigate T cell responses (Th1, Th9, Th17, Th22, and Treg) in H. pylori-infected pediatric patients with gastritis. Moreover, it is to investigate the expression of PD-1 and PDL-1 immune checkpoint inhibitors, which are known to affect tumor formation, in H. pylori-infected pediatric patients. The study included 80 pediatric patients, aged 5 to 18, who applied to Istanbul Sarıyer Hamidiye Etfal Hospital with a variety of complaints and met the inclusion criteria. There were only 30 pediatric patients with H. pylori infection out of the 80 pediatric patients. As the control group, 23 patients with non-infected with H. pylori were included in the study. Two biopsy samples were taken from the antrum part of gastritis into the tube containing the RNA later from the patients who underwent endoscopy. DNA isolation with one of the biopsy samples and RNA isolation with the other were performed simultaneously. Using the isolated DNAs, H. pylori infection was identified by urease PCR assays. Isolated DNAs were amplified with primers specific to H. pylori-specific virulence genes by PCR, then visualized by agarose gel electrophoresis. Thus, the characterization of fifteen different H. pylori specific-virulence genes were achieved. The correlation of H. pylori-specific virulence genes with each other was determined by the Pearson product-moment correlation coefficient. The isolated RNA was used to determine the expression of transcription factors and cytokines, which are markers of T-cell subsets, at the mRNA level by the Real-Time PCR. Thus, the expression of transcription factors (FOXP3) and cytokines (IFNɣ, IL-9, IL-17, IL-22), which are Th1, Th9, Th17, and Th22 markers, were determined by RT-PCR for H. pylori-infected 30 pediatric patients with gastritis. Additionally, the mRNA expression level of PD1 and PDL-1 in H. pylori-infected and non-infected (control) patients were determined by RT- PCR. The conventional urease PCR assay was applied to all of the patients who were determined to be infected or not infected with H. pylori according to the results of the pathology report. According to the urease PCR assay, all patients infected with H. pylori had at least one of the ureA and ureB are virulence genes. Patients not included in the ureA and ureB virulence genes were used as the control group according to the criteria. After the detection of infection with H.pylori, correlations were detected between 13 different virulence genes; significant positive correlation; vacAs1-napA, vacAs1-sabA,vacAs1-iceA1,vacs1-ureB , dupA-napA, dupA -sabA, dupA-iceA2, dupA-ureB, napA-oipA, napA- sabA, napA-iceA2, napA-ureB, oip iceA1, oipA-iceA2, oipA-ureB, oipA - vacAm2, sabAiceA2 , sabA-ureB, iceA1- ureB, iceA2-ureB, iceA2- GGT, ureB- GGT, and babA2-cagA, a significant negative correlation is detected; vacAs1-vacAs2 and vacAm1-vacAm2. The distribution of virulence genes of H. pylori strains in inactive chronic gastritis and active chronic gastritis, which are different gastritis pathologies, was compared. As a result, the oipA and iceA2 genes were found to be positive at a significantly higher rate in pediatric patients with active chronic gastritis than in pediatric patients with inactive chronic gastritis. mRNA expression levels were determined by RT-PCR using T cell markers. As a result, the expression level of IFN-ɣ, IL-9, IL-17, IL-22, FOXP3, and a PDL-1 was significantly higher in H. pylori-infected pediatric patients than in H. pylori-noninfected pediatric patients. As previously mentioned, some of our H. pylori-infected individuals had inactive chronic gastritis whereas others had active chronic gastritis. The levels of IFN-ɣ, IL-9, IL-17, IL-22, and FOXP3 expression were compared between the two distinct gastritis pathogenesis to determine whether there was a statistically significant difference. Consequently, there was no significant difference between H. pylori-infected patients with active chronic gastritis versus inactive chronic gastritis. Surprisingly, when the expression of T cell markers in patients with inactive chronic gastritis was compared with the control group; had significantly higher expression than patients with active chronic gastritis. Furthermore, the relationship between H. pylori density (less-moderate-high), T cell markers, and PD-1, PDL-1 expression was investigated. No significant correlation was detected between the H. pylori density and expression of T cell markers and the expression of PD-1, PDL-1. In this study, the detection of virulence genes in H. pylori-infected Turkish pediatric patients diagnosed with gastritis was investigated in detail, and the expression of T cell responses, PD-1 and PDL-1 were determined. Our study is the first to investigate the expression levels of IL-9, IL-22, PD-1 and PDL-1 and the relationship between H. pylori density and the expression of these cytokines and immune checkpoint inhibitors in H. pylori-infected diagnoses with gastritis Turkish pediatric patient population.
