Gastroenterology and Hepatology from Bed to Bench

Inflammatory bowel disease (IBD) is a chronic disease of unknown etiology which mostly involves the intestine and requires a personalized approach for treatment. IBD represents a heterogeneous group of patients with inherently variable disease courses. Hence, the heterogeneity of patient populations may delay the diagnosis, clinical practice and initiation of appropriate treatment. Use of biomarkers for diagnosis and management of IBD is still necessary. Descriptions of the immunological pathway abnormalities in IBD improve assessment to identify the patient’s disease status, and relative risk of progression to complicated disease behaviors, and this information may ultimately influence therapeutic decisions. In this study, we try to explain the role of biomarkers in early diagnosis, estimating prognosis, and target agents for correct managements of IBD’s patients. This information might be important to provide insight into emerging panels of multiple IBD biomarkers and highlighting the essential role of personalizes panel for each patient.

Aim: The aim was to investigate breath test outcomes in patients with suspected SIBO and indicative symptoms of SIBO, diagnosed by breath testing.

Background: Breath testing is used to detect small intestinal bacterial overgrowth (SIBO) by measuring hydrogen and methane produced by intestinal bacteria.

Methods: This retrospective cross sectional study included 311 patients with gastrointestinal symptoms who underwent the breath test for evaluation of SIBO at Celiac Disease Center at Columbia University, New York, in 2014-2015. The patients were divided into two groups based on the physician’s choice: lactulose breath test group (72%) and glucose breath test group (28%). Among them, 38% had a history of celiac disease or non-celiac gluten sensitivity.

Results: In total, 46% had a positive breath test: 18% were positive for methane, 24 % positive for hydrogen and 4% positive for both gases (p=0.014). Also, 50% had a positive lactulose breath result and 37% had a positive glucose breath result (p=0.036). The most common symptom for performing the breath test was bloating and the only clinical symptom that significantly showed a positive glucose breath test was increased gas (p=0.028).

Conclusion: Lactulose breath test was more often positive than glucose breath test. Positivity for hydrogen was more common than methane. Bloating was the most frequently perceived symptom of the patients undergoing the breath test but the only statistically significant clinical symptom for a positive glucose breath test was increased gas. Furthermore, the results showed that there was no significant association between positive breath test result and gender, age, non-celiac gluten sensitivity or celiac disease.

Keywords: Small intestinal bacterial overgrowth, Lactulose breath test, Glucose breath test, Gastrointestinal symptoms, Adults.

Aim: The inclusion of elderly donors can increase the pool of organs available for transplant.

Background: To compare clinical outcomes and survival rates in patients who received livers from donors aged ? 80 years vs. younger donors.

Methods: We considered all liver transplantations performed in our unit between January 2006 and January 2015. Twelve patients received liver from a cadaveric donor aged ? 80 years (study group) and their outcomes were compared with those of patients who received liver from a younger donor (control group). This study was carried out to analyze the characteristics of donors and recipients, as well as the clinical course and survival of recipients.

Results: Statistically significant differences were observed in donors' age (55.6 ± 14.4 vs. 82.7 ± 2.7 years, p < 0.001), donors' ICU stay (p = 0.008), donors' ALT levels (p = 0.009) and donors' AST levels (p = 0.01). Statistically significant differences were found in ischemia time (p < 0.05). In total, 8.3% of the recipients of liver from a donor aged < 80 required retransplantation vs. 25% of recipients of donor’s ? 80 years. Patient survival at one, three and five years was 89%, 78.6% and 74.5%, respectively vs. 83.4%, 79.4% and 59.6% for the study group.

Conclusion: Livers from older donors can be safely used for transplantation with acceptable patient survival rates. However, graft survival rates are lower for recipients of livers from older donors as compared to younger donors, and survival only increased with retransplantation.

Keywords: Liver transplantation, Older donors, Octogenarians

Aim: Since the impact of H. pylori and its virulence is not clear in GERD, this study aimed to evaluate the prevalence of cag A and cag E gens of H. pylori among Iranian GERD patients.

Background: Gastroesophageal reflux disease (GERD) is defined as a condition of reflux the stomach juice by low pH causes tissue damage. Helicobacter pylori may or may not influence the GERD; however, it is unclear.

Methods: This study was a case-control study performed on patients with GERD who underwent upper gastrointestinal endoscopy at Taleghani Hospital of Tehran, Iran. Prevalence of H. pylori and presence of the cag A and cag E genes in GERD and control group was investigated.

Results: H. pylori was detected in 54% and 62% of GERD and control groups respectively. Prevalence of cag A gene among GERD patients was 44.4% whereas among the control group it was 87%. Prevalence of the cag E among GERD patients and control group was 44.4% and 64% respectively. Coexistence of cag A and cag E in GERD patients was 25.7% and in the control patients it was 54.8%.  

