Interleukin 12B mRNA level and rs3212227 genotyping in peripheral blood mononuclear cells of inflammatory bowel disease patients

Background/aim: Inflammatory bowel disease (IBD) is a multifactorial disorder. Single nucleotide polymorphisms (SNPs) in the IL-12 gene, which are the main factors to regulate the immune reaction, play an important role in the production of IL-12 molecules. The aim of this study was to evaluate the correlation between the SNP on position +1188 of the 3 ' UTR region of the IL-12 p40 subunit gene and expression of the IL-12 p40 gene. Materials and methods: This case-control study was performed with 102 patients with IBD and 107 healthy people. PCR-RFLP and comparative real-time PCR were performed to assess the association between genotype and IL-12 gene expression. Results: The frequency of AA, CA, and CC genotypes of this gene at position +1188 was calculated to be 58.8%, 32.4%, and 8.8% in patients and 61.7%, 26.2%, and 12.1% in the control group, respectively, with no significant difference between the two groups (IL- 12B rs3212227: AA (Reference 1), CA (P = 0.407); OR (95% CI) 0.771 (0.418­1.424), CC (P = 0.561); OR (95% CI) 1.313 (0.524­3.292)). Also, the IL-12B mRNA expression level was compared between IBD patients and healthy controls and demonstrated a significant association (R 2 0.136, 95% CI 1.892­3.872, P < 0.0001). Conclusion: Our results show that IL-12B expression in IBD may be associated with altered immune and inflammatory responses.

Lack of Association between Interleukin 23R (IL-23R) rs10889677 Polymorphism and Inflammatory Bowel Disease Susceptibility In an Iranian Population.

BACKGROUND: Inflammatory bowel diseases (IBDs), which include ulcerative colitis (UC) and Crohn's disease (CD), are inflammatory disorders that affect the gastrointestinal tract. A combination of inflammatory cytokines has an important role in IBD development. Genome-wide association studies have shown that polymorphisms in the interleukin-23R gene (IL-23R) increase susceptibility to IBD. The aim of this study was to investigate the IL-23R 3' UTR SNP to determine a potential association between genotype distribution and IBD. METHODS: The case group included 102 IBD patients and the control group included 107 healthy individuals. IL-23R polymorphisms rs10889677 were genotyped using PCR-RFLP analysis. RFLP results were confirmed by direct sequencing. RESULTS: The allele and genotype frequencies in patients and controls were evaluated and compared, and no significant association between this functional rs10889677 polymorphism and risk of IBD was observed (P=0.587; adjusted OR: 0.89; 95% CI: 0.597-1.339). We also found no significant association between CD (14.71%) and UC (85.29%) patients in allele or genotype levels (P>0.05). CONCLUSION: Our results suggest that the rs10889677 A>C polymorphism is not a potential prognostic marker in Iranian patients with IBD.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

AIM: In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. BACKGROUND: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. METHODS: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. RESULTS: mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). CONCLUSION: Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Comparison of three methods for mitochondria isolation from the human liver cell line (HepG2).

AIM: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application. BACKGROUND: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders. PATIENTS AND METHODS: Mitochondrial isolation from the human liver cell line (HepG2) was performed using two commercially available kits, including Qproteome (Qiagen) and MITOISO2 (Sigma-Aldrich), as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods. RESULTS: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods. CONCLUSION: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria.

Prognostic Significance of Nuclear ß-Catenin Expression in Patients with Colorectal Cancer from Iran.

BACKGROUND: Beta catenin plays a key role in cancer tumorigenesis. However, its prognostic significance in patients with colorectal cancer (CRC) remains controversial. It has been demonstrated that 90% of all tumors have a mutation in individual components of multiple oncogenes in Wnt/ß-catenin pathway. Accumulation of nuclear ß-catenin in cytoplasm leads to uncontrolled cell proliferation. Thus, nuclear ß-catenin accumulation may be a valuable biomarker associated with invasion, metastasis and poor prognosis of CRC. OBJECTIVES: In this study the prognostic value of beta catenin expression in 165 Iranian CRC patients was evaluated. PATIENTS AND METHODS: In this cross sectional retrospective study immunohistochemistry analyses of formalin-fixed paraffin-embedded (FFPE) tumor tissues were performed to characterize the expression of nuclear ß-catenin in a series of 165 Iranian patients with colorectal carcinoma. Heat-induced antigen retrieval using the microwave method was applied for all staining procedures. Staining was scored independently by two observers, and a high level of concordance (90%) was achieved. Statistical analysis was done using the SPSS software for Windows, version 13.0.0 (SPSS Inc., Chicago, IL). Two-tailed P < 0.05 was considered statistically significant. RESULTS: The patients consisted of 85 males and 80 females. Eighty-eight patients had primary tumor of the rectum and sigmoid, while 77 patients had primary tumor of the colon. The mean period of follow-up was 47.2 ± 10 months and the median period of follow-up was 38 months (range 6 - 58) for each patient. Of 165 tumors, 32 tumors (19.39 %) showed expression of ß-catenin and 133 (80.6 %) were negative for ß-catenin expression. Based on our findings the distribution of Microsatellite Instability (MSI) status differed between patients with nuclear ß-catenin positive and negative tumors and this difference was significant (P = 0.001). Patients with nuclear ß-catenin positive expression profile were found to be younger than patients with negative nuclear ß-catenin expression (P = 0.010). Univariate and multivariate analysis showed that tumors with ß-catenin expression had a poorer prognosis compared to tumors without ß-catenin expression. CONCLUSIONS: According to our findings, the distribution of nuclear b-catenin expression is a poor prognostic marker in patients with colon cancer.