Prevention Of Corneal Allograft Rejection Research Articles

Resveratrol regulates macrophage recruitment and M1 macrophage polarization and prevents corneal allograft rejection in rats.

Resveratrol is an immune modulator that can reduce M1 macrophage polarization in vitro. Reducing macrophage recruitment and M1 polarization can prevent corneal allograft rejection (CGR). In this study, rat corneal allograft rejection models were established to explore the effects of resveratrol on CGR and macrophages and the underlying mechanisms after corneal transplantation. Corneal allograft models were established, and 100 mg/kg resveratrol was injected intraperitoneally. The corneal allografts were assessed clinically using the Holland rejection scoring system, anterior segment photography, and anterior segment optical coherence tomography. Corneal macrophages, pro-inflammatory cytokines, and corneal lymphatic vessels were detected using hematoxylin and eosin staining, immunofluorescence staining, and real-time quantitative polymerase chain reaction (qRT-PCR). Dendritic cells (DCs) in cervical lymph nodes were explored using flow cytometry. RNA sequencing experiments were conducted to identify the mechanisms through which resveratrol affected CGR. The results were verified using Simple Western analysis. Pro-inflammatory cytokines by macrophages in vitro were measured using qRT-PCR and enzyme-linked immunosorbent assays. Resveratrol significantly prolonged the survival of corneal grafts and reduced graft edema and central corneal thickness. Corneal macrophage recruitment and M1 macrophage polarization decreased significantly after corneal transplantation in the resveratrol group. Resveratrol also reduced pro-inflammatory cytokines in corneal grafts and suppressed the early generation of cornea lymphatic vessels and the recruitment of cornea inflammatory cells 14 days after surgery. Resveratrol decreased the proportion of DCs in ipsilateral cervical lymph nodes. The effect of resveratrol on CGR was related to the phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) pathway. Resveratrol reduced the secretion of pro-inflammatory cytokines by M1 macrophages in vitro. Our findings suggest that resveratrol can reduce corneal macrophage recruitment and M1 macrophage polarization after corneal transplantation in rats and prevent CGR. The PI3K/Akt pathway may be an important mechanism that warrants further research.

Characterization and Evaluation of Rapamycin-Loaded Nano-Micelle Ophthalmic Solution.

Rapamycin-loaded nano-micelle ophthalmic solution (RAPA-NM) offers a promising application for preventing corneal allograft rejection; however, RAPA-NM has not yet been fully characterized. This study aimed to evaluate the physicochemical properties, biocompatibility, and underlying mechanism of RAPA-NM in inhibiting corneal allograft rejection. An optimized RAPA-NM was successfully prepared using a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (PVCL-PVA-PEG) graft copolymer as the excipient at a PVCL-PVA-PEG/RAPA weight ratio of 18:1. This formulation exhibited high encapsulation efficiency (99.25 ± 0.55%), small micelle size (64.42 ± 1.18 nm), uniform size distribution (polydispersity index = 0.076 ± 0.016), and a zeta potential of 1.67 ± 0.93 mV. The storage stability test showed that RAPA-NM could be stored steadily for 12 weeks. RAPA-NM also displayed satisfactory cytocompatibility and high membrane permeability. Moreover, topical administration of RAPA-NM could effectively prevent corneal allograft rejection. Mechanistically, a transcriptomic analysis revealed that several immune- and inflammation-related Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the downregulated genes in the RAPA-NM-treated allografts compared with the rejected allogenic corneal grafts. Taken together, these findings highlight the potential of RAPA-NM in treating corneal allograft rejection and other ocular inflammatory diseases.

Piperlongumine alleviates corneal allograft rejection via suppressing angiogenesis and inflammation.

