ABSTRACT
Objective:To investigate the inhibitory effect of RMT1-10-induced tolerogenic dendritic cells (Tol-DCs) in vitro on high-risk corneal allograft rejection in mice and its mechanism. Methods:One hundred SPF male BALB/c mice and fifty SPF male C57BL/6 mice were selected.Bone marrow-derived immature dendritic cells (imDCs) obtained from C57BL/6 mice were divided into imDCs group, mature dentritic cells (mDCs) group, RMT1-10 group, and IgG isotype control group.The imDCs in the four groups were cultured with no intervention, lipopolysaccharide, RMT1-10 and lipopolysaccharide, or IgG isotype antibody and lipopolysaccharide for 7 days according to grouping.The expression levels of different phenotypes of DCs including CD11c, CD80, CD86, major histocompatibility complex (MHC)-Ⅱ, T cell immunoglobulin and mucin domain containing molecule (Tim)-4 and CD103 in the four groups were detected by flow cytometry.The concentrations of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the DCs supernatants were determined by enzyme-linked immunosorbent assay.A mixed lymphocyte culture system was established, and the stimulation index (SI) of CD4 + T cell proliferation stimulated with DCs was detected by cell counting kit 8 method.Corneal neovascularization was induced by corneal stromal suture in BALB/c mice, and the 80 mice with neovascularization in 4 quadrants growing into the middle and peripheral cornea were used as recipients.The recipient mice were randomized into imDCs group, mDCs group, RMT1-10 group, and IgG isotype control group using the random number table method, with 20 mice in each group.An injection of corresponding DCs (1×10 6 cells/100 μl) was administered to the recipient mice via the tail vein according to grouping.At 7 days following the injection, C57BL/6 mice were used as donors and penetrating keratoplasty was performed.Within one month after the operation, signs of corneal grafts rejection, including opacity, edema and neovascularization, were observed by slit lamp biomicroscopy and scored every day.At 21 days after the operation, 5 recipients selected from each group were subcutaneously injected with naive C57BL/6 splenocytes (1×10 6 cells/100 μl) behind the ear.The delayed type hypersensitivity (DTH) was evaluated by ear swelling at 24 hours after the subcutaneous injection.The use and care of experimental animals complied with the Regulations on the Management of Experimental Animals promulgated by the State Science and Technology Commission.This study protocol was approved by an Ethics Committee of the Affiliated Hospital of Chengde Medical University (No.CYFYLL2020055). Results:Compared with mDCs group, the expressions of CD80, CD86 and MHC-Ⅱ, and the percentage of Tim-4-positive cells in CD11c-positive cells were significantly decreased in RMT1-10 group, showing statistically significant differences (all at P<0.001). The percentage of Tim-4-positive cells were significantly decreased in RMT1-10 group than imDCs group, and the percentage of CD103-positive cells in RMT1-10 group was significantly higher than imDCs group, mDCs group and IgG isotype control group (all at P<0.001). The concentrations of IL-10 and TGF-β in the cell culture supernatant of RMT1-10 group were significantly higher than those of the other three groups, with statistically significant differences (all at P<0.001). There were statistically significant differences in the SI of CD4 + T cell proliferation simulated by DCs ( Fgroup=1 833.00, P<0.001; Fratio=230.40, P<0.001; Finteraction=3.06, P=0.01). The SI of DCs/CD4 + T cells ratio at 1∶5, 1∶10, 1∶20 and 1∶40 were all significantly lower in imDCs group than mDCs group, and were all significantly lower in RMT1-10 group than imDCs group (all at P<0.05). There was a statistically significant difference in corneal graft survival curve among various groups ( χ2=77.69, P<0.001). The survival rate of RMT1-10 group was significantly higher than that of imDCs group ( χ2=9.74, P=0.002), and the survival rate of imDCs group was significantly higher than that of mDCs group ( χ2=31.02, P<0.001). The ear swelling of recipient mice of positive control group, mDCs group, IgG isotype control group, imDCs group and RMT1-10 group was (503.6±17.2), (475.7±17.6), (456.2±18.8), (225.2±39.4), (118.1±12.6), and (106.4±7.4) μm, with a statistically significant difference among them ( F=377.10, P<0.001). The mice ear swelling was more serious in positive control group than mDCs group, more serious in IgG isotype control group than imDCs group, and more serious in imDCs group than RMT1-10 group (all at P<0.05). Conclusions:RMT1-10 can inhibit the rejection of high-risk corneal transplantation in mice, the mechanism of which may be attributed to inducing imDCs to transform into Tol-DCs in vitro and up-regulating the expression of TGF-β and IL-10, which promotes antigen-specific immune tolerance after adoptive transfer, thereby indirectly prolongs the survival of corneal grafts.
