Development of a gastric cancer risk calculator for questionnaire-based surveillance of Iranian dyspeptic patients.

BACKGROUND: Gastric cancer (GC) is considered a silent killer, taking more than three quarters of a million lives annually. Therefore, prior to further costly and invasive diagnostic approaches, an initial GC risk screening is desperately in demand. METHODS: In order to develop a simple risk scoring system, the demographic and lifestyle indices from 858 GC and 1132 non-ulcer dyspeptic (NUD) patients were analysed. We applied a multivariate logistic regression approach to identify the association between our target predictors and GC versus NUD. The model performance in classification was assessed by receiver operating characteristic (ROC) analysis. Our questionnaire covering 64 predictors, included known risk factors, such as demographic features, dietary habits, self-reported medical status, narcotics use, and SES indicators. RESULTS: Our model segregated GC from NUD patients with the sensitivity, specificity, and accuracy rates of 85.89, 63.9, and 73.03%, respectively, which was confirmed in the development dataset (AUC equal to 86.37%, P < 0.0001). Predictors which contributed most to our GC risk calculator, based on risk scores (RS) and shared percentages (SP), included: 1) older age group [> 70 (RS:+ 241, SP:7.23), 60-70 (RS:+ 221, SP:6.60), 50-60 (RS:+ 134, SP:4.02), 2) history of gastrointestinal cancers (RS:+ 173, SP:5.19), 3) male gender (RS:+ 119, SP:3.55), 4) non-Fars ethnicity (RS:+ 89, SP:2.66), 5) illiteracy of both parents (RS:+ 78, SP:2.38), 6) rural residence (RS:+ 77, SP:2.3), and modifiable dietary behaviors (RS:+ 32 to + 53, SP:0.96 to 1.58). CONCLUSION: Our developed risk calculator provides a primary screening step, prior to the subsequent costly and invasive measures. Furthermore, public awareness regarding modifiable risk predictors may encourage and promote lifestyle adjustments and healthy behaviours.

The role of CEACAMs versus integrins in Helicobacter pylori CagA translocation: a systematic review.

The delivery of Helicobacter pylori CagA into host cells was long believed to occur through the integrin cell surface receptors. However, the role of CEACAM receptors has recently been highlighted, instead. Here, we have categorized the existing experimental evidence according to whether deletion, upregulation, downregulation, or inhibition of the target ligands (T4SS or HopQ) or receptors (integrins or CEACAMs), result in alterations in CagA phosphorylation, cell elongation, or IL-8 production. According to our analysis, the statistics favor the essence of most of the T4SS constituents and the involvement of HopQ adhesin in all three functions. Concerning the integrin family, the collected data is controversial, but yielding towards it being dispensable or involved in CagA translocation. Yet, regarding cell elongation, more events are showing ß1 integrin being involved, than αvß4 being inhibitory. Concerning IL-8 secretion, again there are more events showing α5, ß1 and ß6 integrins to be involved, than those showing inhibitory roles for ß1, ß4 and ß6 integrins. Finally, CEACAM 1, 3, and 5 are identified as mostly essential or involved in CagA phosphorylation, whereasCEACAM 4, 7, and 8 are found dispensable and CEACAM6 is under debate. Conversely, CEACAM1, 5 and 6 appear mostly dispensable for cell elongation. Noteworthy is the choice of cell type, bacterial strain, multiplicity and duration of infection, as well as the sensitivity of the detection methods, all of which can affect the variably obtained results.

A Bayesian latent class extension of naive Bayesian classifier and its application to the classification of gastric cancer patients.

