Objective To investigate the impact of knocking out tumor necrosis factor-related ap-optosis-inducing ligand ( TRAIL) gene ( TRAIL-/-) on colonic inflammation and regulatory T cells ( Treg) in mice with dextran sulfate sodium (DSS)-induced experimental colitis. Methods C57BL/6 mice were ran-domly assigned into four groups with 10 in each group:wild-type ( WT) control, WT colitis, TRAIL-/- con-trol and TRAIL-/- colitis. The mouse model of colitis was induced by oral administration of 3. 5％ DSS and the severity of colonic inflammation was assessed. Peripheral blood mononuclear cells ( PBMCs) and mesen-teric lymph nodes ( MLNs) were collected. The ratios of Treg cells to CD4+T cells in PBMCs were detected by flow cytometry. Expression of Treg cell-associated transcription factor (Foxp3) and cytokine (IL-10) at mRNA level was measured by real-time fluorescent quantitative polymerase chain reaction. Western blot and enzyme-linked immunosorbent assay ( ELISA) were used to detect the expression of Foxp3 and IL-10 at pro-tein level, respectively. Results Compared with the WT control group, the WT colitis group showed signif-icantly decreased proportions of Treg cells in PBMCs [(1. 85±0. 38)％ vs (3. 12±0. 69)％, P<0. 05], but increased proportions in MLNs [(11. 79±1. 18)％ vs (6. 24±1. 04)％, P<0. 05]. Compared with the WT mice with colitis, the TRAIL-/- mice with colitis had more severe colonic inflammation and significantly in-creased proportions of Treg cells in PBMCs [(3. 15±0. 64)％ vs (1. 85±0. 38)％, P<0. 05], but de-creased Treg cells in MLNs [(9. 80±0. 50)％ vs (11. 79±1. 18)％, P<0. 05]. Expression of Foxp3 and IL-10 at mRNA and protein levels in PBMCs of the WT mice with colitis was significantly lower than that in the WT control mice [ Foxp3 mRNA: 0. 48 ± 0. 21 vs 1. 06 ± 0. 31, IL-10 mRNA: 0. 23 ± 0. 07 vs 1. 22 ± 0. 38;Foxp3 protein:0. 68±0. 12 vs 1, IL-10 protein:(4. 91± 0. 72) pg/ml vs (21. 86±2. 40) pg/ml;all P<0. 05], while in MLNs, the expression of Foxp3 and IL-10 at mRNA and protein levels was significantly higher than that of the WT control group [Foxp3 mRNA:3. 71±0. 49 vs 1. 03±0. 15, IL-10 mRNA:11. 98 ±6.10 vs 1. 01±0. 31; Foxp3 protein: 1. 60±0. 03 vs 1, IL-10 protein: (1260. 00±18. 02) pg/ml vs (1184. 00±38. 62) pg/ml;all P<0. 05]. Compared with the WT mice with colitis, the TRAIL-/-mice with colitis showed significantly increased expression of Foxp3 and IL-10 at mRNA and protein levels [ Foxp3 mRNA:1. 80±0. 49 vs 0. 48±0. 21, IL-10 mRNA:1. 67±0. 99 vs 0. 23±0. 07;Foxp3 protein:1. 10±0. 01 vs 0. 68±0. 12, IL-10 protein:(31. 33± 25. 02) pg/ml vs (4. 58±3. 73) pg/ml; all P<0. 05], while de-creased expression in MLNs [ Foxp3 mRNA: 0. 49 ± 0. 21 vs 3. 71 ± 0. 49, IL-10 mRNA: 2. 80 ± 1. 82 vs 11. 98±6. 10; Foxp3 protein: 1. 21±0. 12 vs 1. 60±0. 03, IL-10 protein: (1158. 00±26. 48) pg/ml vs (1190. 00±37. 19) pg/ml;all P<0. 05]. Conclusions Knocking out the expression of TRAIL might af-fect the ratios of Treg cells in peripheral blood and MLNs, thereby aggravating the colitis in mice.

OBJECTIVETo assess the association of vascular endothelial growth factor (VEGF) gene polymorphisms with susceptibility to Crohn's disease (CD) in a Chinese population.

METHODSFor 275 CD patients and 495 controls, the genotypes of VEGF gene rs699947 and rs3025039 loci were determined with a SNaPshot method.

