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Background The progression to gastric cancer has been linked to chronic infection with Helicobacter pylori (H. pylori). Immune checkpoint inhibitors (programmed cell death -1, PD-1; programmed cell death –ligand 1, PD-L1) have a role in cancer immune escape. The relationship between H. pylori virulence factors with PD-1, PD-L1 T helper 1 (Th1), T helper 17 (Th17), and regulatory T cell (Treg) response genes, has not been thoroughly investigated in the development of gastric cancer. Therefore, we evaluated how H. pylori virulence factors influence the expression levels of immune-related genes in the development of gastric immunopathology. Methods A total of 92 gastric tissues of normal controls and patients with gastritis, gastric ulcer, and gastric cancer were examined for the expression of immune-checkpoint inhibitor genes (PD-1 PD-L1), Th1 (interferon- γ, IFN-γ), Th17 (interleukin- 17, IL-17, Retinoic-acid-receptor- related orphan nuclear receptor gamma t, RORγ-t), and Treg (Forkhead box P3, FOXP3) response genes with quantitative real-time PCR (qRT-PCR). Furthermore, correlation of H. pylori virulence factors’ (cytotoxin-associated gene A, cagA; vacuolating cytotoxin gene A, vacA (s1,s2,m1,m2); blood group antigen-binding adhesin gene A, babA, duodenal ulcer promoting gene A, dupA; the putative neuraminyllactose-binding hemagglutinin homolog, hpaA; neutrophil-activating protein A napA; outer inflammatory protein A, oipA; urease A, ureA; and urease B, ureB) genotypes with a degree of inflammation and density of H. pylori were investigated. Next, the relationship between H. pylori virulence factors and immune-checkpoint inhibitor genes, and T-cell response genes was evaluated. Eventually, a decision tree model was developed to determine the clinical outcome of patients using expression data. Results The intensity of PD-1 and PD-L1 mRNA expression was increased significantly in gastric tissue of patients with gastric ulcer (PD-1: 2.3 fold, p=0.01; PD-L1: 2.1 fold, p=0.004), and gastric cancer (PD-1: 2 fold, p= 0.04; PD-L1: 1.8 fold, p=0.05) compared with control subjects. Also, PD-1: PD-L1 expression was significantly higher in patients with gastritis, who were infected with a marked density of H. pylori compared with its mildly infected counterparts. Furthermore, a novel negative correlation was found between PD-1 (r= -0.43) and PD-L1 (r= -0.42) with FOXP3 in patients with gastritis. CagA-positive H. pylori strain's negative association with PD-L1 expression (r=-0.34) was detected in patients with gastritis. Interestingly, PD-1 mRNA expression correlated positively with vacA s2/m2, in gastritis (r=0.43) and ulcer (r=0.43) patients. Furthermore, PD-1: PDL1 expression negatively correlated with vacA m1/m2 (r=-0.43 for PD-1; r=-0.38 for PD-L1) in gastritis patients. Moreover, an inverse correlation of PDL1 was present with vacA m1 (r=0.52) and vacA s1/m1 (r=0.46) versus vacA m2 (r=-0.44) and vacA m1 (r=0.52) and vacA s1/m2 (r=-0.14) in ulcer patients, respectively. Also, a correlation of vacA m2 (r=-0.47) and vacA s1/s2 (r= 0.45) with PD-1 was detected in ulcer patients. In addition, a novel negative correlation between FOXP3 mRNA levels and napA was shown in patients with gastritis and ulcer (r=-0.59). Finally, a computer-based model that was developed showed that knowing the expression levels of PD-L1, RORγ-t, and vacA s1/m2 would be useful to detect the clinical outcome of a patient. Conclusion Our results suggested that PD-1:PD-L1 immune checkpoint inhibitors were increased in gastric pre-cancerous lesions that progress to gastric cancer. Herein, we report the relationship between H. pylori virulence factors and expression of host immune checkpoint inhibitors for diagnostic prediction of gastric malignancies using computer-based models.

ÖZET Helicobacter pylori (H. pylori), insanlarda, gastrit, peptik ülser, gastrik kanser ve mukoza ilintili lenfoid doku lenfoması gibi ciddi mide hastalıklarına yol açan önemli bir hastalık etkenidir. H. pylori, ürettiği üreaz enzimleri sayesinde üreyi, karbondioksit ve amonyağa parçalayarak mide çeperinin asidik koşullarını normal pH’ya getirip hayatta kalabildiği için bu bakterilerle vücudun savaşımı kolay değildir. H. pylori enfeksiyonları için antibiyotikler mevcut olsa da antibiyotik direnci gelişimi nedeniyle bu tedaviler sonuçsuz kalabilmekte ve yeni antibiyotiklere gereksinim her geçen gün artmaktadır. Hidroksisinamik asit türevleri basit fenolik asitler olup meyvelerde, meyve çekirdeklerinde ve sebzelerde bulunmaktadır. Ferulik asit, kafeik asit, p-kumarik asit, sinapik asit, sözü edilen bu fenolik asit grubuna ait olup antioksidan, anti-inflamatuvar, antimikrobiyal özelliklere sahiptir ve bu nedenle bazı bakteri enfeksiyonlarının tedavisinde, ilaçlara seçenek olarak kullanılmaktadır. Bu çalışmamızda, bu üç fenolik asidin H. pylori üzerindeki antimikrobiyal etkinliği ve ürez enzimini engelleme etkisi araştırıldı. Hidroksisinamik asit türevlerinin anti-H. pylori etkisi H. pylori G27 standart suşu üzerinde test edildi. Minimum inhibisyon konsantrasyonu (MİK), değerleri 512 ila 0,5 ug/mL arasında değiştiği seri tüp seyreltme yöntemiyle, minimum bakterisidal konsantrasyon (MBK) değerleri ise MİK içinde kullanılan aynı konsantrasyonlarda canlı ve ölü bakterilerin nispi oranının hesaplanması ile belirlendi. MİK için CLSI M07-A9, MBC için CLSI M26-A protokolleri kullanıldı. Ureaz inhibisyon aktivitesi Helicheck, üreaz aktivitesine özgü indikatörlü besiyerinde ölçülmüştür. H. pylori’ye karşı test edilen tüm bileşikler için MİK 64 ug/mL ve MBK 128 ug/mL idi. Test edilen bileşiklerin H. pylori tarafından salgılanan üreaz enzimi üzerinde hiçbir inhibisyonu saptanmadı. Nükleotid salma deneyi sonuçlarına göre, hidroksisinamik asit türevlerinin bakteri zarında hasara sebep olması ile zarda oluşan deliklerden dışarı salınma eğiliminde olması beklenen herhangi bir nüklotit miktarı ölçülememiştir. Gerçekleştirilen bu çalışma, literatür taramalarımız doğrulutusunda, hidroksisinamik asit türevlerinin anti-H. pylori aktivitesini gösteren ilk çalışmadır. Bu bileşiklerin anti-H. pylori üzerindeki etki mekanizmasını anlamak için daha ileri analizlere ihtiyaç vardır.