BACKGROUND: The Naive Bayes (NB) classifier is a powerful supervised algorithm widely used in Machine Learning (ML). However, its effectiveness relies on a strict assumption of conditional independence, which is often violated in real-world scenarios. To address this limitation, various studies have explored extensions of NB that tackle the issue of non-conditional independence in the data. These approaches can be broadly categorized into two main categories: feature selection and structure expansion. In this particular study, we propose a novel approach to enhancing NB by introducing a latent variable as the parent of the attributes. We define this latent variable using a flexible technique called Bayesian Latent Class Analysis (BLCA). As a result, our final model combines the strengths of NB and BLCA, giving rise to what we refer to as NB-BLCA. By incorporating the latent variable, we aim to capture complex dependencies among the attributes and improve the overall performance of the classifier. METHODS: Both Expectation-Maximization (EM) algorithm and the Gibbs sampling approach were offered for parameter learning. A simulation study was conducted to evaluate the classification of the model in comparison with the ordinary NB model. In addition, real-world data related to 976 Gastric Cancer (GC) and 1189 Non-ulcer dyspepsia (NUD) patients was used to show the model's performance in an actual application. The validity of models was evaluated using the 10-fold cross-validation. RESULTS: The presented model was superior to ordinary NB in all the simulation scenarios according to higher classification sensitivity and specificity in test data. The NB-BLCA model using Gibbs sampling accuracy was 87.77 (95% CI: 84.87-90.29). This index was estimated at 77.22 (95% CI: 73.64-80.53) and 74.71 (95% CI: 71.02-78.15) for the NB-BLCA model using the EM algorithm and ordinary NB classifier, respectively. CONCLUSIONS: When considering the modification of the NB classifier, incorporating a latent component into the model offers numerous advantages, particularly within medical and health-related contexts. By doing so, the researchers can bypass the extensive search algorithm and structure learning required in the local learning and structure extension approach. The inclusion of latent class variables allows for the integration of all attributes during model construction. Consequently, the NB-BLCA model serves as a suitable alternative to conventional NB classifiers when the assumption of independence is violated, especially in domains pertaining to health and medicine.

Chronic Obstructive Pulmonary Disease: Novel Genes Detection with Penalized Logistic Regression.

OBJECTIVE: This study aimed to introduce novel techniques for identifying the genes associated with developing chronic obstructive pulmonary disease (COPD) and to prioritize COPD candidate genes using regression methods. MATERIALS AND METHODS: This is a secondary analysis of the data from an experimental study. We used penalized logistic regressions with three different types of penalties included least absolute shrinkage and selection operator (LASSO), minimax concave penalty (MCP), and smoothly clipped absolute deviation (SCAD). The models were trained using genome-wide expression profiling to define gene networks relevant to the COPD stages. A 10-fold cross-validation scheme was used to evaluate the performance of the methods. In addition, we validate our results by the external validity approach. We reported the sensitivity, specificity, and area under curve (AUC) of the models. RESULTS: There were 21, 22, and 18 significantly associated genes for LASSO, SCAD, and MCP models, respectively. The most statistically conservative method (detecting less significant features) was MCP detected 18 genes that were all detected by the other two approaches. The most appropriate approach was a SCAD penalized logistic regression (AUC= 96.26, sensitivity= 94.2, specificity= 86.96). In this study, we have a common panel of 18 genes in all three models that show a significant positive and negative correlation with COPD, in which RNF130, STX6, PLCB1, CACNA1G, LARP4B, LOC100507634, SLC38A2, and STIM2 showed the odds ratio (OR) more than 1. However, there was a slight difference between penalized methods. CONCLUSION: Regularization solves the serious dimensionality problem in using this kind of regression. More exploration of how these genes affect the outcome and mechanism is possible more quickly in this manner. The regression-based approaches we present could apply to overcoming this issue.

Assessment of the mixed origin of the gastric epithelial extracellular vesicles in acellular transfer of Helicobacter pylori toxins and a systematic review.

BACKGROUND: H. pylori are generally considered as extracellular organisms, with exclusive colonization of the gastric milieu. Yet, several extra gastric manifestations are associated with this infection. The aim of the present study was to investigate the feasibility of toxin transfer by extracellular vesicles, from bacterial and epithelial origins. METHODS: Tox-positive H. pylori and its two cagA and vacA mutant strains were used to produce bacterial vesicles (BVs) and to infect AGS cells. The produced BVs and the infected cell vesicles (ICVs) were collected by ultracentrifugation and evaluated by western blotting, DLS and electron microscopy. These two sets of vesicles were applied to a second set of recipient AGS cells, in which the acellular transfer of toxins, IL-8 production and downstream morphologic changes were assessed, by western blotting, ELISA and light microscopy, respectively. RESULTS: The BVs were positive for H. pylori membrane markers (BabA and UreB), VacA and CagA toxins, except for from the corresponding mutant strains. The ICVs were larger in size and positive for bacterial markers, as well as epithelial markers of CD9, LGR5, but negative for nuclear (Ki76) or cytoplasmic (ß-actin) markers. Bacteria-independent transfer of CagA and VacA into the recipient cells occurred upon treatment of cells with BVs and ICVs, followed by cellular vacuolation and elongation. IL-8 production was induced in recipient AGS cells, treated with BVs (1279.4 ± 19.79 pg/106 cells), early (8 h, 1171.4 ± 11.31 pg/106 cells) and late (48 h, 965.4 ± 36.77 pg/106 cells) ICVs (P < 0.0001). CONCLUSION: Our data indicates that ICVs, with mixed bacterial and epithelial constituents, similar to BVs, are capable of transferring bacterial toxins into the recipient cells, inducing IL-8 production and subsequent morphologic changes, in an acellular manner.