  • Öge
    Investigation of the mitochondrial metabolism of Helicobacter-activated B cells
    (Graduate School, 2022) Şentürk, Zeynep Nur ; Yazgan Sayı, Ayça ; Molecular Biology-Genetics and Biotechnology Programme
    Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, and spiral-shaped bacterium and a member of the Helicobacteraceae family. H. pylori was discovered in 1982 by Warren and Marshall. Helicobacter infection can lead to multiple gastro pathologies such as chronic gastritis, gastric cancer, peptic ulcer disease, and mucosa-associated lymphoid tissue lymphoma. Whereas more than 50% of the world population has been infected with H. pylori, 80% of them are asymptomatic. Similar to H. pylori; H. felis is a gram-negative, urease-positive, spiral-shaped, and microaerophilic bacteria. Studies have shown that H. felis can induce gastric atrophy, metaplasia, dysplasia, chronic and persistent inflammatory response, and gastric cancer in mice models. Because H. pylori have less capacity to activate an efficient immune response in mice compared to Helicobacter felis (H. felis); H. felis is used to generate mice models for studying this pathogen. B cells play critical roles in adaptive immunity with antigen presentation to T cells and antibody production. Recently, new B cell subsets have been shown to exert anti-inflammatory and immune suppressive properties were discovered. These B cells are termed regulatory B cells (Bregs) by Bhan and colleagues. In mice; mainly CD19+CD21hiCD23hiCD24hi transitional 2 marginal-zone precursor cells (T2-MZP), IL-10 producing CD1dhiCD5+ B10 cells, CD19+CD21hi CD23- marginal-zone (MZ) B cells, Tim-1+ B cells, CD19+CD5+ B1a cells, CD9+ B cells, CD138+ plasma B cells, and CD138+CD44hi plasma blasts are identified as Breg subsets. For maintaining host immune tolerance and balance effector immune responses, these Breg cells secrete IL-10, IL-35, and TGF-ꞵ cytokines. In addition to cytokines, Breg cells also use cell membrane-bound molecules such as CD39, CD73, programmed death-ligand 1(PD-L1), or aryl hydrocarbon receptors for their functions. Stimulation of B cells with H. felis; signals via TLR2 and MyD88 and results with IL-10 producing Bregs. Bregs can induce differentiation of naive CD4+ T cells to IL-10-producing regulatory Tr1 cells by direct B and T cell interactions for suppressing Helicobacter-associated pathologies. Metabolism is the collection of all anabolic and catabolic reactions which are the generation and breakdown of cellular substances respectively. Oxidative phosphorylation (OXPHOS) is one of the major metabolic pathways inside the cell. It occurs in the mitochondria and consists of the tricarboxylic acid cycle (TCA) and electron transport chain reactions for generating ATP. Shortly, in OXPHOS electrons formed from the tricarboxylic acid cycle (TCA); are combined with molecular oxygen (final acceptor of electron transport chain) and this results in many oxidation/reduction reactions where energy is released for the production of ATP from ADP. Mitochondria are double membrane organelles that have both outer and inner mitochondrial membranes (OMM and IMM) and are found in eukaryotic cells. Membrane transporters and electron transport chain (ETC) complexes of mitochondria localize on the inner mitochondrial membrane. IMM encloses a viscous structure called a mitochondrial matrix. Enzymes, mitochondrial DNA (mtDNA), ribosomes, and nucleotides are placed in the mitochondrial matrix. Mitochondrial DNA encodes 37 mitochondrial genes including 22 transfer RNAs, 2 ribosomal RNAs, and 13 important oxidative phosphorylation polypeptides: ND1, ND2, ND3, ND4L, ND4, ND5, ND6 (parts of Complex I); Cytochrome b (parts of Complex III), COI, COII, COIII (parts of Complex IV) and ATP6, ATP8 (parts of Complex V). To provide expression of a mitochondrial gene, mitochondrial DNA needs to be transcribed by mitochondrial transcription factor A (Tfam), mitochondrial RNA polymerase (POLRMT), and mitochondrial