Neovascularization and inflammatory response are two essential features of corneal allograft rejection. Here, we investigated the impact of Piperlongumine (PL) on alleviating corneal allograft rejection, primarily focusing on pathological angiogenesis and inflammation. A murine corneal allograft transplantation model was utilized to investigate the role of PL in preventing corneal allograft rejection. PL (10 mg/kg) or vehicle was intraperitoneally injected daily into BALB/c recipients from day -3 to day 14. The clinical signs of the corneal grafts were monitored for 30 days. Corneal neovascularization and inflammatory cell infiltration were detected by immunofluorescence staining and immunohistochemistry. The proportion of CD4+ T cells and macrophages in the draining lymph nodes (DLNs) was examined by flow cytometry. In vitro, HUVECs were cultured under hypoxia or incubated with TNF-α to mimic the hypoxic and inflammatory microenvironment favoring neovascularization in corneal allograft rejection. Multiple angiogenic processes including proliferation, migration, invasion and tube formation of HUVECs in hypoxia with or without PL treatment were routinely evaluated. The influence of PL treatment on TNF-α-induced pro-inflammation in HUVECs was investigated by real-time PCR and ELISA. In vivo, PL treatment effectively attenuated corneal allograft rejection, paralleled by coincident suppression of neovascularization and alleviation of inflammatory response. In vitro, PL distinctively inhibited hypoxia-induced angiogenic processes in HUVECs. Two key players in hypoxia-induced angiogenesis, HIF-1α and VEGF-A were significantly suppressed by PL treatment. Also, TNF-α-induced pro-inflammation in HUVECs was hampered by PL treatment, along with a pronounced reduction in ICAM-1, VCAM-1, CCL2, and CXCL5 expression. The current study demonstrated that PL could exhibit both anti-angiogenic and anti-inflammatory effects in preventing corneal allograft rejection, highlighting the potential therapeutic applications of PL in clinical strategy.

Local and Systemic Injections of Human Cord Blood Myeloid-Derived Suppressor Cells to Prevent Graft Rejection in Corneal Transplantation.

Myeloid-derived suppressor cells (MDSCs) are therapeutic agents to prevent graft rejection in organ transplants by modulating inflammation. Herein, the immunosuppressive effect of human cord blood MDSCs on corneal allograft models was confirmed. CB-MDSCs were locally (subconjuctival, 5 × 105) or systemically (intravenous, 1 × 106) injected twice on days 0 and 7. A corneal transplantation model was established using C57BL/6 and BALB/c mice, and corneal graft opacity was measured to evaluate graft rejection up to 6 weeks. Results showed that graft survival in the MDSCs groups increased compared to vehicle groups after 42 days. Systemic and local MDSC administration inhibited the maturation (MHC-IIhi CD11c+) of dendritic cells (DCs) and the differentiation of interferon γ+ CD4+ Th1 in draining lymph nodes (LNs). However, vehicle groups increased the infiltration of CD3+ T cells and F4/80+ macrophages and produced prominent neovascular and lymphatic vessels into the graft site with increased mRNA expression of VEGF-A/C and VEGFR-1/R-3. Local MDSCs administration showed prominent anti-angiogenic/anti-lymphangiogenic effects even at lower MDSCs doses. Thus, CB-MDSCs could relatively suppress the infiltration of pathological T cells/macrophages into the corneas and the migration of mature DCs into draining LNs Therefore, ocular and systemic MDSCs administration showed therapeutic potential for preventing corneal allograft rejection.

Role of Immune Cell Diversity and Heterogeneity in Corneal Graft Survival: A Systematic Review and Meta-Analysis.

Corneal transplantation is one of the most successful forms of solid organ transplantation; however, immune rejection is still a major cause of corneal graft failure. Both innate and adaptive immunity play a significant role in allograft tolerance. Therefore, immune cells, cytokines, and signal-transduction pathways are critical therapeutic targets. In this analysis, we aimed to review the current literature on various immunotherapeutic approaches for corneal-allograft rejection using the PubMed, EMBASE, Web of Science, Cochrane, and China National Knowledge Infrastructure. Retrievable data for meta-analysis were screened and assessed. The review, which evaluated multiple immunotherapeutic approaches to prevent corneal allograft rejection, showed extensive involvement of innate and adaptive immunity components. Understanding the contribution of this immune diversity to the ocular surface is critical for ensuring corneal allograft survival.