A Novel Anti-CD22 scFv.Bim Fusion Protein Effectively Induces Apoptosis in Malignant B cells and Promotes Cytotoxicity.

CD22 is a B-cell surface antigen which is highly expressed in cancerous B-cell lineages. Anti-CD22 antibodies are currently under focus as promising biologics against hematologic B-cell malignancies. Herein, we introduce a novel active recombinant anti-CD22 scFv.Bim fusion protein for targeting this cancerous antigen. An expression cassette encoding anti-CD22 scFv.Bim fusion protein was expressed in Pichia pastoris. The binding ability, cytotoxicity, and apoptotic activity of the purified recombinant protein against CD22+ Raji cell line were assessed by flow cytometry, microscopy, and MTT assay. Using bioinformatics, the 3D structure of the fusion protein and its interaction with CD22 were assessed. The in vitro binding analysis by immunofluorescence microscopy and flow cytometry demonstrated the specific binding of scFv.Bim to CD22+ Raji cells but not to CD22- Jurkat cells. MTT data and Annexin V/PI flow cytometry analysis confirmed the apoptotic activity of anti-CD22 scFv.Bim against Raji cells but not Jurkat cells. In silico analysis also revealed the satisfactory stereochemical quality of the 3D model and molecular interactions toward CD22. This novel recombinant anti-CD22 scFv.Bim fusion protein could successfully deliver the pro-apoptotic peptide, BIM, to the target cells and thus nominates it as a promising molecule in treating B-cell malignancies.

Helicobacter pylori acquisition rates and the associated risk factors amongst newlywed couples; a prospective cohort study in Tehran, Iran.

BACKGROUND: The rates and routes of Helicobacter pylori transmission, in a high-prevalent country like Iran, with gastric cancer as the leading cause of male cancer mortality, are of great essence. Here, we have studied the H. pylori-associated risk factors and the likelihood of interspousal transmission. METHODS: In a cohort of 686 young prewed couples, questionnaires were self-administered and serum samples were collected, for assessment of risk factors and H. pylori serostatus, at baseline and follow-up. Of the 475 H. pylori single- or double-seronegative couples, 201 returned for follow-up. The average follow-up duration was 2.2 (SD 0.6) years, with a total of 560.1 person-years. Logistic regression and Cox regression models were used to estimate the odds ratios (ORs) and hazard ratios (HRs). RESULTS: The risk of infection was higher in men than women (OR: 1.3, 95% CI: 1.0-1.8) and among metropolitan than rural residents (OR = 1.4, 95% CI: 1.1-1.9). It was also significantly higher among those with three (OR = 1.6, 95% CI: 1.1-2.2), and four or more siblings (OR = 1.4, 95% CI: 1.0-1.9), in reference to those with one or no siblings. Adult H. pylori acquisition occurred in 10.9% (27/247) of the seronegative participants. The risk of the acquisition was significantly associated with age (P value for trend=0,000). It was also significantly lower among participants who had various degrees of education as compared to illiterate subjects (HR = 0.2, 95% CI: 0.1-0.9). Nevertheless, our analysis did not find any evidence for interspousal transmission (HR = 1.0, 95% CI: 0.4-2.2). CONCLUSION: Whilst H. pylori acquisition was detected in the young adult Iranian population, our findings did not support interspousal transmission, as a mode of acquisition.

Infection with a hypervirulent strain of Helicobacter pylori primes gastric cells toward intestinal transdifferentiation.

BACKGROUND: Intestinal metaplasia, gastric-to-intestinal transdifferentiation, occurs as a result of the misexpression of certain regulatory factors, leading to genetic reprogramming. Here, we have evaluated the H. pylori-induced expression patterns of these candidate genes. METHODS: The expression levels of 1) tissue-specific transcription factors (RUNX3, KLF5, SOX2, SALL4, CDX1 and CDX2), 2) stemness factors (TNFRSF19, LGR5, VIL1) and 3) tissue-specific mucins (MUC5AC, MUC2) were evaluated by quantitative real-time PCR in gastric primary cells (GPCs), in parallel with two gastric cancer (MKN45 and AGS) cell lines, up to 96h following H. pylori infection. RESULTS: Following H. pylori infection of GPCs, RUNX3 declined at 24h post infection (-6.2 ± 0.3) and remained downregulated for up to 96h. Subsequently, overexpression of self-renewal and pluripotency transcription factors, KLF5 (3.6 ± 0.2), SOX2 (7.6 ± 0.5) and SALL4 (4.3 ± 0.2) occurred. The expression of TNFRSF19 and LGR5, demonstrated opposing trends, with an early rise of the former (4.5 ± 0.3) at 8h, and a simultaneous fall of the latter (-1.8 ± 0.5). This trend was reversed at 96h, with the decline in TNFRSF19 (-5.5 ± 0.2), and escalation of LGR5 (2.6 ± 0.2) and VIL1 (1.8 ± 0.3). Ultimately, CDX1 and CDX2 were upregulated by 1.9 and 4.7-fold, respectively. The above scenario was, variably observed in MKN45 and AGS cells. CONCLUSION: Our data suggests an interdependent gene regulatory network, induced by H. pylori infection. This interaction begins with the downregulation of RUNX3, upregulation of self-renewal and pluripotency transcription factors, KLF5, SOX2 and SALL4, leading to the downregulation of TNFRSF19, upregulation of LGR5 and aberrant expression of intestine-specific transcription factors, potentially facilitating the process of gastric-to-intestinal transdifferentiation.

