Corneal Allograft Rejection Research Articles

Functional Tumor Necrosis Factor Alpha Polymorphisms and Haplotype Analysis in High-Risk Corneal Transplantation

BackgroundTumor necrosis factor alpha (TNF-α) plays a critical role in diverse cellular processes including ocular immune tolerance, inflammation, and allograft rejection. The ubiquitous transcription factor nuclear factor kappa B (NF-κB) regulates expression of numerous genes. Induction of the TNF-α pathway is involved in the inflammatory response and loss of transplant tolerance. ObjectivesWe investigated functional single nucleotide polymorphisms (SNPs) in the promoter region of TNF-α and an insertion/deletion (indel) polymorphism of NF-κB1 in corneal transplant recipients considered to be at increased risk of immunological rejection (ie, high-risk corneal transplantations) and looked for any associations with corneal transplantation outcome. Patients and MethodsThree hundred eighty-four full thickness corneal transplant recipients were followed for 3 years and episodes of reversible and irreversible allograft rejection were recorded. Using DNA obtained from these patients, 5 SNPs located in the promoter region −1031 T/C rs1799964, −863 C/A (rs1800630), −857 C/T (rs1799724), −308 G/A (rs1800629), and −238 G/A (rs361525), and one SNP upstream from the transcription start site (+489) rs1800610 of TNF-α were analyzed using induced heteroduplex generation. A functional NF-κB1 indel (−94) was also investigated. Haplotypes were inferred by PHASE and associations with rejection were determined by chi-square analysis. ResultsThe TNF-α haplotype TCTGGA was significantly associated with reduced risk of corneal graft rejection (Pc < .005) and TCTAGA was associated with increased risk of rejection (Pc < .005) in high-risk corneal transplants. There was no association with the NF-κB1 indel (Pc > .05). ConclusionAccording to haplotype frequencies, our results suggest that the TCTGGA haplotype may confer additional protection against risk of immunological rejection whereas TCTAGA may increase risk of corneal allograft rejection in the high-risk setting. However, both haplotypes were relatively rare and thus would not warrant genotyping for individual patient selection for anti-TNF therapy.

Haplotype Analysis on Chromosome 6p of Tumor Necrosis Factor Alpha, Vascular Endothelial Growth Factor A, and Interleukin-17F Alleles Associated With Corneal Transplant Rejection

ObjectiveThe aim of this work was to investigate single-nucleotide polymorphisms (SNPs) in multiple genes on chromosome 6p in corneal transplant recipients known to be at increased risk of failure through immunologic rejection (ie, “high-risk” corneal transplants). Tumor necrosis factor alpha (TNF-α) is a key immunoregulatory cytokine in the ocular environment, interacting with a variety of factors in a synergistic way and playing a crucial role in many stages of the inflammatory response. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors, supporting both hemangiogenesis and lymphangiogenesis, both key in transplant tolerance and rejection. Interleukin-17 (IL-17) is a multifunctional cytokine produced by T-helper 17 cells, exerting specific effector functions during an immune response. Association of SNPs in all 3 genes with corneal transplant outcome was therefore investigated. MethodsThree hundred five corneal transplant recipients were followed for 3 years, and episodes of allograft rejection were recorded. With the use of patient DNA, 6 SNPs of 3 different genes on chromosome 6p were investigated. The TNF-α promoter SNP −308 G/A (rs1800629) was analyzed with the use of induced heteroduplex generation; 2 VEGF-A functional variants were analyzed, −2578 (rs699947) C/A and −1154 (rs1570360) G/A, with the use of Taqman genotyping assays; and 3 nonsynonymous IL-17F SNPs in exon 3 (negative strand), (rs2397084) A/G, (rs11465553) G/A, and (rs763780) A/G, were investigated with the use of direct sequencing. Haplotypes were inferred with the use of PHASE using positive strand alleles, and exact measures of association were determined with the use of Mid-P exact chi-square. ResultsSix common haplotypes were inferred, with the haplotype TNF-α (rs1800629), VEGF-A (rs699947), (rs1570360), IL-17F (rs763780), (rs11465553), and (rs2397084) ACGTCT having a significant association with corneal transplant rejection (odds ratio, 1.78; 95% confidence interval, 1.01–3.11; P = .04). ConclusionsThe results suggest that patients carrying a combination of SNPs for TNF-α, VEGF-A, and IL-17F of ACGTCT haplotype may have an increased risk of corneal allograft rejection compared with patients carrying other haplotypes.