The aim of this study is to evaluate the association between seven important H. pylori virulence factors and antibiotic resistance in patients with gastritis. H. pylori strains isolated from 33 patients with gastritis were examined. Antimicrobial susceptibilities were tested by GenoType® HelicoDR (Hain Life Science, Germany) test kit and RT-PCR. The virulence-factors were determined using conventional PCR. 39% of patients were resistant for clarithromycin and 27% of patients were resistant for fluoroquinolone. 15% of patients were resistant to both clarithromycin and fluoroquinolone. The H. pylori vacA m1/s2 genotype was the most frequent allelic combination. Patients were possessed the vacA s1, m1 (6.1%); s1, m2 (6.1%); s2, m1 (15.1%); and s2, m2 (3.0%) genotypes. 94% of patients with gastritis were positive for H. pylori napA gene. Also, there were no dupA gene-positive gastritis patients. There was no significant correlation between the vacA, cagA, oipA, hpaA, babA, napA, dupA, ureA, ureB virulence genes, clarithromycin, and fluoroquinolone resistance. Herein, we report that the relationship between the H. pylori napA gene and gastritis. Although we found a correlation between H. pylori virulence factor and clinical outcome, there is a need for further studies to enlighten the relation between H. pylori virulence genes and antibiotic resistance.

Purpose: To develop a multiplex-urease PCR assay specific for ureA and ureB genes directly from gastric biopsy specimens and compare the results of multiplex-urease PCR assay with rapid urease test (RUT) and histopathology. Methodology: This study was conducted on 109 patients. A RUT was run; histopathological staining and multiplex urease PCR were applied to biopsy specimen to detect H. pylori. Multiplex-urease PCR, RUT and histopathology results were compared by calculating Cohen’s kappa coefficient. Results: A multiplex-urease PCR assay specific for ureA and ureB genes was developed to detect H. pylori directly from biopsy specimens. Cohen's kappa coefficient results of histopathological staining and multiplex-urease PCR indicate a substantial agreement. There was a moderate agreement between the results of histopathological staining and RUT results. There is a fair agreement between the multiplex urease PCR and the RUT results. Furthermore, the multiplex-urease PCR can detect H. pylori in some samples that are identified as negative by the rapid urease test and histopathological staining method. Moreover, in some patient samples ureA could not be detected while ureB detected. Conclusion: Multiplex-urease PCR assay was developed to detect ureA and ureB genes of H. pylori directly from gastric biopsy specimens. Comparison results indicated that detection rate of H. pylori with multiplex-urease PCR and histopathological staining is higher than the RUT. Moreover, it is critical to apply RUT test to biopsy specimens taken from both antrum and corpus part as in multiplex urease PCR assay. Furthermore, developing a multiplex PCR for both ureA and ureB is essential to detect active H. pylori infection.

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer-based models.

Bu calisma kapsaminda, L-metiyonin’den hareketle bazi yeni 1,3,4-tiyadiazol [5-8] ve 1,2,4-triazol [9-12] turevi bilesikler sentezlenmistir. Ilgili tiyosemikarbazit’in siklokondensasyonu ile elde edilen 1,3,4-tiyadiazol ve 1,2,4-triazol bilesikleri, yeni tiyoure turevlerinin sentez baslangic maddelerini de olusturmaktadir. Sentezlenen bilesiklerin [4-12] yapilari IR, 1H-NMR ve 13C-NMR spektroskopisi yontemleriyle aydinlatilmistir. Elde edilen bilesikler antiviral ve antibakteriyel etkinlikleri acisindan degerlendirilmislerdir. N-{3-(Metilsulfanil)-1-[5-(fenilamino)-1,3,4-tiyadiazol-2-il] propil}benzamit’in [5], Influenza A H3N2 virusune karsi EC50: 31.4 μM derisimde etkinlik gosterdigi saptanmis ve devam eden calismalarda gelistirilmek uzere onder bilesik olarak tanimlanmistir. Antituberkuloz etki calismalari sonucunda 1-[4-(4-kloro-(3-trifluorometil)fenil]-3-[3-(metilsulfanil)-1-(4- fenil-5-tiyokso-4,5-dihidro-1H-1,2,4-triazol-3-il)propil] tiyoure’nin [12] M. tuberculosis H37Rv susuna karsi MIK : 30.88 µM derisimde etkinlik gosterdigi belirlenmistir.