Conclusion: We did not find correlation between H. pylori existence in GERD patients in comparison to the control group. Similar to other Asian studies, the presence of the cag A in control group was more than GERD patients significantly. The co-existence of cag A and cag E was also more in control group significantly.

Keywords: Cag A, Cag E, Helicobacter pylori, GERD, Iran.

Aim: The main goal of this analysis was prioritization of co-expressed genes and miRNAs that are thought to have important influences in the pathogenesis of colon and lung cancers.

Background: MicroRNAs (miRNAs) as small and endogenous noncoding RNAs which regulate gene expression by repressing mRNA translation or decreasing stability of mRNAs; they have proven pivotal roles in different types of cancers. Accumulating evidence indicates the role of miRNAs in a wide range of biological processes from oncogenesis and tumor suppressors to contribution to tumor progression. Colon and lung cancers are frequently encountered challenging types of cancers; therefore, exploring trade-off among underlying biological units such as miRNA with mRNAs will probably lead to identification of promising biomarkers involved in these malignancies.

Methods: Colon cancer and lung cancer expression data were downloaded from Firehose and TCGA databases and varied genes extracted by DCGL software were subjected to build two gene regulatory networks by parmigene R package. Afterwards, a network-driven integrative analysis was performed to explore prognosticates genes, miRNAs and underlying pathways.

Results: A total of 192 differentially expressed miRNAs and their target genes within gene regulatory networks were derived by ARACNE algorithm. BTF3, TP53, MYC, CALR, NEM2, miR-29b-3p and miR-145 were identified as bottleneck nodes and enriched via biological gene ontology (GO) terms and pathways chiefly in biosynthesis and signaling pathways by further screening.

Conclusion: Our study uncovered correlated alterations in gene expression that may relate with colon and lung cancers and highlighted the potent common biomarker candidates for the two diseases.

Keywords: Colon cancer, Gene regulatory network, Lung cancer, miRNA

Aim: Analysis reconstruction networks from two diseases, IBD and NASH and their relationship, based on systems biology methods.

Background: IBD and NASH are two complex diseases, with progressive prevalence and high cost for countries. There are some reports on co-existence of these two diseases. In addition, they have some similar risk factors such as age, obesity, and insulin resistance. Therefore, systems biology approach can help to discover their relationship.

Methods: DisGeNET and STRING databases were sources of disease genes and constructing networks. Three plugins of Cytoscape software, including ClusterONE, ClueGO and CluePedia, were used to analyze and cluster networks and enrichment of pathways. Based on degree and Betweenness, hubs and bottleneck nodes were defined.

Results: Common genes between IBD and NASH construct a network with 99 nodes. Common genes between IBD and NASH were extracted and imported to STRING database to construct PPI network. The resulting network contained 99 nodes and 333 edges. Five genes were selected as hubs: JAK2, TLR2, TP53, TLR4 and STAT3 and five genes were selected as bottleneck including: JAK2, TP53, AGT, CYP3A4 and TLR4. These genes were hubs in analysis network that was constructed from hubs of NASH and IBD networks.

Conclusion: Systems biology methods, specifically PPI networks, can be useful for analyzing complicated related diseases. Finding Hub and bottleneck proteins should be the goal of drug designing and introducing disease markers.

Keywords: In?ammatory bowel diseases (IBD), Non-alcoholic steatohepatitis (NASH), Protein-protein interaction (PPI) network analysis, Hub-bottlenecks, Protein clusters.

Aim: Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs.

Background: Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation.

Methods: We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines.

Results: Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively.

Conclusion: Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression

Keywords microRNA regulation; miR-122a; HSVtk gene; ganciclovir; cancer therapy

Aim: We describe the minimum requirements and a simplified method for isolation and characterization of mesenchymal stem cells (MSCs) from human bone marrow.

Background: MSCs are well known adult stem cells present in many tissues such as adipocytes, chondrocytes, osteoblasts, and neurons. Many isolations and characterization methods have emerged to apply MSCs in the clinical applications, which many of them are expensive and time-consuming.

Methods: MSC isolation was carried out from human bone marrow, and cultured in defined medium. Cultures were maintained at 370C in a humidified atmosphere containing 5% CO2 for 48h. The medium was exchanged every 3-4 days. Adherent cells were characterized according to main criteria defined by ISCT, such as differentiation capability to adipocyte and osteoblast using specific differentiation mediums; also, flow cytometry verified MSC specific markers.

Results: Isolated MSCs had a fibroblastic-like appearance with adherent property to the culture plate. Differentiation function was proved with the formation of lipid drops and calcium oxalates on the differentiated MSCs and finally, purified MSCs from bone marrow were positive for cell surface markers, CD73, CD90, and CD105 while being negative for CD34 and CD45.

Conclusion: These findings confirm that the represented method is capable of isolating MSCs from bone marrow with proven results according to all minimum criteria defined by the International Society for Cellular Therapy (ISCT).