RESULTSThe allelic and genotypic frequencies of the rs699947 and rs3025039 loci did not differ between the two groups (all P>0.05). By stratification analysis, allele A and genotype CA+AA of rs699947 were more frequent in patients with colonic CD compared with the controls (P=0.006, 95%CI:1.143-2.234; P=0.005, 95%CI:1.203-2.900, respectively). Compared with the controls, the allele A and genotype CA+AA of rs699947 were less frequent in patients with ileal lesions including ileal CD and ileocolonic CD (P=0.033, 95%CI:0.524-0.974;P=0.043, 95%CI:0.481-0.989, respectively). The frequency of TT homozygote of rs3025039 was lower in patients with non-stricturing and non-penetrating CD compared with the controls (P=0.036, 95%CI:0.016-0.870).

CONCLUSIONPolymorphisms of the VEGF gene rs699947 locus may contribute to an increased risk for colonic CD, but may play a protective role in patients with ileal lesion. Individuals carrying the TT genotype for VEGF rs3025039 locus may be less susceptible to non-stricturing and non-penetrating CD.

Objective To explore the relationship of Crohn's disease (CD) susceptibility to aryl hydrocarbon receptor (AhR) polymorphisms and haplotypes in Han population in Wenzhou city, China. Methods A total of 310 CD patients and 573 age-and sex-matched healthy controls were enrolled in our study. Three single nucleotide polymorphisms (SNPs) of AhR(rs10249788,rs2158041,rs2066853) were determined by the improved multiple ligase detection reaction technique. Unconditional logistic regression analyses was applied to analyze the allelic and genotypic differences of each SNP between CD patients and controls, as well as their influence on the clinicopathologic characteristics in CD patients. Analyses of linkage disequilibrium and haplotype were performed by Haploview 4.2 software in all study subjects. Results Compared with the controls, the variant allele (T) and genotype (CT+TT) of (rs2158041) were evidently decreased among CD patients (19.52% vs. 25.04%, P=0.009; 34.19% vs. 44.68%, P=0.003). According to"the Montreal Classification Standards", CD patients were divided into different subgroups. The variant allele(T)and genotype(CT+TT)of(rs2158041)were significantly lower in patients with terminal ileum CD than in controls (16.79% vs. 25.04%, P=0.005; 28.24% vs. 44.68%, P=0.001). Similar conclusions were also drawn in patients with constricting disease when compared with the controls(15.20%vs.25.04%,P=0.003;28.43% vs.44.68%,P=0.003).The three SNPs above were shown to be in a linkage disequilibrium.Compared with the controls respectively,the frequency of haplotype(CCG)was increased in CD patients (44.73% vs. 39.60%, P=0.039), whereas that of haplotype (CTG) was decreased (18.02% vs. 22.78%, P=0.047). Conclusions AhR (rs2158041) variation might influence the risk as well as the location and behavior of CD. The haplotype (CCG) possibly increase the risk of CD development, whereas haplotype(CTG)might decrease it.

Objective To investigate the association of ulcerative colitis (UC) with fork head/ winged helix transcription factor-3 (Foxp3) polymorphisms in Han population in Zhejiang province,China.Methods A total of 381 UC patients and 490 healthy controls were enrolled in this study.The four single nucleotide polymorphisms (SNPs) of Foxp3 (rs3761547,rs2232365,rs2294021,rs3761548) were examined by SNaPshot.The analyses of linkage disequilibrium (LD) and haplotype were also performed in all study subjects.Results When male and female UC patients were compared with their corresponding controls respectively,the alleles and genotypes of the four SNPs were not statistically different (all P ＞0.05).According to severity and location of the disease,the UC patients were divided into different subgroups.The alleles (C,G,A) of (rs2232365,rs2294021,rs3761548) were more frequent in male patients with severe UC than in the male controls (69.6％ vs 34.3％,P =0.001;69.6％ vs 34.3％,P =0.001;39.1％ vs 14.4％,P =0.002,respectively).As compared with the female controls,the alleles (C,G,A) and genotypes (TC + CC,AG + GG,CA + AA) of (rs2232365,rs2294021,rs3761548) were significantly increased in the female patients with severe UC (51.9％ vs 38.0％,63.5％ vs 39.2％,53.8％ vs21.4％,80.8％ vs57.7％,84.6％ vs58.4％,76.9％ vs34.7％,all P＜0.05).The four SNPs above were shown to be in a strong LD both in male and in female subjects.When male and female UC patients were compared with their corresponding controls respectively,nevertheless,each haplotype frequency was not statistically different (all P ＞ 0.05).Conclusions Foxp3 (rs2232365,rs2294021,rs3761548) variations might engender the increased risk of severe UC in Chinese Han patients.

OBJECTIVETo assess the association of single nucleotide polymorphisms (SNPs) and haplotypes of solute-linked carrier family 26 member A3 (SLC26A3) gene with ulcerative colitis (UC) among Chinese patients.