Subconjunctival administration of low-dose murine allogeneic mesenchymal stromal cells promotes corneal allograft survival in mice

BackgroundSystemic administration of mesenchymal stromal cells (MSCs) has been efficacious in many inflammatory disease settings; however, little data are available on the potential immunomodulatory effects following local MSC administration in the context of corneal transplantation. The purpose of this study was to assess the potential of subconjunctival injection of MSCs to promote corneal allograft survival.MethodsMSCs were isolated from female C57BL/6 (H-2k) or Balb/c (H-2d) mice and extensively characterized. An allogeneic mouse corneal transplant model was used with Balb/c mice as recipients of C57BL/6 grafts. A dose-finding study starting with 5 × 105 MSCs injected subconjunctivally at day − 7 was tested first followed by a more clinically translatable low-dose single or dual injection strategy on day − 1 and day + 1 before/after transplantation. Graft transparency served as the primary indicator of transplant rejection while neovascularization was also recorded. Lymphocytes (from draining lymph nodes) and splenocytes were isolated from treatment groups on day 2 post-transplantation and characterized by flow cytometry and qRT-PCR.ResultsBoth high- and low-dose injection of allogeneic MSCs on day − 7 led to 100% graft survival over the observation period. Moreover, low-dose dual subconjunctival injection of 5 × 104 allogeneic MSCs on day − 1 or day + 1 led to 100% allograft survival in transplant recipients (n = 7). We also demonstrate that single administration of allogeneic MSCs on either day − 1 or day + 1 promotes rejection-free graft survival in 100% (n = 8) and 86% (n = 7) of transplanted mice, respectively. Early time point ex vivo analysis suggests modulation of innate immune responses towards anti-inflammatory, pro-repair responses by local MSC administration.ConclusionThis work demonstrates that low-dose subconjunctival injection of allogeneic MSCs successfully promotes corneal allograft survival and may contribute to refining future MSC immunotherapies for prevention of corneal allograft rejection.

Topical 0.03% tacrolimus versus systemic mycophenolate mofetil as adjuncts to systemic corticosteroids for preventing graft rejection after repeat keratoplasty: one-year results of a randomized clinical trial.

To compare the efficacy of topical 0.03% tacrolimus in combination with systemic corticosteroids versus systemic mycophenolate mofetil (MMF) and corticosteroids in preventing corneal allograft rejection after repeat keratoplasty. This prospective, randomized clinical trial enrolled 63 consecutive eyes of 63 patients who underwent repeat keratoplasty after a failed penetrating keratoplasty. Group 1 (32 eyes) received MMF orally 1 g twice daily for the first 6 months and then 1 g daily for the next 6 months, and group 2 (31 eyes) received topical 0.03% tacrolimus four times a day for 12 months. All patients were treated with topical and oral corticosteroids postoperatively. The participants were observed closely for signs of graft rejection, and the rates of rejection-free graft survival were calculated and compared between the two groups at postoperative month 12. The groups were balanced in patient's age and risk factors for graft rejection (e.g., original diagnosis, number of previous grafts, and quadrants of corneal vascularization). Endothelial graft rejection occurred in 5 eyes (15.6%) of group 1 and 6 eyes (19.4%) of group 2 (P = 0.75). Irreversible endothelial graft rejection resulting in graft failure occurred in 3 eyes of each group (P = 0.99). The rate of rejection-free graft survival was 84.4% in group 1 and 80.6% in group 2 at postoperative month 12 (P = 0.74). Topical 0.03% tacrolimus was as effective as systemic MMF as adjuncts to topical and systemic corticosteroids in reducing endothelial graft rejection with 12 months follow up after repeat keratoplasty.

The NLRP3 inflammasome regulates corneal allograft rejection through enhanced phosphorylation of STAT3.

The success of corneal transplantation is limited by allograft rejection, but the pathogenic mechanisms of this disease remain poorly defined. In this study, we showed that the NOD, LRR-and pyrin domain-containing protein3 (NLRP3) inflammasome-mediated interleukin-1β (IL-1β) production exacerbated corneal allograft rejection. Extracellular ATP contributed to the NLRP3 inflammasome-mediated IL-1β release, which in turn was preferentially skewed toward Th17 differentiation via enhanced phosphorylation of STAT3. Pharmacological inhibition of IL-1β/IL-6-STAT3 signaling significantly delayed corneal allograft rejection. Thus, the identification of NLRP3 inflammasome's key role sheds new light on the pathogenesis of corneal allograft rejection and opens a potential new avenue for treating or preventing corneal allograft rejection.

Hemin Promotes Corneal Allograft Survival Through the Suppression of Macrophage Recruitment and Activation.