transcription factor B1 and B2 (Tfb1m and Tfb2m). Two of the most critical features of multicellular life are metabolism and immunity. These can be explained as the need to distribute nutrients across cells, tissues & organs and protect from injury and inflammation. In recent years, studies have focused on elucidating the metabolism of immune cells in the context of their survival, activation, differentiation, and functions. For the activation of immune cells, signals which are triggered by metabolic intermediates and ATP molecules are required. In order to maintain proper immune cell activation, differentiation, and function, mitochondrial metabolism which generates energy plays a critical role. Studies have demonstrated that B cell activation with B cell receptor (BCR) or different Toll-like receptor (TLR) ligands changes mitochondrial dynamics. LPS-stimulated B cells enhance mitochondrial mass, and co-stimulation of B cells with BCR ligand IgM and TLR9 ligand CpG increases mitochondrial biogenesis. In addition to that, anti-CD-40 and IL-4 stimulated B cells to undergo OXPHOS. However, there is no information in the literature about the mitochondrial metabolism of Helicobacter-activated B cells. The main aim of this study is to elucidate the mitochondrial metabolism of Helicobacter-infected B cells. For this purpose, B cells were magnetically isolated from spleens of C57BL6 mice and treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Afterwards, cells were collected at respective time points for mitochondrial mass and membrane potential staining by using Mitoview Green and Mitoview 633 or TMRE dyes respectively in the flow cytometry. The supernatant of these cells is used for the IL-10 ELISA experiments for checking their IL-10 secretion. H. felis, PAM3CSK4, and LPS-stimulated B cells increased their IL-10 production most noticeably at 24h and 48h indicating the suppressive capacity of that cells. Also, compared to the unstimulated control group, all of the stimulant groups increased both the mass and membrane potential of mitochondria at 24h and 48h time points. The second aim of our study was to investigate whether B cells with high mitochondrial membrane potential (Mitoview 633+ B cells) produce IL-10 or not. For that, after B cells were isolated from IL-10 GFP reporter (VertX IL10 egfp) mice, they were treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Mitochondrial membrane potential were analyzed by flow cytometry. Afterwards, at 6h, 24h and 48h we evaluated mitochondrial membrane potential of the IL-10 producing B cells with quadrant analysis using flow cytometry. At 6h time there were no significant changes on IL-10+ Mitoview 633+ B cells. But at 24h and 48h time points; H.felis, PAM3CKS4, and LPS-stimulated B cells increased IL-10+ Mitoview 633+ B cells. These data show that in all stimulated B cell groups; high portion of the IL-10 producing B cells also have high mitochondrial potential. Our data shows, H. felis, PAM3CSK4, and LPS-stimulated Mitoview 633 + B cells increased their IL-10 GFP signal both at 24h and 48h time points compared to the unstimulated control group. The third aim of our study was to investigate mitochondrial biogenesis markers: mtDNA:nDNA ratio and mitochondrial transcription factor A (Tfam) gene expression. To perform Q-PCR experiments isolated and treated B cells were collected for DNA and RNA isolation at 6h, 24h, and 48h. For mtDNA:nDNA ratio analysis in Q-PCR; respectively cytochrome c oxidase subunit I (COX1) and 18S ribosomal subunit gene (RPS18) were targeted by using gene-specific primers. While H. felis, PAM3CSK4, and LPS-stimulated B cells decreased their mtDNA:nDNA ratio, they increased Tfam expression level compared to the unstimulated control group. This study showed that H. felis-activated B cells have active/functional mitochondria, and increased oxidative phosphorylation for energy production and their IL-10 production can be related to their mitochondrial metabolism.