METHODSFor 416 UC patients and 584 controls, 5 SNPs of the SLC26A3 gene (rs17154444, rs7810937, rs7785539, rs2108225 and rs6951457) were determined with a SNaPshot method. Linkage disequilibrium (LD) and haplotype were analyzed for all subjects.

RESULTSThe G allele and AG+GG genotype of rs2108225 were more prevalent in UC patients compared with the controls (65.14% vs. 58.65%, P=0.030; 87.02% vs. 81.85%, P=0.012, respectively). The C allele and TC+CC genotype of rs17154444 were more prevalent in patients with severe UC than in other patients (14.00% vs. 6.01%, P<0.01; 28.00% vs. 11.48%, all P<0.01). Similar conclusion may also be drawn for C allele and GC+CC genotype of rs7785539 (8.00% vs. 7.38%, P=0.011; 16.00% vs. 13.93%, P=0.017, respectively). The SNPs rs17154444, rs7810937, rs7785539 and rs2108225 were found to be in strong LD. Compared with the controls, the T-A-G-G haplotype was more prevalent in UC patients (62.60% vs. 58.20%, P=0.017), whereas the T-G-G-A haplotype was less common in UC patients (27.40% vs. 31.60%, P=0.041).

CONCLUSIONVariations of the SLC26A3 rs2108225 may enhance the risk of UC. The rs17154444 and rs7785539 polymorphisms of the SLC26A3 gene are correlated with the severity of UC. The T-A-G-G haplotype formed by rs17154444, rs781093, rs7785539 and rs2108225 of the SLC26A3 gene may increase the risk for UC, whereas the T-G-G-A haplotype may decrease this risk.

Adult ; Asian Continental Ancestry Group ; genetics ; China ; Chloride-Bicarbonate Antiporters ; genetics ; Colitis, Ulcerative ; genetics ; Female ; Genotype ; Haplotypes ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVETo assess the association of transcobalamine II (TCN2) gene polymorphisms and serum levels of homocysteine (Hcy), vitamin Band folate with ulcerative colitis (UC) among Chinese patients.

METHODSFor 397 UC patients and 574 controls, two single nucleotide polymorphisms of the TCN2 gene (rs1801198, rs9606756) were tested with an improved multiple ligase detection reaction method. Serum Hcy, vitamin Band folate were measured with an enzymatic cycling assay and an chemiluminescence immunoassay, respectively.

RESULTSThe allelic and genotypic frequencies of rs1801198 and rs9606756 did not differ significantly between the two groups (all P> 0.05). Compared with those of the control group, the frequencies of G allele and CG+GG genotype of rs1801198 were greater in patients with moderate and severe UC (both P< 0.05). The same conclusion may also be drawn for the G allele and AG genotype of rs9606756 (both P< 0.05). Compared with the controls, average Hcy level was enhanced in UC patients (P< 0.01), whereas average vitamin Band folate levels were decreased in UC patients (both P< 0.01). In both groups, the average level of Hcy was lower in individuals carrying CC of (rs1801198) than in those with CG+GG (both P< 0.05). A similar conclusion was also drawn for individuals with AA of rs9606756 when compared with those carrying AG(both P< 0.05). Compared with patients with mild UC, average Hcy level was increased in those with moderate and severe UC (P< 0.01), while average vitamin Band folate levels were decreased in those with moderate and severe UC (both P< 0.01). The prevalence of hyperhomocysteinemia(HHcy), vitamin Bdeficiency and folate deficiency was greater in UC patients than in controls (all P< 0.01). In UC patients, the level of Hcy was negatively correlated with those of vitamin B(P< 0.01), albumin(P< 0.01), red blood cells(P< 0.01) and platelet (P< 0.05), but positively correlated with white blood cells(P< 0.01) and Mayo score (P< 0.01). Both HHcy and folate deficiency were independent risk factors for UC (OR=4.173, OR=5.206, both P< 0.01).

CONCLUSIONTCN2 (rs1801198, rs9606756) variations, as well as serum levels of Hcy, vitamin Band folate, are correlated with UC. Both HHcy and folate deficiency are independent risk factors for UC.