To explore the roles of hemin in preventing corneal allograft rejection (CGR) and the underlying mechanisms. Hemin (30 mg/kg) was intraperitoneally injected into rats with a corneal allograft on alternate days, from the day of transplantation until euthanasia. The clinical signs of the corneal allografts were evaluated and recorded according to a previously published system. Corneal edema, macrophage infiltration, and phenotype, and the expression of chemokines, cytokines, and heme oxygenase (HO)-1 were detected by histology, real-time PCR, and Western blot. The rat macrophage cell line NR8383 was used to explore the mechanisms of action of hemin in vitro. Treatment with hemin significantly prolonged corneal allograft survival, with decreased corneal edema and fewer macrophages. Moreover, hemin treatment alleviated inflammation in the corneal grafts, as characterized by downregulated mRNA levels of proinflammatory mediators. In addition, hemin administration reduced the proportion of proinflammatory M1 macrophages and increased the proportion of anti-inflammatory M2 macrophages in the corneal grafts. Hemin treatment induced HO-1 expression in vivo and in vitro, whereas co-administration of zinc protoporphyrin IX (ZnPP), an HO-1 inhibitor, blocked the beneficial effects of hemin in preventing CGR. Our results are the first to demonstrate that hemin, a Food and Drug Administration-approved drug, promotes corneal allograft survival. These findings indicate that hemin might be a potential alternative treatment for CGR.

Cyclosporine a drug-delivery system for high-risk penetrating keratoplasty: Stabilizing the intraocular immune microenvironment.

Cyclosporine A (CsA) is an essential medication used to prevent corneal allograft rejection. Our preliminary studies revealed that CsA drug-delivery system (DDS) was more effective in preventing high-risk corneal allograft rejection than topical CsA application. However, the impacts of CsA DDS on the intraocular immune microenvironment were not fully elucidated. In the present study, we investigated the effect of CsA DDS on the cornea allograft, aqueous humor, and iris-ciliary body using a rabbit model of high-risk penetrating keratoplasty. New Zealand white rabbits were divided into four groups: a normal control group, an untreated group, a CsA eye drop group and a CsA DDS group. Graft survival was monitored for 12 weeks, and the therapeutic effects of CsA DDS were evaluated at 3 and 12 weeks after high-risk keratoplasty. In the CsA DDS group, the mean graft survival time was significantly prolonged when compared with the untreated and CsA eye drop groups. At all time-points, Langerhans cell density, inflammatory cell density, and central corneal thickness in the CsA DDS group were much lower(all p < 0.01) than the untreated and CsA eye drop groups, in which their parameters were significantly higher than the normal control group (all p < 0.01). Compared with the untreated and CsA eye drop groups, an implanted CsA DDS markedly decreased the CD11b+ and CD8+ T cell infiltration in the corneal grafts. CsA DDS treatment also greatly reduced the CD4+ T cell density and the expression of interferon-gamma, interleukin-2 (IL-2), IL-6, CD80, and CD86 mRNA both in the corneal graft and iris-ciliary body (all p < 0.01). Moreover, CsA DDS significantly reduced the IL-2 level in aqueous humor (p < 0.01). Taken together, our results suggest that CsA DDS implanted into the anterior chamber create a relative immunosuppressive microenvironment in the corneal graft, iris-ciliary body, and aqueous humor. Stabilizing the intraocular immune microenvironment could partially elucidate the mechanism of CsA DDS in suppressing corneal graft rejection.

Inhibition of RelA expression via RNA interference induces immune tolerance in a rat keratoplasty model.

