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Objective:To explore the therapeutic effect of anti-interleukin (IL)-12/IL-23 p40 antibody on experimental autoimmune uveitis (EAU) and its mechanism.Methods:Sixty-six SPF female C57BL/6N mice aged 6-8 weeks were selected.EAU model was established in 24 mice through immunization with the interphotoreceptor retinoid-binding protein (IRBP) 651-670.The 24 mice were sacrificed before immunization, and on the 3rd, 12th, and 18th day after immunization, with 6 at each time point.Flow cytometry was used to detect the proportion of IL-17A + interferon-γ (IFN-γ) + CD4 + T cells in the spleen, lymph nodes and eyeballs.Another 6 mice were selected to establish EAU model, and fundus images of the mice were taken with a small animal imaging instrument and optical coherence tomography (OCT) 18 days after immunization.The 6 mice were sacrificed after OCT examination and the eyeballs were collected.Hematoxylin-eosin staining was used to observe the retinal inflammation and morphological changes in tissue structure.Flow cytometry was employed to detect the proportion of IL-17A + IFN-γ + CD4 + T cells in lymph nodes.The 6 mice were divided into IL-17A + IFN-γ + highly expressed group and IL-17A + IFN-γ + lowly expressed group according to flow cytometry results, and the retinal injury was compared between the two groups.EAU model was established in another 36 mice, which were divided into anti-IL-12/IL-23 p40 group and IgG group by random number table method, with 18 mice in each group.Anti-IL-12/IL-23 p40 or IgG was injected by tail vein at a 3-day inteval according to grouping.On the 12th and 18th day after immunization, 6 mice were selected from each group to collect lymph nodes and eyeballs, and the proportion of T cell subsets was detected by flow cytometry.Eyeballs of 6 mice in each group were extracted on the 24th day after immunization and retinal damage was observed by hematoxylin-eosin staining.The induced differentiation of CD4 + T cells in vitro was assayed by flow cytometry.The expressions of IL-17 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA) after induced differentiation of IL-17A + IFN-γ + CD4 + T cells.The relative expression levels of Th1 transcription factor T-bet and Th17 transcription factor retinoid acid-related orphan nuclear receptor γt (ROR-γt) after induced differentiation of IL-17A + IFN-γ + CD4 + T cells were detected by real-time quantitative PCR.The use and care of animals followed the ARVO statement and this study protocol was approved by an Ethics Committee of Experimental Animals of Tianjin Medical University Eye Hospital (No.TJYY2019111019). Results:There were significant differences in the proportion of IL-17A + IFN-γ + CD4 + T cells in lymph nodes, spleen and eyeballs between wild-type mice and EAU mice at the 3rd, 12th and 18th day after immunization ( H=9.642, 16.531, 10.385; all at P<0.05). Compared with before immunization, the proportion of IL-17A + IFN-γ + CD4 + T cells was significantly increased in lymph nodes of EAU mice on the 12th day following immunization and was significantly increased in spleen and lymph nodes on day 18 after immunization (all at P<0.05). Severe retinal exudation, retinal detachment, severe inflammatory cell infiltration and extensive retinal folds were detected in IL-17A + IFN-γ + highly expressed mice.Mild retinal edema, focal inflammatory cell infiltration and mild retinal folds were found in IL-17A + IFN-γ + lowly expressed mice.The proportion of CD3 and IL-17A + IFN-γ + CD4 + T cells in the eyeballs of anti-IL-12/IL-23 p40 group was lower than that in IgG group at the 18th day after immunization, and the differences were statistically significant ( t=15.304, 8.080; both at P<0.05). On day 12 after immunization, the percentage of IL-17A + IFN-γ + CD4 + T cells in anti-IL-12/IL-23 p40 group was (0.33±0.18)%, which was significantly lower than (4.83±0.45)% in IgG group ( t=15.974, P<0.001). Compared with IgG group, the percentage of Th1, Th17, IL-17A + IFN-γ + CD4 + T cells and the expression levels of IL-17, IFN-γ, T-bet, ROR-γt in anti-IL-12/IL-23 p40 group were significantly decreased, with statistical significances (all at P<0.05). Conclusions:Anti-IL-12/IL-23 p40 has a therapeutic effect on EAU by inhibiting IL-17A + IFN-γ + CD4 + T cells.
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Objective To investigate the expression of microRNA (miRNA)-10a in the intestinal mucosa,serum and peripheral blood mononuclear cell (PBMC) of patients with inflammatory bowel disease (IBD) and explore its role and relevance in the pathogenesis of the disease.Methods The intestinal or colonic mucosal biopsy specimens of nine active ulcerative colitis (UC) patients,11 active Crohn's disease (CD) patients and eight patients with negative colonoscopy result as control were collected.The sera of 12 active UC patients,13 active CD patients and nine healthy controls were collected.The PBMC of nine active UC patients,11 active CD patients and eight healthy controls were collected.The expression of miRNA-10a in the intestinal mucosa,sera and PBMC and the expression of IL-12/IL-23 p40 in the intestinal mucosa were detected by real-time polymerase chain reaction (PCR).Each 8 cases of active UC and CD patients were collected.The intestinal mucosa before infliximab (IFX) treatment and six weeks after three times of IFX treatment were collected.And at same time,the intestinal mucosa of 11 active UC patients and 10 active CD patients were collected and cultured for 18 hours stimulated with IFX in vitro and then the expression of miRNA-10a in the intestinal mucosa was tested.One-way analysis of variance was used for comparison in three samples.Paired t-test was used for two samples comparison.Spearman test was used for correlation analysis.Results Compared with healthy controls,the expression of miRNA-10a in the intestinal mucosa,serum and PBMC of UC and CD patients significantly decreased (F=38.45,30.46 and 14.74,all P＜0.05).There was no statistic significance between UC and CD groups.The expression of IL-12/IL-23 p40 in the intestinal mucosa of UC and CD patients significantly increased (F=32.90,P＜0.05).The expression of IL-12/IL-23 p40 was negatively correlated with the expression of miRNA-10a in the intestinal mucosa of CD patients.After three times of IFX treatment,the expression of miR-10a in the intestinal mucosa of IBD patients significantly increased (t=3.341,3.382,both P＜0.05).After stimulated with IFX in vitro,the expression of miRNA-10a in the intestinal mucosa significantly increased (t=3.095,7.193,both P＜0.05).Conclusions miRNA-10a was closely correlated with the inflammation of IBD patients and with the role of targeting IL-12/IL-23 p40.miRNA-10a might be a new target for the IBD treatment.