Adult ; Colitis, Ulcerative ; blood ; etiology ; genetics ; Female ; Folic Acid ; blood ; Genotype ; Homocysteine ; blood ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Transcobalamins ; genetics ; Vitamin B 12 ; blood

Objective To explore the association of Crohn's disease (CD) with T cell immunoglobulin and mucin domain 3 (Tim-3) gene polymorphisms in patients of Zhejiang Han population in China.Methods A total of 308 CD patients and 573 age-and sex-matched healthy controls were enrolled in our study.Two single nucleotide polymorphisms (SNPs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique (iMLDR).Analyses of linkage disequilibrium and haplotype were also performed by Haploview 4.2 software in all study subjects.Results In general,the allele and genotype frequencies of Tim-3 (rs1036199 and rs10515746) were not statistically different between CD patients and the controls (all P ＞0.05).According to the Montreal Classification,CD patients were divided into different subgroups.The variant allele (C) and genotype (AC + CC) of rs1036199 were more frequent in CD patients with penetrating diseases than in the controls (10.4％ vs 1.7％,P =0.002;20.8％ vs 3.5％,P =0.023).Similar conclusions were also drawn for the variant allele (A) and genotype (CA + AA) of rs10515746 in patients with penetrating diseases when compared with the controls (10.4％ vs 2.2％,P =0.000;20.8％ vs 4.2％,P =0.033,respectively).The two SNPs of Tim-3 were in strong linkage disequilibrium (D'=1.0,r2 =0.928).The haplotype (AC) formed by their wild-type alleles (A) and (C) was decreased in patients with penetrating CD compared with the controls (89.6％ vs 98.3％,P =0.000).However,the haplotype (CA) formed by their variant alleles was more frequent in patients with penetrating CD than in the controls (10.4％ vs 1.6％,P =0.000).Conclusions Tim-3 (rs1036199 and rs10515746) variations might be correlated with the enhanced risk of penetrating diseases in CD patients.Furthermore,the haplotype (AC) and (CA) formed by the two SNPs might be a protective and a risky factor for penetrating CD respectively.

Dysregulated immune response is crucial for the pathogenesis of inflammatory bowel disease.Recent studies suggested that imbalance among Th1, Th2 and Th17 cells was closely related to the aberrant intestinal immune response.Aims: To investigate the association of imbalance among Th1, Th2 and Th17 cells and Crohn''s disease (CD).Methods: Thirty-six CD patients admitted from Jan.2013 to Dec.2014 at the Second Affiliated Hospital of Wenzhou Medical University were enrolled;40 patients undergoing intestinal polypectomy were collected as controls.Inflamed and normal mucosal tissues were obtained from CD patients and the controls, respectively;expressions of Th1, Th2 and Th17 cells related transcriptional factors (T-bet, GATA-3 and RORγt) and cytokines (IFN-γ, IL-4 and IL-17A) were determined by real-time PCR and immunohistochemistry.Results: In comparison with the controls, mRNA expression of T-bet and RORγt, mRNA and protein expressions of IFN-γ and IL-17A, as well as the ratios for T-bet/GATA-3 and RORγt/GATA-3, which represented balance of Th1/Th2 and Th17/Th2, respectively, were all significantly increased in inflamed mucosal tissue in CD patients (P<0.05).Expression of each of the three transcriptional factors was positively correlated with its corresponding cytokine (P<0.05).IFN-γ+ and IL-17A+ cells mainly located in the intestinal epithelial layer and lamina propria with cytoplasmic immunoreactivities in CD patients.In the stratified analysis, all the above-mentioned parameters were significantly higher in active CD than in inactive CD (P<0.05);furthermore, the ratio for RORγt/T-bet, which represented balance of Th17/Th1, was also increased significantly in active CD (P<0.05).Conclusions: Imbalance among Th1, Th2 and Th17 cells in intestinal mucosal tissue was closely related with CD.Polarization of Th1 and Th17 cells are involved in this process, and Th17 polarization is predominant in active disease.

Objective To investigate the relationship between gene polymorphisms of T cell immunoglobulin domain and mucin domain protein-3 (Tim-3) and ulcerative colitis (UC) in Han nationality of Zhejiang.Methods A total of 391 UC patients and 573 healthy controls were recruited.Two single nucleotide polymorphisms (SPNs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique.Chi-square test or Fisher's exact test was performed to analyze the differences in the distribution of Tim-3 gene polymorphisms and its influence on the location and severity.Haploview 4.2 software was used to analyze linkage disequilibrium (LD) and haplotype.Results The frequencies of genotype CA+AA and mutant allele A of rs10515746 in UC were lower than those in healthy controls (1.79％,7/391 vs 4.19％,24/573;0.90％,7/782 vs 2.18％,25/1 146;x2=4.295 and 4.712,P=0.038 and 0.030).However,there was no significant differences in frequencies of genotype CA+ CC and mutant allele C of gene rs1036199 between UC patients and the healthy controls (1.79％,7/891 vs 8.49％,20/578;0.90％,7/782 vs 1.74％,20/1 146;both P＞0.05).The frequencies of genotype CA+AA and mutant allele A of rs10515746 in mild and moderate UC patients were both higher than those in severe UC patients (2.87 ％,7/244 vs 0;1.43 ％,7/488 vs 0),and the differences were statistically significant (Fisher's exact test,P=0.049 and 0.048).The analysis for LD indicated that rs1036199andrs10515746 were closeLD (D'=0.92,r2=0.72).Furthermore,the frequency of haplotype CA formed by the mutant alleles C and A of these two SNPs was lower in UC patients than that in healthy controls (0.64％,5/782 vs 1.74％,20/1 146),and the difference was statistically significant (x2 =4.441,P=0.035).Conclusions Tim-3 (rs10515746) gene mutation may not only decrease the incidence,but also reduce the severity of UC.Moreover,the haplotype CA formed by the mutant alleles of rs1036199 and rs10515746 may also reduce the incidence of UC.