Mitochondrial DNA Copy Number Variations and Serum Pepsinogen Levels for Risk Assessment in Gastric Cancer

Background: Variations in mitochondrial DNA copy number (mtDNA-CN) of peripheral blood leukocytes (PBLs), as a potential biomarker for gastric cancer (GC) screening has currently been subject to controversy. Herein, we have assessed its efficiency in GC screening, in parallel and in combination with serum pepsinogen (sPG) I/II ratio, as an established indicator of gastric atrophy. Methods: The study population included GC (n = 53) and non-GC (n = 207) dyspeptic patients. The non-GC group was histologically categorized into CG (n = 104) and NM (n = 103) subgroups. The MtDNA-CN of PBLs was measured by quantitative real-time PCR. The sPG I and II levels and anti-H. pylori serum IgG were measured by ELISA. Results: The mtDNA-CN was found significantly higher in GC vs. non-GC (OR = 3.0; 95% CI = 1.4, 6.4) subjects. Conversely, GC patients had significantly lower sPG I/II ratio than the non-GC (OR = 3.2; CI = 1.4, 7.2) subjects. The combination of these two biomarkers yielded a dramatic amplification of the odds of GC risk in double-positive (high mtDNA-CN-low sPGI/II) subjects, in reference to double-negatives (low mtDNA-CN-high sPGI/II), when assessed against non-GC (OR = 27.1; CI = 5.0, 147.3), CG (OR = 13.1; CI = 2.4, 72.6), or NM (OR = 49.5; CI = 7.9, 311.6) groups. Conclusion: The combination of these two biomarkers, namely mtDNA-CN in PBLs and serum PG I/II ratio, drastically enhanced the efficiency of GC risk assessment, which calls for further validations.

Identification of potential microRNA panels for pancreatic cancer diagnosis using microarray datasets and bioinformatics methods.

Pancreatic cancer (PC) is a malignancy with little/no warning signs before the disease reaches its ultimate stages. Currently early detection of PC is very difficult because most patients have non-specific symptoms leading to postponing the correct diagnosis. In this study, using multiple bioinformatics tools, we integrated various serum expression profiles of miRNAs to find the most significant miRNA signatures helpful in diagnosis of PC and constructed novel miRNA diagnosis models for PC. Altogether, 27 differentially expressed miRNAs (DEMs) showed area under curve (AUC) score >80%. The most promising miRNAs, miR-1469 and miR-4530, were individually able to distinguish two groups with the highest specificity and sensitivity. By using multivariate cox regression analyses, 5 diagnostic models consisting of different combinations of miRNAs, based on their significant expression algorithms and functional properties were introduced. The correlation model consisting of miR-125a-3p, miR-5100 and miR-642b-3p was the most promising model in the diagnosis of PC patients from healthy controls with an AUC of 0.95, Sensitivity 0.98 and Specificity 0.97. Validation analysis was conducted for considered miRNAs on a final cohort consist of the microarray data from two other datasets (GSE112264 & GSE124158) . These results provide some potential biomarkers for PC diagnosis after testing in large case-control and cohort studies.

Research Performance in Stem Cell Science and Regenerative Medicine in Iran: A National Comprehensive Observation.