Asymptomatic ocular herpes infections precipitate rapid rejection of corneal allografts (TRAN3P.917)

Abstract Most herpes simplex virus type 1 (HSV-1) infected individuals are asymptomatic despite harboring latent virus in their trigeminal ganglia (TG). To determine if these ‘quiet’ infections influence corneal transplant survival we infected the corneas of C57Bl/6 mice with an HSV-1 strain that causes a latent TG infection but no overt corneal pathology. Infected mice received a corneal transplant from syngeneic mice that express ovalbumin (OVA) as a surrogate alloantigen. Graft survival was 9% in infected mice as compared to 58% in non-infected mice. Transcripts for chemokines including CXCL10, CXCL9, CCL5, and CCL2 were significantly elevated, and were significantly reduced by CD4 T cell depletion prior to infection. To assess CD4 T cell antigen specificity irradiated wild type C57BL/6 mice were reconstituted with mixed BM from CD4 KO mice and RAG-/- OT-II transgenic mice so all CD4 T cells were OVA-specific. The chimeras were not infected or received a quiet HSV-1 infection with or without transfer of polyclonal CD4 T cells prior to transplantation of OVA expressing grafts. Graft survival in chimeras was: 100% (non-infected), 60% (quiet infection, no T cell transfer), and 20% (quiet infection with CD4 T cell transfer). We conclude that quiet HSV-1 corneal infections can augment CD4 T cell mediated corneal allograft rejection by increasing chemokine expression in the graft bed and possibly recruiting HSV-specific CD4 T cells into the rejection response.

Thrombospondin-1 Polymorphisms Influence Risk of Corneal Allograft Rejection

We investigated single nucleotide polymorphisms (SNPs) of thrombospondin-1 (TSP-1) on the risk of corneal allograft rejection. The TSP-1 is known to be involved in the immune response of the anterior chamber of the eye, activating TGF-β2, promoting peripheral and systemic tolerance, and counteracting the proangiogenic activity of VEGF. Three tagging SNPs spanning the TSP-1 region (rs1478604, A>G; rs2228261, C>T; and rs2228262, A>G) were genotyped. Association with risk of rejection was investigated in a group of 378 corneal transplant recipients with risk factors for allograft rejection. Transplant recipients had completed 3-year follow-up. The TSP-1 rs1478604 A SNP was associated significantly with an increased risk of corneal allograft rejection (odds ratio [OR], 1.58; 95% confidence interval [CI], 1.02-2.45; P = 0.04) and there was a trend toward the rs1478604, rs2228261, rs2228262 ACA haplotype increasing risk of rejection. The results suggest that TSP-1 rs1478604 AA homozygotes may be at increased risk of corneal transplant rejection, especially if they carry the ACA haplotype.

Rapamycin-loaded poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) nanoparticles: preparation, characterization and potential application in corneal transplantation

Allograft rejection is the major cause of corneal graft failure. To inhibit corneal allograft rejection, rapamycin as a novel immunosuppressive agent has been discovered. However, the high water insolubility and low bioavailability of rapamycin has strongly hindered its application in the clinical setting. In this paper, we attempted to develop a novel rapamycin nano-formulation using poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) nanoparticles as carrier by an emulsion evaporation method for potential application in corneal transplantation. The solubility of rapamycin in the nano-formulation was determined and in-vitro release studies were performed. The developed rapamycin-loaded PCEC nanoparticles were further characterized by dynamic light scattering, transmission electron microscopy, X-ray diffraction and differential scanning calorimetery. Toxicity studies were performed in eye-related cell lines. The rapamycin in nano-formulation exhibited ∼10³-fold increased solubility as compared with native rapamycin. According to the results of the in-vitro cytotoxicity assay, the developed PCEC nanoparticles did not exhibit any apparent cytotoxicity against various eye-related cell lines with PCEC nanoparticle concentrations in the range of 0.05-10 mg/ml. In-vitro release study showed that the release of rapamycin was sustained from PCEC nanoparticles over a period of 48 h. All the results suggested that the developed rapamycin-loaded PCEC nanoparticles might be suitable for immunosuppression in corneal transplantation by instillation administration.