RelA, the most important regulator of NF-kB activity, and its mechanisms in keratoplasty immune rejection have not been fully investigated. In the present study, lentivirus-mediated silencing of RelA expression in a bone marrow-derived dendritic cell (BMDC) model was tested. The BMDCs were transfected with RelA-shRNA to induce an immature, maturation-resistant and tolerogenic phenotype, while not significantly changing IFN-γ, IL-10 and IL-17 expression. A fully allogeneic rat cornea transplant model was established for in vivo studies. The allograft mean survival time (MST) of lv-shRelA-DC injection groups were significantly longer than the untreated BMDC group and control group. The corneal opacity and neovascularization scale of the lv-shRelA-DC injection groups were slight compared to pair control others. Postoperative flow cytometric analysis revealed that the percentage of Treg positive cells was dramatically increased in animals that received an lv-shRelA-DC injection. ELISA and qRT-PCR analyses of serum showed that IFN-γ and IL-17 expression were suppressed by lv-shRelA-DC treatement. In vivo experiments demonstrated that IL-10 induced immunosuppression was partly attributed to injection of lv-shRelA-DC throughout the experiment, differing from the general anti-inflammatory factors. Luciferase and Chromatin IP evaluation showed that RelA knockdown in BMDCs significantly reduces DNA binding to IFN-γ, IL-10 and the IL-17 promoter and inhibited of transcriptional activity. Taken together, this study illustrates a significant role of RelA in mediating the corneal neovascularization by affecting IL-17 expression. Our comprehensive analysis shows that the significant role of RelA provides a novel and feasible therapeutic approach for the prevention of corneal allograft rejection.

Hyaluronic acid-coated niosomes facilitate tacrolimus ocular delivery: Mucoadhesion, precorneal retention, aqueous humor pharmacokinetics, and transcorneal permeability

Tacrolimus (FK506) was used to prevent corneal allograft rejection in patients who were resistant to steroids and cyclosporine. However, the formulation for FK506 ocular delivery remained a challenge due to the drug’s high hydrophobicity, high molecular weight, and eye’s physiological and anatomical constraints. The aim of this project is to develop an ocular delivery system for FK506 based on a combined strategy of niosomes and mucoadhesive hyaluronic acid (HA), i.e., FK506HA-coated niosomes, which exploits virtues of both niosomes and HA to synergistically improve ophthalmic bioavailability. The FK506HA-coated niosomes were characterized with particle size, zeta potential, and rheology behavior. Mucoadhesion of FK506HA-coated niosomes to mucin was investigated through surface plasmon resonance in comparison with non-coated niosomes and HA solution. The results showed that niosomes possessed adhesion to mucin, and HA coating enhanced the adhesion. The in vivo precorneal retention was evaluated in rabbit, and the results showed that HA-coated niosomes prolonged the residence of FK506 significantly in comparison with non-coated niosomes or suspension. Aqueous humor pharmacokinetics test showed that area under curve of HA-coated niosomes was 2.3-fold and 1.2-fold as that of suspension and non-coated niosomes, respectively. Moreover, the synergetic corneal permeability enhancement of the hybrid delivery system on FK506 was visualized and confirmed by confocal laser scanning microscope. Overall, the results indicated that the hybrid system facilitated FK506 ocular delivery on mucoadhesion, precorneal retention, aqueous humor pharmacokinetics and transcorneal permeability. Therefore, HA-coated niosomes may be a promising approach for ocular targeting delivery of FK506.

Donor Bone Marrow–derived Dendritic Cells Prolong Corneal Allograft Survival and Promote an Intragraft Immunoregulatory Milieu

Investigations into cell therapies for application in organ transplantation have grown. Here, we describe the ex vivo generation of donor bone marrow-derived dendritic cells (BMDCs) and glucocorticoid-treated BMDCs with potent immunomodulatory properties for application in allogeneic transplantation. BMDCs were treated with dexamethasone (Dexa) to induce an immature, maturation-resistant phenotype. BMDC and Dexa BMDC phenotype, antigen presenting cell function, and immunomodulatory properties were fully characterized. Both populations display significant immunomodulatory properties, including, but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. BMDCs and Dexa BMDCs display a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model, injection of donor-derived, untreated BMDC or Dexa BMDCs (1 × 10(6) cells, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. Although neovascularization was not reduced and evidence of donor-specific alloantibody response was detected, a significant reduction in allograft cellular infiltration combined with a significant increase in the ratio of intragraft FoxP3-expressing regulatory cells was observed. Our comprehensive analysis demonstrates the novel cellular therapeutic approach and significant effect of donor-derived, untreated BMDCs and Dexa BMDCs in preventing corneal allograft rejection.

Graft rejection in keratoplasty ‐ what are the benefits of systemic immune modulation?