BACKGROUND: Accomplishments in stem cell science and tissue engineering have resulted in a remarkable revolution in the context of future medicine. However, a general insight over the progress of stem cell research in Iran is still lacking. A better understanding of current needs in this field is required to design a better national roadmap. METHODS: In this study, we defined the geographical regions based on the significance of their contribution to stem cell research; then, using the Scopus database, we retrieved reports from Iran and other countries concerning stem cell science and regenerative medicine published from 1994 until the end of 2016. RESULTS: A significant number of citations belong to North America (6554029 citations equal to 49% of the total citations) and Europe (4425465 citations equal to 33% of the total citations) and the rest of citations were related to Asia (2423352 citations equal to 18% of the total citations). East Asian (2168472 citations equal to 76% of the total citations related to Asia) documents were cited more than those from the Middle East (ME) (494141 citations equal to 17% of the total citations related to Asia) and North and Central Asia (196382 citations equal to 7% of the total citations related to Asia). Iran as a country in the ME attracted 17% of the total citations related to the Asian countries winning the second position in this regard. The overview of total number of citations showed a sharp increase and upward trend in citation numbers for all the Iranian institutes from 2007 that resulted in the expansion of stem cell science in all major cities of Iran such as Shiraz (8%), Mashhad (5%), Isfahan (5%) and Ahvaz (5%). H-index of Tehran University of Medical Science, which has the highest total citations and document numbers, is the highest among all Iranian research institutes. Citation per paper of Royan Institute (RI) is the highest among the top 10 Iranian institutes, by 13 citations per paper. CONCLUSION: Stem cell research in Iran is rapidly developing. Since 2007, the number of published documents in major research institutes increased; thus, there is necessity for analysis of the status of publications in this field and choosing a better direction based on needs. Furthermore, it is necessary to expand and organize international collaborations to enrich our research and benefit from different team experiences.

Autologous Bone Marrow Stem Cell Transplantation in Liver Cirrhosis after Correcting Nutritional Anomalies, A Controlled Clinical Study.

OBJECTIVE: Liver transplantation is the gold standard approach for decompensated liver cirrhosis. In recent years, stem cell therapy has raised hopes that adjusting some clinical and laboratory parameters could lead to successful treatments for this disease. Cirrhotic patients may have multiple systemic abnormalities in peripheral blood and irregular cell populations in bone marrow (BM). Correcting these abnormalities before BM aspiration may improve the effectiveness of cell-based therapy of liver cirrhosis. MATERIALS AND METHODS: In this controlled clinical trial study, 20 patients with decompensated liver cirrhosis were enrolled. Patients were randomly assigned to control and experimental groups. Blood samples were obtained to measure vitamin B12, folate, serum iron, total iron bonding capacity (TIBC) and ferritin before any intervention. Furthermore, the iron storage and fibrosis level in BM biopsies, as well as the percentage of different cell populations, were evaluated. Prior to cell isolation for transplantation, we performed palliative supplement therapy followed by a correction of nutritional deficiencies. Mononuclear cells (MNCs) were then isolated from BM aspirates and transfused through peripheral vein in patients in the experimental group. The model of end-stage liver disease (MELD) score, The international normalized ratio (INR), serum albumin and bilirubin levels were assessed at 0 (baseline), 3 and 6 months after cell transplantation. RESULTS: The MELD score (P=0.0001), INR (P=0.012), bilirubin (P<0.0001) and total albumin (P<0.0001) levels improved significantly in the experimental group after cell transplantation compared to the baseline and control groups. Moreover, the increase in serum albumin levels of patients in the experimental group was statistically significant 6 months after transplantation. CONCLUSION: We have successfully improved the conditions of preparing -BM-derived stem cells for transplantation. Although these cells are relatively safe and have been shown to improve some clinical signs and symptoms temporarily, there need to be more basic studies regarding the preparation steps for effective clinical use (Registration number: IRCT2014091919217N1).

The Inhibitory Effect of Mouse Gastric DNA on Amplification of Helicobacter pylori Genomic DNA in Quantitative PCR

Background: Quantitation of Helicobacter pylori (Hp) in the gastric tissue is essential for assessment of vaccination/therapeutic regimens. Materials & Results: Here, the inhibitory effect of mouse gastric DNA (MgDNA) on amplification of Hp genomic DNA (HpDNA) was evaluated by spiking HpDNA with serial dilutions of MgDNA, which yielded concentrations of >10 ng/µl and >0.63-10 ng/µl of MgDNA, as inhibition and interference zones, respectively. Mice were then inoculated with varying doses of Hp and assessed at the inhibition-free concentration of 0.63 ng/µl. The average Hp copy numbers per microgram of gastric tissue discriminated mice having received high vs. low dose inoculums (p < 0.001). Secondly, Hp copy numbers were quantitated in immunized mice, which demonstrated significantly lower numbers, in reference to controls (p = 0.006). Conclusion: Our method, bypassing the inhibition and/or interference imposed by MgDNA, was able to quantitate gastric tissue-colonizing Hp, segregating mice inoculated with low vs. high doses of Hp, as well as those immunized from controls.