Exogenous IL-10 induces corneal transplantation immune tolerance by a mechanism associated with the altered Th1/Th2 cytokine ratio and the increased expression of TGF-β

The aim of the present study was to examine the effect of exogenous IL-10 transfected rat dendritic cells (DCs) in corneal allografts. Rat lymphocyte separation medium and a cytokine induction method was used to extract and culture precursor cells of rat bone marrow-derived dendritic cells. A corneal transplant model was established, with Sprague-Dawley (SD) rats as the recipients and Wistar rats as the donors. Flow cytometry (FCM) was used to detect the expression of CD83, which indicates a mature dendritic cell, and a specific cell surface stimulatory molecule CD86. Western blot analysis was used to detect interleukin (IL)-10 protein expression and RT-PCR was used to detect cytokine IL-10, IL-2 and TGF-β mRNA expression in each group. Compared with the other groups, the survival time of corneal grafts in the IL-10-green fluorescent protein (GFP)-DC group was significantly prolonged and H&E staining demonstrated mild graft edema and inflammatory cell infiltration. There was a high expression of IL-10 and a low expression of the surface antigens, CD83 and CD86, with a lower proliferation of T lymphocytes in the IL-10-GFP-DC group. The expression of IL-10 and TGF-β in the IL-10-GFP-DC group was higher than that in the other groups, while the expression of IL-2 was lower. The transfection of the IL-10 gene inhibited dendritic cell maturation. IL-10 gene-modified immature dendritic cells (imDC) were able to inhibit corneal allograft rejection and induce immune tolerance to prolong the survival time of the corneal graft. The Th1/Th2 deviation and the high expression of TGF-β may lead to immune tolerance.

In Vivo Downregulation of Innate and Adaptive Immune Responses in Corneal Allograft Rejection by HC-HA/PTX3 Complex Purified From Amniotic Membrane

Heavy chain-hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection. The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4(+) T cells, or IFN-γ/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence-labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification. In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-γ, IL-2, CD25, and CD69 in activated CD4(+) T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12. HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4(+) T cells.

Tolerogenic dendritic cells suppress murine corneal allograft rejection by modulating CD28/CTLA‐4 expression on regulatory T cells

Anterior chamber-associated immune deviation (ACAID) is initiated when dendritic cells (DCs) capture intraocular antigen and induce regulatory T cells (Tregs) in the spleen. We investigated whether DCs could be used as an immunotherapy to prevent murine corneal allograft rejection by inducing Tregs. Bone marrow-derived DCs (BMDCs) were differentiated with transforming growth factor-β2 (TGF-β2) in the presence of donor-derived allopeptide in vitro (TGFβ2-DCs), and adoptively transferred to BALB/c mice. Three days later a corneal allograft transplant was carried out. Graft rejection, as well as the number and the phenotype of splenic Tregs, were determined after transplant. CD86, CD80, CD40 were present, and MHC class II expression were significantly reduced on TGFβ2-DCs compared to BMDCs not exposed to TGF-β2. TGFβ2-DCs increased the number of splenic Tregs (CD4(+)CD25(+)) in recipient mice prior to transplant by modulating CD28/CTLA-4 expression. These induced Tregs suppressed proliferation of CD4(+)CD25(-) T cells ex vivo. TGFβ2-DCs delayed corneal allograft rejection and TGFβ2-DC recipient mice had significantly more splenic Tregs expressing Foxp3 and CTLA-4, but fewer CD28+ Tregs. Expression of GITR, CD69, and CD45RB were similar in TGFβ2-DC and control mice. Therefore, the phenotype of TGFβ2-DCs suggests they are tolerogenic DCs (tolDCs). In vivo, these cells increased the number and function of Tregs by modulating CD28/CTLA-4 expression. The suppressor Tregs induced by TGFβ2-DCs may be involved in the induction of ACAID, which helps to suppress corneal allograft rejection. Our results provide proof of principle for the use of tolDCs as immunotherapy during transplant.

IFN-γ Blocks CD4+CD25+ Tregs and Abolishes Immune Privilege of Minor Histocompatibility Mismatched Corneal Allografts

Th1 CD4+ cells are believed to be the primary mediators of corneal allograft rejection. However, rejection of fully allogeneic C57BL/6 corneal allografts soared from 50% to 90% in both interferon-gamma (IFN-γ)(-/-) and anti-IFN-γ-treated BALB/c mice. In contrast, similar deficits in IFN-γ in BALB/c hosts enhanced immune privilege of BALB.B (minor histocompatibility [minor H] antigen-matched, major histocompatibility complex [MHC]-mismatched) and NZB (MHC-matched, minor H antigen-mismatched) corneal allografts-decreasing rejection from 80% to ~20%. This effect of IFN-γ was independent of CD4+ T cell lineage commitment as both anti-IFN-γ-treated acceptor and rejector mice displayed a Th2 cytokine profile. The presence of IFN-γ prevented the generation of alloantigen-specific CD4+CD25+ T regulatory cells (Tregs) in hosts receiving either MHC only mismatched BALB.B or minor only histocompatibility (minor H)-mismatched NZB corneal allografts. Tregs in these hosts promoted corneal allograft survival by suppressing Th2 effector cells. By contrast, IFN-γ was necessary for the generation of CD4+CD25+ Tregs that prevented rejection of fully allogeneic C57BL/6 corneal allografts in BALB/c hosts. These findings suggest that MHC-matching in combination with blockade of IFN-γ holds promise as a means of enhancing corneal allograft survival.