ABSTRACT
Objective To investigate the association of Tim-1 protein expression and its gene polymorphism with nutritional parameters in patients with systemic lupus erythematosus (SLE).Methods Peripheral blood samples were collected from 126 patients with SLE.Serum Tim-1 protein levels were detected by ELISA,and the Tim-1 gene-416G＞C,-1454G＞A polymorphism was detected by PCR-RFLP.Prealbumin,ceruloplasmin,and retinol conjugated protein levels were determined by immunoturbidimetry.Ferritin and 1,25-dihydroxy vitamin D3 levels were detected by electrochemiluminescence.Results Concentrations of serum Tim-1 protein,prealbumin,ceruloplasmin,retinol binding protein,ferritin,and 1,25-dihydroxy vitamin D3 were 249.7±30.2 pg/mL,226±42 μg/mL,363±95 μg/mL,29.4± 13.2 μg/mL,355± 164 ng/mL,and 26.4-± 11.5 ng/mL,respectively.In the-416G＞C site,GG,GC,and CC genotypes accounted for 11.9％,57.1％,and 31.0％,respectively.In the-1454G＞A site,GG,GA,and AA genotypes accounted for 67.5％,26.2％,and 6.3％,respectively.The Tim-1 protein concentration did not differ significantly between the different genotypes of the-416G＞C site (F=0.575,P=0.564) or-1454G＞A site (F=1.255,P=0.289).Tim-1 level was significandy negatively correlated with prealbumin (r =-0.176,P =0.033),and positively correlated with ceruloplasmin (r =0.205,P =0.014) and 1,25-dihydroxy vitamin D3 (r=0.166,P=0.042).The serum prealbumin level decreased significantly (P=0.027) in patients harboring the GG genotype in the-1454G＞A site,whereas the serum 1,25-dihydroxy vitamin D3 level decreased significandy (P =0.024) in patients with the AA genotype in the-1454G＞A site.Conclusion Serum Tim-1 protein level and the-1454G＞A polymorphism of Tim-1 gene are associated with the nutritional function of patients with SLE.
ABSTRACT
Background Corneal transplantation is the most reliable and effective means to treat the corneal blindness in the clinical,immune rejection is a major cause of corneal graft failure after the keratoplasty.Objective This study aimed to investigate the role of Tim-1 in the immune reaction following corneal transplantation in rats.Methods Forty clean female Wistar rats were randomized into normal control group,autologous corneal transplantation group and allogeneic corneal transplantation group.Penetrating corneal transplantation was performed with the Wistar rat donors and Wistar rat receipts in the autologous corneal transplantation group,while with the SD rat donors and Wistar rat receipts in the allogeneic corneal transplantation group.The corneal graft diameter was 3.5 mm and the plant bed diameter was 3.0 mm.The inflammatory response of the grafts was examined under the slit lamp microscope 7 days and 14 days after operation and scored based on the criteria of Larkin.Rejection index (RI),mean survival time and survival rate were calculated.The histopathological examination was performed 7 days and 14 days after surgery to evaluate the inflammatory manifestation,and the expressions of Tim-1 protein and mRNA were assayed by immnunochemistry and real-time fluorescence quantitative PCR (RT-qPCR)in the time points mentioned above.Results Mild edema of the grafts were found 7 days after operation in both the autologous corneal transplantation group and the allogeneic corneal transplantation group.In postoperative 14 days,the grafts were clear in the autologous corneal transplantation group,but the thickening,neovacularization and cloudy of the grafts were exhibited in the allogeneic corneal transplantation group.The survival rate of the grafts was 100％ in the autologous corneal transplantation group and that of the allogeneic corneal transplantation group was 0 with the survival time of (9.8±1.2) days.Histopathological examination revealed the stromal infiltration of inflammatory cells in both the autologous and allogeneic corneal transplantation groups in the seventh day,however,the inflammatory cells were obvious decreased in the autologous group but increased in the allogeneic corneal transplantation group in the fourteenth day.Immunochemistry showed a gradually declined positive cells for Tim-1 protein in the autologous corneal transplantation group,but the positive cells were exactly elevated in the allogeneic corneal transplantation group from 7 days through 14 days after operation;While only few positive cells were seen in the normal control group.The expression levels of Tim-1 mRNA in the grafts were 1.24 ± 0.03,5.85 ± 0.08 and 6.54 ± 0.20 in the normal control group,autologous corneal transplantation group and that of the allogeneic corneal transplantation group,respectively,in the seventh day,and in the fourteen day after operation,the expression level declined to 1.54 ±0.10 in the autologous corneal transplantation group and elevated to 8.62±0.24 in the allogeneic corneal transplantation group,showing significant differences among the different groups and various time points (Fgroup =3 277.590,P =0.000 ; Ftime =136.000,P =0.000).Conclusions Tim-1 may play an important role not only in the inflammatory response but also in the rejection reaction of the corneal transplantation.