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Objective To analyze the correlation of interleukin(IL)-12B gene single nucleotide polymorphism(SNP)rs6887695 with clinical phenotypes(including age at onset,family history,clinical types,gender)of psoriasis vulgaris in Chinese Han population.Methods This study recruited 575 patients with psoriasis vulgaris and 1403 healthy controls.DNA samples were obtained from these subjects.PCR with Taqman fluorescent probe(ABI 7900 system)was performed to analyze the genotype of SNP rs6887695 in IL-12B gene.Statistical analysis was carried out by using the software SPSS 14.0,and Chi-square test was conducted to compare the frequency of the SNP rs6887695 genotypes and alleles between the patients and controls as well as between patients with different clinical phenotypes of psoriasis.Results The frequency of GG,GC and CC genotype of the SNP rs6887695 was 42.61％,45.39％ and 12.0％ respectively in the patients,compared to 34.42％,47.83％ and 17.75％ in the healthy controls(x2 =16.31,P ＜ 0.01);the frequency of G and C allele of the SNP rs6887695 was 65.30％ and 34.70％ respectively in the patients,compared to 58.34％ and 41.66％ respectively in the healthy controls(x2 =16.54,P＜0.01).Significant differences were observed in the distribution of genotypes and alleles of the SNP rs6887695 between patients with chronic plaque psoriasis(n =543)and those with acute guttate psoriasis(n =32,x2 =18.11,12.19,both P ＜ 0.01).Increased frequency of G allele and GG genotype of the SNP rs6887695 were noted in the patients with psoriasis vulgaris compared with the healthy controls,and in the patients with plaque psoriasis compared with those with guttate psoriasis.However,there was no statistical difference in the distribution of SNP rs6887695 genotypes or alleles between 540 patients with adult onset psoriasis and 35 patients with child onset psoriasis,between 102 patients with family history and 440 patients without family history,or between 341 male patients and 234 female patients(all P ＞ 0.05).Conclusions The IL-12B SNP rs6887695 may be associated with the susceptibility to psoriasis vulgaris in Chinese Han population,especially with the susceptibility to plaque psoriasis,but seems unassociated with the age at onset,family history or gender of patients.
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Objective To investigate the association between single nucleotide polymorphism (SNP) of interleukin-12 (IL-12) p40 3'untranslated region rs3212227 site and hepatitis C virus (HCV) infection. Methods Patients with hepatitis C (n=127) were genotyped and analyzed for the SNP of IL-12 p40 rs3212227 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. The serum HCV RNA levels of patients with hepatitis C were detected using real time fluorescence quantitative-polymerase chain reaction (FQPCR). Inter-group comparisons of genotype and allele frequency were analyzed using chi-square test.Results In patients with hepatitis C, the frequencies of AA, AC and CC genotypes of IL-12 p40 rs3212227 site were 34. 6% ,40. 9% and 24. 4% , respectively,and the frequencies of allele A, C of IL12 p40 rs3212227 site were 55.1% and 44. 9%, respectively. The frequency of rs3212227 C allele in patients with HCV RNA ≥2. 0× 106 copy/mL was higher than that in patients with HCV RNA <2. 0 ×106 copy/mL (χ2 =7. 367, P = 0. 007). The frequency of rs3212227 C allele in responders to interferon (IFN) therapy was lower than that in patients with nonresponse to IFN therapy (χ2 =4. 942,P=0. 026). Conclusions The SNP of rs3212227 is correlated with HCV infection. The carriers with C allele may be susceptible to HCV infection, while resistant to IFN therapy.
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Objective To investigate the asseciation of 1188A/C polyrnorphism of Interlenkin-12B gene (IL-12B) with remit-relapse multiple sclerosis (RRMS) in the southern Chinese population.Methods Ninety-four patients with RRMS and 145 age- and-sex-matched normal controls were recnfited in this study.The polymorphism was detected by using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assay in these subjects.The frequencies of the alleles in each group were statistically analyzed.Results The frequency of the allele A increased significantly in RRMS patients (64.4%) compared with that in healthy controls (53.8%, χ2=5.228, P=0.022).An increased risk for MS was suggested in carriers of the A allele (OR=1.551, 95% CI=1.064-2.262).Conclusions The A allele in 1188A/C polymorphism of IL-12B gene may be a risk factor for RRMS in southern Chinese population.