AbstractPurpose Graft rejection is one of the major causes of corneal transplant failure after keratoplasty, especially in case of an ocular or systemic inflammatory disease. To reduce the patient’s immune reaction against the corneal allograft, topical or systemic immunosuppression is a clinical necessity. This talk provides an overview of the benefits of systemic immunosuppression and of its indications.Methods The advantages and disadvantages of the use of systemic immunomodulatory drugs in the prevention of corneal allograft rejection after keratoplasty will be analyzed and demonstrated.Results Various systemic immunomodulatory drugs are available to prevent or to reduce the risk of corneal allograft rejection. The use of systemic immunosuppressive drugs may be limited by side effects.Conclusion Systemic immunosuppression prolongs corneal allograft survival and reduces the risk of graft rejections. While the use of systemic immunosuppressive drugs is beneficial in the perioperative stage, the long‐term‐use is restricted to high‐risk settings.

Donor bone marrow derived dendritic cells promote corneal allograft survival in the rat

AbstractPurpose Glucocorticoid treatment of ex‐vivo generated donor bone marrow derived dendritic cells (BMDCs) will promote graft survival upon injection into corneal transplant recipients.Methods BMDCs were propagated from Dark Agouti (DA) rat BM precursor cells and for glucocorticoid treated BMDCs, dexamethasone (Dexa) 10e‐6M was added to the culture. BMDCs +/‐ Dexa phenotype, APC function and immunostimulatory capacity were examined. A fully allogeneic rat corneal transplantation model (DA to LEW) was used for in‐vivo studies. BMDCs +/‐ Dexa were harvested and 1x10e6 cells injected intravenously into recipients 7 days prior to corneal transplantation. Graft survival and development of opacity, edema and neovascularisation were monitored. On the average day of rejection graft infiltrating cell populations were analysed.Results Ex‐vivo generated BMDCs have a semi‐mature phenotype and can be treated with Dexa to maintain their immature phenotype (n=5 p&lt;0.05). BMDCs are capable of activating allogeneic lymphocytes, efficiently presenting antigen and activating antigen specific T cells however, there is a reduction in the level of proliferation (n=4, p&lt;0.05). When applied in‐vivo BMDCs and Dexa BMDCs significantly prolong corneal allograft survival (MST&gt; 30d, n=14 p&lt;0.0004 and n=24 p&lt;0.0001 resp.) compared to untreated allogeneic controls (MST 18d, n=11). A significant reduction in the total number of graft infiltrating cells was observed for both treated groups (p&lt;0.05).Conclusion Our results demonstrate a significant therapeutic effect of donor‐derived BMDCs with and without glucocorticoid treatment to prolong corneal transplant survival which represents a novel therapeutic approach for the prevention of corneal allograft rejection.

The observation of the effects of a micromolecular compound J2 on rat corneal allograft using atomic force microscopy