Immunohistochemical Analysis of LGR5 and TROY Expression in Gastric Carcinogenesis Demonstrates an Inverse Trend

Background: Two of the Wnt signaling pathway target genes, tumor necrosis factor receptor family member (TROY) and leucine-rich G-protein coupled receptor (LGR5), are involved in the generation and maintenance of gastrointestinal epithelium. A negative modulatory role has recently been assigned to TROY, in this pathway. Here, we have examined their simultaneous expression in gastric carcinogenesis. Methods: Tumor and paired adjacent tissues of intestinal-type gastric cancer (GC) patients (n = 30) were evaluated for LGR5 and TROY expression by immunohistochemistry. The combination of the percentage of positively¬ stained cells and the intensity of staining was defined as the composite score and compared between groups. The obtained findings were re-evaluated in a mouse model. Results: TROY expression in the tumor tissue was significantly lower than that of the adjacent tissue (2.5 ± 0.9 vs. 3.3 ± 0.9, p = 0.004), which was coincident with higher LGR5 expression (3.6 ± 1.1 vs. 2.7 ± 0.9, p = 0.001). This observation was prominent at stages II/III of GC, leading to a statistically significant mean difference of expression between these two molecules (p = 0.005). In the H. pylori infected-mouse model, this inverse expression was observed in transition from early (8-16 w) to late (26-50 w) time points, post treatment (p = 0.002). Conclusion: Our data demonstrates an inverse trend between TROY down-regulation and LGR5 up-regulation in GC tumors, as well as in response to H. pylori infection in mice. These findings support a potential negative modulatory role for TROY on LGR5 expression.

A simple and cost-efficient adherent culture platform for human gastric primary cells, as an in vitro model for Helicobacter pylori infection.

BACKGROUND: Most two- dimensional in vitro models for studying host- H. pylori interactions rely on tumor-derived cell lines, which harbor malignant alterations. The recent development of human gastric organoids has overcome this limitation and provides a highly sophisticated, yet costly, short-term model for H. pylori infection, with restricted use in low-budget centers. METHOD: Tissue specimens from upper, middle, and lower stomachs of H. pylori-negative volunteers were collectively dispersed and cultured on mouse embryonic fibroblast (MEF) or collagen-coated plates. Gastric primary cells (GPCs) were evaluated by light microscopy, immunostaining, qRT-PCR and ELISA analysis of cellular secretions, before and after H. pylori infection. RESULTS: The formation and long-term (up to 1 year) maintenance of GPCs was highly dependent on adherent inactivated MEF cells, cultured in enriched media. These cells were multipassageable and able to undergo stable freezer storage and subsequent revival. The cellular composition of GPCs included the combination of cytokeratin 18 (CK18) and E-cadherin (E-cad)-positive epithelial cells, MUC5AC-positive gastric cells, and leucine-rich repeat containing G protein-coupled receptor 5 (LGR5)-positive progenitor cells. These cells produced significant amounts of gastric pepsinogens I and II. GPCs also allowed for extended (up to 96 hours) H. pylori infection, during which they underwent morphological alterations (cellular vacuolation and elongation) and hyperproduction of gastric pepsinogens and inflammatory cytokines (IL-8 and TNF-α). CONCLUSION: We, hereby, present a simple, consistent, and cost-efficient gastric cell culture system, which provides a suitable model for extended in vitro infection of H. pylori. This platform can be employed for a variety of gastric-related research.

Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis - A simple and cost-efficient method.