Analysis of Macrophage Phenotype in Rejected Corneal Allografts

We investigated the phenotype of macrophages infiltrating rejected corneal allografts. We performed allogeneic or syngeneic corneal transplantation in mice, and humanely killed animals at day 28 during allograft rejection when 60% of corneal allografts were rejected. We divided allografts into two groups: grafts with rejection as rejectors and grafts without rejection as nonrejectors, and analyzed for macrophage infiltration and their phenotype using immunohistochemistry. In addition, we investigated the time course of proinflammatory cytokines and chemokines by analyzing corneal grafts at days 7, 28, and 42 using real-time RT-PCR. Also, we assayed human corneal allografts with chronic graft failure. We found that a large number of CD11b(+), F4/80(+), or inducible nitrous oxide synthase cells (iNOS(+)) infiltrated corneal allografts during rejection in mice, while the cells were found rarely in syngeneic or allogeneic grafts that were not rejected. There were rare CD11c(+) cells in rejectors and nonrejectors. Many mannose receptor cells (MRC(+)) were present in nonrejectors, but not in rejectors. The levels of Th1 cytokines, IFN-γ, and IL-2 were highly increased in rejectors at day 28, indicating immune rejection. Also, the levels of IL-12a, IL-1β, TNF-α, CCL3, and iNOS that are produced by activated macrophages were markedly increased in rejectors at day 28, compared to syngeneic grafts or nonrejectors. Similarly, human corneal allografts with chronic graft failure had higher levels of IL-12a, IL-1β, CCL3, and iNOS than controls. Increased numbers of macrophages in rejected corneal allografts implicate that these cells might contribute to the immunopathogenesis of corneal graft rejection.

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.

Systemic application of sphingosine 1‐phosphate receptor 1 immunomodulator inhibits corneal allograft rejection in mice

This study aims to investigate the effects of systemic application of sphingosine 1-phosphate receptor 1(S1P1) on allogeneic corneal transplantation in mice. A total of 112 BALB/c mice received corneal grafts from C57BL/6 donors. Recipients were randomly divided into seven groups and treated with intraperitoneal injections of S1P1 (5 mg/kg/days), cyclosporine A (5 mg/kg/days), dexamethasone (1 mg/kg/days) and rapamycin (2 mg/kg/days). S1P1was combined with rapamycin or cyclosporine A, and saline served as negative control. Serum levels of IL-2, IL-10, TGF-β1 and IFN-γ were measured by Elisa. The numbers of CD4+ T and regulatory (Treg) cell phenotype were measured by flow cytometry. Cytokine mRNA expression was analysed by real-time quantitative PCR. CD4+ T cells and cytokines were histologically identified by immunofluorescence staining. Corneal graft survival was prolonged by intraperitoneal injections in S1P1 alone (mean survival time MST, 35.3 ± 5.6 days), S1P1 combined with rapamycin (MST, 38.7 ± 6.5 days) or S1P1 and cyclosporine A (MST, 32.7 ± 4.8 days) compared with the controls (MST, 14.6 ± 0.2 days; n = 5, p < 0.01). S1P1 alone increased CD4+ T (p < 0.01) and Treg cells (p < 0.01; n = 5) in the cervical and mesenteric lymph nodes compared with the controls and S1P1 + rapamycin (p < 0.05; n = 5). TGF-β1 and IL-10 mRNA transcriptions in corneal grafts following S1P1+ rapamycin increased (both p < 0.01; n = 3), and TGF-β1 and IL-10 in the serum level following S1P1 alone increased (both p < 0.01; n = 3). These results paralleled the findings obtained from immunofluorescence. S1P1 has significant effect in corneal allograft rejection inhibition. The combined treatment of S1P1 and rapamycin results in synergistic effect.

