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Evaluation of thrombospondin-1 gene polymorphisms in corneal allograft rejection in Asian Indian patients.

ABSTRACT: Purpose:To evaluate the frequency and the association of Thrombospondin 1 (THBS1) gene single nucleotide polymorphisms (SNPs) in Asian Indian patients with optical full thickness corneal grafting surgery. Methods:Prospective case-control analysis of optical penetrating keratoplasty patients with and without immune rejection and controls for genotyping of 3 THBS1 gene SNPs (rs1478604 A>G; rs2228261 C>T; rs2228262 A>G) by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS PCR). Results:Among 58 patients [45 with immune allograft rejection (DNA isolation was possible in 38 samples) and 13 without immune corneal allograft rejection] and 65 controls, allele frequencies observed for rs1478604 (A>G) are A: 69.7% and 72.6%, G: 30.2% and 27.3%; for rs2228261 (C>T) are T: 70.2% and 62.3%, C: 29.7% and 37.6%; and for rs2228262 (A>G) A: 97.4% and 98.4%; G 2.5% and 1.5% respectively. Genotype frequencies were rs1478604 (A>G) AA: 57.8% and 59.3%, AG 23.6% and 26.5%; GG 18.4% and 14%; for rs2228261 (C>T) TT: 40.5% and 33.8%, TC: 59% and 56.9%, CC: 0% and 9.2%; for rs2228262 (A>G) AA: 94.8% and 96.8%, AG: 5.1% and 3.1% in rejection and controls respectively. The allele and genotype frequency for the 3 described THSB1 SNPs did not show any difference between the corneal graft immune rejection patients and controls. Conclusion:Asian Indian population evaluated for THBS1 gene SNPs by ARMS PCR genotyping in Asian Indian population did not show any genetic association to immune rejection occurrence in our study.

SUBMITTER: Vanathi M

PROVIDER: S-EPMC7210848 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Evaluation of thrombospondin-1 gene polymorphisms in corneal allograft rejection in Asian Indian patients.

Vanathi Murugesan M Shukla Rashmi R Balakrishnan Prahlad P Dwivedi Roopa R Gupta Noopur N Tandon Radhika R

Indian journal of ophthalmology 20200401 4

<h4>Purpose</h4>To evaluate the frequency and the association of Thrombospondin 1 (THBS1) gene single nucleotide polymorphisms (SNPs) in Asian Indian patients with optical full thickness corneal grafting surgery.<h4>Methods</h4>Prospective case-control analysis of optical penetrating keratoplasty patients with and without immune rejection and controls for genotyping of 3 THBS1 gene SNPs (rs1478604 A>G; rs2228261 C>T; rs2228262 A>G) by Amplification Refractory Mutation System-Polymerase Chain Rea ...[more]

PMID: 32174570

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Project description:BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-? expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8?±?1.2 days). Injection twice of MSCs (MST 12.6?±?1.4 days) was more effective than a single injection (MST 10.8?±?1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4?+?T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-? expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Project description:Rationale: Corneal transplantation is an effective treatment to corneal blindness. However, the immune rejection imperils corneal allograft survival. An interventional modality is urgently needed to inhibit immune rejection and promote allograft survival. In our previous study, subconjunctival injections of bone marrow-derived mesenchymal stem cells (BM-MSCs) into a rat model of corneal allograft rejection extended allograft survival for 2 d. In this study, we sought to generate IL-10-overexpressing BM-MSCs, aiming to boost the survival-promoting effects of BM-MSCs on corneal allografts and explore the molecular and cellular mechanisms underlying augmented protection. Methods: A population of IL-10-overexpressing BM-MSCs (designated as IL-10-BM-MSCs) were generated by lentivirus transduction and FACS purification. The self-renewal, multi-differentiation, and immunoinhibitory capabilities of IL-10-BM-MSCs were examined by conventional assays. The IL-10-BM-MSCs were subconjunctivally injected into the model of corneal allograft rejection, and the allografts were monitored on a daily basis. The expression profiling of long noncoding RNA (lncRNA) in the allografts was revealed by RNA sequencing and verified by quantitative real-time PCR. The infiltrating immune cell type predominantly upregulating the lncRNA expression was identified by RNAscope in situ hybridization. The function of the upregulated lncRNA was proved by loss- and gain-of-function experiments both in vivo and in vitro. Results: The IL-10-BM-MSCs possessed an enhanced immunoinhibitory capability and unabated self-renewal and multi-differentiation potentials as compared to plain BM-MSCs. The subconjunctivally injected IL-10-BM-MSCs reduced immune cell infiltration and doubled allograft survival time (20 d) as compared to IL-10 protein or plain BM-MSCs in the corneal allograft rejection model. Further, IL-10-BM-MSCs significantly upregulated lncRNA 003946 expression in CD68+ macrophages infiltrating corneal allografts. Silencing and overexpressing lncRNA 003946 in macrophage cultures abolished and mimicked the IL-10-BM-MSCs' suppressing effects on the macrophages' antigen presentation, respectively. In parallel, knocking down and overexpressing the lncRNA in vivo abrogated and simulated the survival-promoting effects of IL-10-BM-MSCs on corneal allografts, respectively. Conclusion: The remarkable protective effects of IL-10-BM-MSCs support further developing them into an effective interventional modality against corneal allograft rejection. IL-10-BM-MSCs promote corneal allograft survival mainly through upregulating a novel lncRNA expression in graft-infiltrating CD68+ macrophages. LncRNA, for the first time, is integrated into an IL-10-BM-MSC-driven immunomodulatory axis against the immune rejection to corneal allograft.

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Evaluation of thrombospondin-1 gene polymorphisms in corneal allograft rejection in Asian Indian patients.

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Evaluation of thrombospondin-1 gene polymorphisms in corneal allograft rejection in Asian Indian patients.

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