INTRODUCTION: Helicobacter pylori express a large array of antigens, each of which is duly responsible for successful colonization and pathogenesis. Here, we have studied host serum antibody responses to four of its immunodominant antigens in association with the infection status and the resulting clinical outcomes. METHODS: For this purpose, four individual H. pylori proteins (UreB, CagA, Tip-α and HP0175) were produced in recombinant forms. Serum antibody responses of 246 (75 GC and 171 NUD) patients, against the above antigens, were evaluated by multiplex immunoblotting. The associations between the resulting data and the infection status, as well as clinical outcomes were evaluated using logistic regression models. RESULTS: Serum antibodies to all four recombinant antigens increased the chances of detecting screening ELISA-positive subjects, in an escalating dose-dependent manner, ranging from 2.6 (1.5-4.7) for HP0175 to 14.3 for UreB (4.3-50.7), exhibiting the lowest and highest odds ratios, respectively (PAdj ≤ 0.001), such that 98.2% of the subjects with antibodies to all four antigens, were also positive by the screening ELISA (P < 0.0001). Among the screening ELISA-positive subjects, the three antigens of CagA, Tip-α, and HP0175 were able to segregate current from past H. pylori infection (P < 0.05). Accordingly, subjects with antibodies to one or more antigen(s) were at 5.4 (95% CI: 1.8-16.4) folds increased chances of having current infection, as compared to triple negatives (PAdj = 0.003). In reference to the clinical outcomes, those with serum antibodies to CagA were more prevalent among gastric cancer, as compared to NUD patients (ORAdj: 5.4, 95% CI: 2.4-12.2, PAdj < 0.0001). When NUD patients were categorized according to their histopathologic status, multiple antigen analysis revealed that subjects with serum antibodies to one or more of the 3 current infection-positive antigens (CagA, Tip-α, and HP0175) were at 9.7 (95% CI: 2.1-44.9, P = 0.004) folds increased risk of atrophic gastritis, in reference to triple negatives. CONCLUSION: The non-invasive multiplex serology assay, presented here, was able to not only detect subjects with current H. pylori infection, it could also screen dyspeptic patients for the presence of gastric atrophy. This simple and cost-efficient method can supplement routine screening ELISAs, to increase the chances of detecting current infections as well as atrophic gastritis.

Serum Antibodies against Helicobacter pylori Neutrophil Activating Protein in Carriers of IL-4 C-590T Genetic Polymorphism Amplify the Risk of Gastritis and Gastric Cancer.

BACKGROUND: Gastric cancer arises, mainly, on an inflammatory background. Helicobacter pylori neutrophil activating (HP-NAP) protein functions as a potent pro-inflammatory mediator. Similarly, IL-4 plays a critical role in the inflammation pathway, the levels of which are altered by C to T transition at position -590 in its promoter region. Here, we have aimed to assess the risk of gastritis and gastric cancer in the co-presence of these two inflammation modulating mediators. METHODS: Gastritis (n=58) and gastric cancer (n=31) patients were evaluated and compared with H. pylori-positive asymptomatic controls (n=46), for serum antibodies against recombinant HP-NAP and IL-4 C-590T single nucleotide polymorphism using immunoblotting and PCR-RFLP, respectively. Multivariable logistic regression, adjusting for age, gender and ethnicity, was used for data analysis. RESULTS: In terms of susceptibility to gastritis, seropositivity to HP-NAP projected a risk impact of 4.62 fold (OR=4.62, 95% CI=1.50-14.22), which when present in IL-4 -590 T carriers augmented the risk up to 9.7 fold (OR=9.70, 95% CI=2.06-45.69). A similar pattern, but of a stronger magnitude, occurred for the risk of gastric cancer, which was estimated at 9.07 fold (OR=9.07, 95% CI=1.99-42.0) for HP-NAP-seropositive subjects and was drastically amplified (OR=33.64, 95% CI=2.06-548.68), when double-positive (HP-NAP seropositive/IL-4 -590 T carrier) subjects were examined against double negatives (HP-NAP seronegative/IL-4 -590 CC). CONCLUSION: Our preliminary data indicate that serum antibodies against HP-NAP represent a state of risk, which is further exacerbated in IL-4 -590 T carriers. These biomarkers, if validated in larger prospective studies, can be used to screen for gastric cancer susceptibility.

Helicobacter pylori Peptidyl Prolyl Isomerase Expression Is Associated with the Severity of Gastritis.

PURPOSE: Helicobacter pylori secretory peptidyl prolyl isomerase, HP0175, is progressively identified as a pro-inflammatory and pro-carcinogenic protein, which serves to link H. pylori infection to its more severe clinical outcomes. Here, we have analyzed host HP0175-specific antibody responses in relation to the severity of gastritis. METHODS: The HP0175 gene fragment was PCR-amplified, cloned, expressed and purified by Ni-NTA affinity chromatography. Serum antigen-specific antibody responses of non-ulcer dyspeptic patients (N = 176) against recombinant HP0175 were detected by western blotting. The infection status of these subjects was determined by rapid urease test, culture, histology, and serology. The grade of inflammation and stage of atrophy were scored blindly according to the OLGA staging system. RESULTS: The recombinant HP0175 (rHP0175) was expressed as a ~35 kDa protein and its identity was confirmed by western blotting using anti-6X His tag antibody and pooled H. pylori-positive sera. Serum IgG antibodies against rHP0175 segregated our patients into two similar-sized groups of sero-positives (90/176, 51.1 %) and sero-negatives (86/176, 48.9 %). The former presented with higher grades of gastric inflammation (OR = 4.4, 95 % CI = 1.9-9.9, P = 0.001) and stages of gastric atrophy (OR = 18.3, 95 %CI = 1.4-246.6, P = 0.028). CONCLUSION: Our findings lend further support to the pro-inflammatory nature of H. pylori peptidyl prolyl isomerase (HP0175) and recommends this antigen as a non-invasive serum biomarker of the severity of H. pylori-associated gastritis.