To study the cell membrane of corneal endothelium with a micromolecular compound J2 in corneal allograft of rat using atomic force microscopy (AFM). Cohort study. Subjects were divided into two groups: group A (n = 15): experimental group; group B (n = 15): placebo control group. At the fifth, tenth, fifteen, twentieth, twenty-fifth day after penetrating keratoplasty, the donor implant was separated from receipt bed, one part of which was stained by HE and the others fixed into AFM sample. Amplitude and height images were obtained in the tapping mode with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis was performed using single-factor analysis of variance and P value was calculated. The average transplant survival time in group A was (33.12 ± 6.80) d, and those in group B was (18.87 ± 4.19) d. There were significant difference between two group (F = 47.7449, P = 0.00). There were obvious differences on ultrastructure measured by AFM between two groups. At the fifth day after penetrating keratoplasty, regular hexagonal structure of corneal endothelium was observed by AFM in both two group. The diameter of corneal endothelium was about 15 µm, uneven microstructure of cell could be found. The time being, different changes were arose in two group: a clear microstructure could be found in group A, however the microstructure of cell could not be recognized in group B. One way analysis of variance showed that significant differences on parameters (Ra, Rp and Rv) were found between two groups (P < 0.05). At the fifth day after penetrating keratoplasty, group A: Ra (97.64 ± 31.58) nm, Rp (297.79 ± 25.19) nm, Rv (545.55 ± 25.83) nm; group B: Ra (112.61 ± 34.29) nm, Rp (265.06 ± 24.17) nm, Rv (544.41 ± 21.78) nm (Fa = 30.9416, P = 0.0000; Fp = 263.6018, P = 0.0000; Pv = 1.2013, P = 0.2735). At the tenth day after penetrating keratoplasty, group A: Ra (102.98 ± 32.98) nm, Rp (711.38 ± 21.94) nm, Rv (639.89 ± 22.58) nm; group B: Ra (222.85 ± 31.28) nm, Rp (111.22 ± 20.35) nm, Rv (746.49 ± 23.17) nm (Fa = 2086.4535, P = 0.0000; Fp = 53768.4676, P = 0.0000; Pv = 3257.3178, P = 0.0000). At the fifteenth day after penetrating keratoplasty, group A: Ra (87.44 ± 34.97) nm, Rp (344.18 ± 21.09) nm, Rv (482.61 ± 22.27) nm; group B: Ra (197.64 ± 35.72) nm, Rp (510.76 ± 24.98) nm, Rv (545.62 ± 23.17) nm (Fa = 1458.1057, P = 0.0000; Fp = 7788.6963, P = 0.0000; Pv = 1153.2860, P = 0.0000). At the twentieth day after penetrating keratoplasty, group A: Ra (85.85 ± 32.53) nm, Rp (348.69 ± 21.26) nm, Rv (367.65 ± 23.12) nm; group B: Ra (201.36 ± 34.12) nm, Rp (788.58 ± 20.34) nm, Rv (563.33 ± 21.01) nm (Fa = 1801.1215, P = 0.0000; Fp = 67 057.9516, P = 0.0000; Fv = 11 770.2195, P = 0.0000). At the twenty-fifth day after penetrating keratoplasty, group A: Ra (104.97 ± 32.47) nm, Rp (395.05 ± 20.38) nm, Rv (396.17 ± 21.59) nm; group B: Ra (43.85 ± 31.28) nm, Rp (249.88 ± 20.79) nm, Rv (154.88 ± 22.37) nm (Fa = 551.4134, P = 0.0000; Fp = 7458.9255, P = 0.0000; Pv = 18 070.5189, P = 0.0000). The morphology and ultrastructure of corneal endothelium in group A with J2 were different from group B by observation with AFM. J2 was an effect micromolecular in prevention of corneal allograft rejection.

Immune regulatory effect of rapamycin and cyclosporine A on experimental corneal allograft survival

Abstract Purpose To analyze the efficiency of low dose systemic treatment with rapamycin (Rapa) alone or in combination with cyclosporin A (CsA) in preventing corneal allograft rejection in a high responder strain combination. Methods A total of 80 C57BL/6 mice received corneal grafts of BALB/c donors. Recipients were treated with CsA 3mg/kg/day or Rapa 0.5mg/kg/day monotherapy or received combined treatment. Intraperitoneal injections were started on the day of surgery and continued for 14 days. In addition, corneal samples were subjected to real time RT‐PCR analysis for cytokine transcription. The frequency of CD4+CD25+Foxp3+ T regulatory cells (Treg) in secondary lymphoid organs were measured by flow cytometry. Memory T cells were estimated by Elispot. Results Monotherapy with Rapa significantly delayed allograft rejection (p=0.03). However, the combination of low dose Rapa and CsA prolonged corneal allografts survival at a significantly higher level (MST=17.1 ± 1.37 days, p=0.000004). Addition of CsA to Rapa resulted in down‐regulation of intragraft CD3, IL‐2, IFN‐γ and IL‐10 transcription (p=0.028, p= 0.027, p=0.028 and p= 0.027 respectively). Rapa alone increased the frequency of CD4+CD25+Foxp3+ Tregs in draining lymph nodes, whereas addition of CsA reduced Tregs. Rapa monotherapy as well as combined treatment prevented development of alloantigen specific IFN‐γ producing memory T cells in spleen. Conclusion Combined treatment with low dose CsA and Rapa resulted in superior graft survival and effectively modulated mRNA expression of inflammation and infiltration markers. Supported by DFG (150/14‐2) and SFB650 TP14