Objective To explore the relation between genetic polymorphisms and the expression in colonic tissues of solute-linked carrier family 26 member A3 (SLC26A3) and susceptibility of Crohn's disease (CD) in Han population of Zhejiang Province.Methods A total of 265 CD patients and 566 gender-and age-matched healthy individuals were enrolled.Alleles and genotypes of SLC26A3 (rs17154444,rs7810937,rs7785539,rs2108225,rs6951457) were examined by SNaPshot.The linkage disequilibrium (LD) and haplotype were also analyzed.Eight patients with colonic CD and eight genderand age-matched patients with benign colonic polyps (control group) were selected.The expression level of SLC26A3 protein in the colonic tissue was detected by immunohistochemistry.T test and rank-sum test were performed for statistical analysis.Unconditional Logistic regression analysis was used to analyze the distributions of SLC26A3 polymorphisms and their effects on the clinicopathological features of CD patients.Results The frequencies of mutant allele of rs2108225,rs7785539 and rs6951457 of the CD group were 53.77％ (285/530),4.72％ (25/530) and 2.83％ (15/530),and the frequencies of mutant genotype were 76.23 ％ (202/265),9.43 ％ (25/265) and 5.66 ％ (15/265),which were lower than those of the control group (60.95％,690/1 132;8.13％,92/1 132;6.10％,69/1 132;83.92％,475/566;15.37％,87/566 and 11.84％,67/566),and the differences were statistically significant (all P＜0.05).The frequencies of mutant allele of rs17154444 and rs7810937 of the CD group were 10.19％ (54/530) and 34.91 ％ (185/530),and the frequencies of mutant genotype were 18.49 ％ (49/265) and 56.23 ％ (149/ 265),compared with those of the control group (8.30％,94/1 132;30.92％,350/1 132;15.55％,88/566 and 51.77％,293/566),the differences were not statistically significant (all P＞0.05).The frequency of mutant allele G of rs2108225 in patients with ileal CD was 47.89 ％ (91/190),and the frequency of mutant genotypeAG+GG was 65.26％(62/95),which were both lower than those of colonic CD (61.62％,122/198 and 85.86％,85/99),and the differences were statistically significant (both P＜0.012 5).rs7810937,rs7785539 and rs2108225 were in a strong linkage disequilibrium.The frequencies of haplotypes AGG and ACA of the CD group were 53.96％ (286/530) and 4.34％ (23/530),which were lower than those of the control group (60.07％,680/1 132 and 7.51％,85/1 132),and the differences were statistically significant (52 =5.534,P=0.019;x2 =5.967,P=0.015).And the frequency of haplotype AGA of the CD group was 8.30％ (44/530),which was higher than that of the control group (1.15％,13/1 132),and the difference was statistically significant (x2 =7.793,P＜0.01).Furthermore,the expression level of SLC26A3 protein in colonic tissues of eight colonic CD patients was 0.19±0.07,which was lower than that of patients with benign colonic polyps (0.26 ±-0.03),and the difference was statistically significant (t=2.55,P=0.023).In addition,the expression levels of SLC26A3 protein in patients carrying genotype GG or AG of rs2108225 were 0.19±0.03 and 0.10±0.01,respectively,which were lower than that of patients carrying genotype AA (0.26± 0.02),and the differences were statistically significant (t=3.19,P=0.033;t=9.06,P=0.003).Conclusions The genetic polymorphismns and their haplotypes of SLC26A3 (rs7785539,rs2108225 and rs6951457) are associated with the susceptibility of CD,and SLC26A3 (rs2108225) polymorphism may affect the expression level of SLC26A3 protein in the colonic tissues.