Helicobacter pylori Strains from Duodenal Ulcer Patients Exhibit Mixed babA/B Genotypes with Low Levels of BabA Adhesin and Lewis b Binding.

BACKGROUND: BabA is a Helicobacter pylori cell surface adhesin, which binds to the ABO/Le(b) histo-blood group antigens (Le(b)) and serves as a virulence factor. METHODS: H. pylori single colonies were isolated from 156 [non-ulcer dyspepsia (NUD) = 97, duodenal ulcer (DU) = 34, gastric cancer (GC) = 25)] patients. babA and babB genes were evaluated by gene/locus-specific PCR. BabA protein expression and Le(b) binding activity were determined by immunoblotting and ELISA, respectively. RESULTS: The combined categorization of H. pylori strains based on high, low or no levels of BabA expression and Le(b) binding, produced 4 groups: (I) BabA-high/Le(b)-high (36 %), (II) BabA-low/Le(b)-low (26 %), (III) BabA-neg/Le(b)-low (30 %) and (IV) BabA-neg/Le(b)-neg (8 %) strains. The majority (63 %) of the BabA-low/Le(b)-low strains exhibited mixed babA/B genotypes as compared to merely 18 % of the BabA-high/Le(b)-high, 15 % of the BabA-neg/Le(b)-neg and 11 % of the BabA-neg/Le(b)-low (P = 0.0001) strains. In contrast to NUD strains, the great majority (70 %) of DU strains were BabA-low/Le(b)-low (11 %, P = 0.0001), which compared to NUD strains, enhanced the risk of DU by 18.8-fold. In parallel, infection with babA/B mixed genotype strains amplified the risk of DU by 3.6-fold (vs. babA-positive: P = 0.01) to 6.9-fold (vs. babA-negative: P = 0.007). CONCLUSIONS: Here, we show higher prevalence of mixed babA/B genotypes among BabA-low/Le(b)-low clinical strains. Recombination of babA and babB genes across their loci may yield lower BabA expression and lower Le(b) binding activity. We conclude that H. pylori strains with lower Le(b) binding activity are better adapted for colonization of the gastric metaplastic patches in the duodenum and enhance the risk of duodenal ulcers.

Distribution of cytokine gene single nucleotide polymorphisms among a multi-ethnic Iranian population.

BACKGROUND: Cytokine gene single nucleotide polymorphisms (SNPs) are widely used to study susceptibility to complex diseases and as a tool for anthropological studies. MATERIALS AND METHODS: To investigate cytokine SNPs in an Iranian multi-ethnic population, we have investigated 10 interleukin (IL) SNPs (IL-1ß (C-511T, T-31C), IL-2 (G-384T), IL-4 (C-590T), IL-6 (G-174C), IL-8 (T-251A), IL-10 (G-1082A, C-819T, C-592A) and tumor necrosis factor-alpha (TNF-α) (G-308A) in 415 Iranian subjects comprising of 6 different ethnicities. Allelic and genotypic frequencies as well as Hardy-Weinberg equilibrium (HWE) were calculated by PyPop software. Population genetic indices including observed heterozygosity (Ho), expected heterozygosity (He), fixation index (FIS), the effective number of alleles (N e) and polymorphism information content (PIC) were derived using Popgene 32 software. Multidimensional scaling (MDS) was constructed using Reynold's genetic distance obtained from the frequencies of cytokine gene polymorphism. RESULTS: Genotypic distributions were consistent with the HWE assumptions, except for 3 loci (IL-4-590, IL-8-251 and IL-10-819) in Fars and 4 loci (IL-4-590, IL-6-174, IL-10-1082 and TNF-α-308) in Turks. Pairwise assessment of allelic frequencies, detected differences at the IL-4-590 locus in Gilakis versus Kurds (P = 0.028) and Lurs (P = 0.022). Mazanis and Gilakis displayed the highest (Ho= 0.50 ± 0.24) and lowest (Ho= 0.34 ± 0.16) mean observed heterozygosity, respectively. CONCLUSIONS: MDS analysis of our study population, in comparison with others, revealed that Iranian ethnicities except Kurds and Mazanis were tightly located within a single cluster with closest genetic affinity to Europeans.