ABSTRACT
The T-cell immunoglobulin- and mucin-domain-containing molecules (Tim) have been implicated in the pathogenesis of immune diseases. In this study, we used quantitative realtime reverse transcription-polymerase chain reaction to examine the Tim-1 mRNA expression in peripheral blood mononuclear cells from Henoch-Schönlein purpura patients. The results showed that Tim-1 mRNA expression was significantly higher in patients, which was closely correlated with serum TNF-α, IL-4 levels, IgA1 levels.
ABSTRACT
Objective To detect the levels of the mRNA expression of TIM-3,TIM-1,T-bet,GATA-3,IFN-γ,IL-4 and Galectin-9 in the peripheral blood monocytes (PBMCs) of the patients with Graves disease(GD),and to explore their potential role in the pathogenesis of GD.Methods We used fluorescence quantitative real-time reverse transcription-polymerase chain reaction to measure the mRNA expression of TIM-3,TIM-1 and other associated genes in PBMCs of 70 patients with GD and 22 healthy controls.In addition,we analyzed the relationship of TIM-3,TIM-1 and other associated genes.Results The expression of TIM-3 and TIM-1 mRNA in the PBMCs from GD patients were abnormally higher,the GD patients with Graves' ophthalmopathy group had significantly higher level of TIM-3 mRNA expression than that of GD patients without Graves' ophthalmopathy group,but no statistically significant difference was found in the expression of TIM-1 mRNA.Untreated GD patients had significantly higher level of TIM-3 mRNA expression than that of GD patients in recurrence group,however the expression of TIM-1 mRAN was opposite.But no statistically significant difference was found in TIM-3 mRNA expression of recovery GD patient and healthy control group.Though the expression of TIM-1 mRNA was significantly decreased,it was still higher than that of the normal control group.Conclusion TIM-3 and TIM-1 may participate in the occurrence,development and turnover of GD.TIM-3 or TIM-1 may prove to be an important target for developing new drugs and treatments to GD.
ABSTRACT
Objective: To investigate the association between three single nucleotide polymorphisms-2562G＞A,-416C＞G and-232G＞A in Tim-3(T cell immunoglobulin domain and mucin domain protein 3)gene promoter region and child allergic asthma in Chinese Han population by using family-based association study.Methods: Genotypes of 3 SNPs(-2562G＞A,-416C＞G and-232G＞A)in 118 allergic asthma nuclear pedigrees were analyzed by restriction fragment length polymorphism.The genotype data were analyzed by using the family-based transmission disequilibrium test(TDT).Haplotypes and their frequencies were established and analyzed by TRANSMIT software.Results: ①No transmission disequilibrium was found at the-2562G＞A and-232G＞A sites from heterozygous parents onto patients in 118 trios analyzed by TDT(P＞0.05);However,at the-416 C＞G locus,the observed values of G allele from heterozygous parents to offspring were significantly higher than the expected values(P＜0.05)②The haplotype TDT analysis by TRANSMIT showed the observed and the expected value in GCA and GGA haplotype from parents to the affected offsprings had significant difference respectively(P＜0.05).The Global X~2 test results also showed that Tim-1 haplotype were associated with child allergic asthma(X~2 = 17.26, P＜0.01).Conclusion: Tim-1 gene promoter-416C＞G locus are associated with allergic asthma susceptibility in Hubei Chinese Han population and the haplotypes constructed by-416C＞G are also associated with asthma.Tim-1 genetic polymorphism may play an important role in the pathogenesis of asthma.