Prevention of mouse corneal allograft rejection by micromolecular compound J2

To investigate the effects of J2 on the prevention of corneal allograft rejection in mice. Randomized control design was applied. One hundred C57BL/6 and thirty eight BALB/c mice were used as donors and recipients, respectively, to establish a corneal allograft model. These mice were randomly divided into A, B, C and D groups. There were 25 mice in each group. Group A, autograft control; Group B, allograft control (placebo was used in this group); Group C, allograft and treated with intraperitoneal ciclosporin A (CsA, 10 mg * kg(-1) * d(-1)) and Group D, allograft and treated with intraperitoneal J2 (15 mg * kg(-1) * d(-1)). All medications were applied on the day of transplantation and were continuously administrated for 12 days. Graft survival index, paraffin sections with HE staining and immunohistochemical staining of frozen sections were observed in each group 1, 2, 3 and 4 weeks after operation. IL-2 and IL-10 expression of cornea was detected by RT-PCR. One-Way ANOVO analysis was used to compare transplant survival time of the corneal allograft of each group. The average transplant survival time in the corneal allograft controls was (18.88 +/- 4.19) d. Treatment with J2 resulted in a significant increase of grafts' survival time (33.62 +/- 6.80) d (q = 0.85, P < 0.01). There was no significant difference between J2 and CsA groups. Histological examination showed that numerous lymphocytes infiltrated into the grafts 21 days after the operation in the control groups. Mild lymphocytes infiltration was found in both CsA and J2 groups. Immunohistochemical studies showed that large amount of CD4(+) and CD8(+) T-lymphocytes infiltrated into the grafts of the placebo group at 21 days. J2 and CsA groups only showed a small number of CD4(+) and CD8(+) T-lymphocytes in the grafts. RT-PCR showed that few IL-2 and IL-10 genes were expressed in the autologous transplantation group in the first week, and the expression of these two cytokines showed no significant increase during following three weeks. Both genes were expressed from the first week in the allogeneic transplantation group and significantly increased in the third week. Both genes were expressed from the first week in CsA and J2 group, expression was reduced in the second and third weeks. Expression of these cytokines in the fourth week was stronger than those in previous three weeks. J2 can inhibit corneal rejection in allograft mice model and its effect is similar to that of CsA.

Immune modulation in corneal transplantation

Allograft rejection is the most common reason for corneal transplant failure, despite the immunologic privilege of both the graft and the anterior chamber. To prevent corneal allograft rejection, various immunomodulatory strategies have been used in experimental corneal transplantation. These include (1) anti-T-cell receptor and T-cell depletion therapy; (2) manipulation of costimulatory molecule function, including both down-regulation of positive stimulatory molecules and/or up-regulation of inhibitory molecules and overproduction of tumor necrosis factor-related, apoptosis-induced ligand; (3) modulation of cytokine production by reducing proinflammatory cytokines (tumor necrosis factor alpha, interleukin [IL]-12, and IL-1) and/or increasing immunoregulatory cytokines (IL-10 and IL-4); (4) macrophage depletion; and (5) overexpression of the immunomodulatory molecule indoleamine 2,3-dioxygenase. Although these approaches appear promising in animal corneal transplantation models, there has been very little translation of these immunomodulatory approaches in human corneal transplantation.

Pharmacologic strategies in the prevention and treatment of corneal transplant rejection

Corneal transplantation remains one of the most successful organ transplantation procedures in humans. The unique structure of the cornea, with its absence of blood vessels and corneal lymphatic, allows the survival of corneal allograft. Recent advances in sutures, storage media, microsurgical instrumentation, and new pharmacological strategies have greatly improved the success of corneal transplantation and the prevention of corneal allograft rejection. Our strategies in the management and prevention of corneal graft rejection can modify and improve the survival of corneal allografts. Preoperative evaluation, understanding the risk factors, and management of ocular surface disorders may greatly improve the survival of the corneal transplant. Early recognition of corneal allograft rejection and aggressive treatment may improve the survival of the corneal graft. Furthermore, patients who undergo corneal transplantation should be maintained under close ophthalmic surveillance and patients should be informed to report immediately whenever symptoms of corneal graft rejection occur. The mainstay of therapy is topical corticosteroids. In severe cases, periocular, intravenous, and oral corticosteroids therapy can be rendered. New therapeutic modalities such as cyclosporine, tacrolimus, daclizumab, mycophenolate mofetil, leflunomide, rapamycin, and others may prove to be of help in the prevention and treatment of corneal graft rejection. Early recognition of corneal graft rejection and prompt treatment are mandatory for the successful survival of the corneal allograft.