Keywords: Mesenchymal Stromal Cell, Flow cytometry, Differentiation

Aim: The aim of this study was to find the relationship between rs1799964 in TNF-a gene as well as rs1051208 of RAF1 gene SNPs on GC in an Iranian population.

Background: Gastric cancer (GC) is the second leading cause of cancer-related death worldwide after lung cancer. Tumor necrosis factor (TNF) is one of the most important factors in the pathogenesis of this cancer. Single nucleotide polymorphisms have a principle role in gene expression of TNF-a and miRNAs which may lead to gastric cancer.

Methods: In a case-control study, we investigated the risk of GC in 198 Iranians. For this purpose, 5 mL of peripheral blood was collected in EDTA –containing tube and genomic DNA was isolated. Genotyping of SNPs was also performed by PCR-RFLP; to approve the outcome, 10% of genotyping results with RFLP were sequenced.

Results: The comparison between case and control groups revealed a significant association between the rs1051208 C allele of RAF1 gene and GC (P = 0.04). We did not observe any remarkable association between TNF-a -1031 in gastric cancer patients and the healthy control group.

Conclusion: The results indicated that C allele in RAF1 gene plays a role in susceptibility to gastric cancer. Therefore, SNPs are among notable biomarkers for predicting susceptibility to dreadful diseases, especially cancers.

Keywords: single nucleoid polymorphism, Gastric cancer, Tumor necrosis factor, microRNA

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Aim: This study evaluated the frequency of C. difficile and CDAD in the ICU of Shahid Bahonhar Hospital, Kerman, Iran.

Background: Clostridium difficile (C. difficile) is the most important antibiotic associated diarrhea agent in intensive care unit (ICU) patients. Based on its toxin producing ability, C .difficile is divided to toxigenic and non-toxigenic strains.

Methods: A total of 233 diarrheal samples were collected from ICU patients. The samples were cultured on Clostridium difficile medium with 5% defibrinated sheep blood containing cycloserine (500 mg/L), cefoxitin (16 mg/L) and lysozyme (5mg/L). The isolates were confirmed as C. difficile by polymerase chain reaction (PCR) of 16s rRNA gene and the presence of toxins genes (tcdA, tcdB, cdtA and cdtB) was also confirmed. Then, the toxin production of isolates was evaluated using ELISA.

Results: C. difficile was isolated from 49 (21%) out of 233 samples. The total isolates fell into the A-/B-/CDT- (48.97%), A+/B-/CDT- (28%), A+/B+/CDT- (20.4%) and A+/B+/CDT+ (2%) types. Both types of C.difficile, A-/B-/CDT- and A+/B-/CDT-, which account for 77.5% of all isolates, were unable to produce the toxin (nontoxigenic). On the other hand, A+/B+/CDT+ and A+/B+/CDT- (22.5%), were able to produce toxin or were toxigenic.

Conclusion: The frequency of C. difficile was about 21% and only 22.4% of C. difficile isolates were able to produce toxins. It is expected that C. difficile A+/B+/CDT± are toxigenic and related to C. difficile associated diarrhea (CDAD). Additionally, about 4.7% of hospitalized patients in ICU suffered from CDAD, which is higher than the rates reported from industrialized countries. Notably, 28% of isolates were C. difficile A+/B-/CDT- which only carries tcdA genes without toxin production.

Keywords: Clostridium difficile, Intensive Care Unit, CDAD.

Aim: The aim of the present study was to determine the prevalence and subtype distribution of Blastocystis and its relation with demographic data and symptoms in humans referred to medical centers in Ahvaz 2014-2015.

Background: Infections with intestinal parasites are one of the most important threats to human health worldwide, especially in tropical and subtropical areas. Blastocystis sp. is a common parasite of humans with a vast variety of non-human hosts. We aimed to study the prevalence and subtypes of Blastocystis sp. in individuals referred to medical laboratories in Ahvaz city, southwest Iran.

Methods: From September 2014 to September 2015, 618 stool samples were collected from 16 medical laboratories in Ahvaz, and examined using direct wet mount, formalin-ether concentration, a modified version of the Ziehl–Neelsen staining technique, and cultivation in xenic HSr + S medium. Subtypes of positive Blastocysts sp. were obtained using the “barcoding” method. The results were analyzed using SPSS software, version 16, with Chi-square and Fisher’s exact test.

Results: Totally, 325 (52.6%) of the referred individuals were men and 293 (47.4%) were women. Blastocystis sp. was observed in 146 (23.6%) samples. Co-infections with other intestinal parasites were found in 32 (5.17%) cases. Out of the 146 positive isolates, 20.83%, 20.83% and 58.34% belonged to ST1, ST2, ST3 respectively.

Conclusion: Blastocystis sp. was quite common in the study population, with a carrier rate corresponding to nearly one in every four individuals. The subtype distribution identified in the present study was largely identical to that reported from other studies in Iran, with ST3 being the most common.

Keywords: Blastocystis, Prevalence, Subtypes. South western Iran.

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