Clinical results of 40 years of paediatric keratoplasty in a single university eye clinic

To evaluate indications for paediatric keratoplasty in recipients aged ≤16 years and assess long-term clinical outcome. Recipients were identified from records of the Danish Cornea Bank. Data were collected from patient journals, clinical follow-up examinations and questionnaires and stratified into pre-, peri- and postoperative variables. Diagnoses were classified into acquired traumatic, acquired nontraumatic and congenital groups. Recipients were divided into groups of <8 and ≥8 years. Data were analyzed using relative percentages and Kaplan-Meier survival plots. Thirty-three out of sixty identified recipients (73 keratoplasties in 63 eyes) were invited. Twenty-four accepted, seven still attended follow-up in our clinic. Follow-up data reached 95% of the eligible recipients (median follow-up 11 years). Twenty-three per cent were <8 years and 77%≤8 years. Diagnoses were mainly acquired nontraumatic (69%), acquired traumatic (12%) and congenital (7%). Indications were primarily optical (52%) or tectonic (41%). Graft survival was best in the acquired nontraumatic group (except regrafts) (median survival 15-20 years) and poorest in the regraft subgroup as well as the acquired traumatic and congenital groups (median survival 1-2 years). Graft failure was higher in the youngest with predisposing risk factors and in combined procedures. In terms of indications, visual improvement and eye preservation was achieved in 70%. Paediatric keratoplasty was successful regarding indication. Graft survival was best in the acquired nontraumatic group and poorest in the congenital group. Vascularization and/or combined risk factors, additional surgeries and young recipient age influenced negatively on graft survival.

Role of T Cell Recruitment and Chemokine-Regulated Intra-Graft T Cell Motility Patterns in Corneal Allograft Rejection

Keratoplasty is the primary treatment to cure blindness due to corneal opacification. However, immune-mediated rejection remains the leading cause of keratoplasty failure. Here, we utilize an in vivo imaging approach to monitor, track, and characterize in real-time the recruitment of GFP-labeled allo-specific activated (Bonzo) T cells during corneal allograft rejection. We show that the recruitment of effector T cells to the site of transplantation determined the fate of corneal allografts, and that local intra-graft production of CCL5 and CXCL9/10 regulated motility patterns of effector T cells in situ, and correlated with allograft rejection. We also show that different motility patterns associate with distinct in vivo phenotypes (round, elongated, and ruffled) of graft-infiltrating effector T cells with varying proportions during progression of rejection. The ruffled phenotype was characteristic of activated effectors T cells and predominated during ongoing rejection, which associated with significantly increased T cell dynamics within the allografts. Importantly, CCR5/CXCR3 blockade decreased the motility, size, and number of infiltrating T cells and significantly prolonged allograft survival. Our findings indicate that chemokines produced locally within corneal allografts play an important role in the in situ activation and dynamic behavior of infiltrating effector T cells, and may guide targeted interventions to promote graft survival.

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T-cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL-4, but not IL-5 or IL-13, prevented Treg suppression of CD4(+) effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4(+) effector T-cell proliferation. In addition, IL-4 did not inhibit Treg suppression of IL-4Rα(-/-) CD4(+) T-cell responses, suggesting that IL-4 rendered effector T cells resistant to Tregs. SRW-sensitized IL-4Rα(-/-) mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL-4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti-IL-4 antibody. Thus, allergy-induced exacerbation of corneal graft rejection is due to the production of IL-4, which renders effector T cells resistant to Treg suppression of alloimmune responses.

Effect of recombinant adeno-associated virus mediated transforming growth factor-beta1 on corneal allograft survival after high-risk penetrating keratoplasty

Corneal transplantation is one of the most common and successful transplant surgeries performed around the world. However, the high-risk corneal transplantation remains a high level of corneal graft failure. Gene transfer of immunomodulatory molecules is considered as one potential strategy in preventing allograft rejection. It is worthy evaluating the effects of the immunemodulating agent on corneal allograft rejection. The purpose of this paper is to investigate the effects and mechanisms of recombinant adeno-associated virus mediated transforming growth factor-beta1 (rAAV-TGF-beta1) on corneal allograft survival using a high-risk rat model after penetrating keratoplasty (PKP). The mean survival time (MST) of corneal grafts was observed and immuno-histochemical staining of TGF-beta1 and Ox-62 was performed in the study. The MST showed significant prolongation in the rAAV-TGF-beta1 group compared to the allograft group. The rejection index (RI) at day 10 revealed was significantly greater in the allograft group than that of the other two groups. Besides the increase of TGF-beta1, the expression of Ox-62 decreasing in rAAV-TGF-beta1 transplanted recipients was detected after transplantation. In short, treatment with rAAV-TGF-beta1 prolongs corneal allograft survival and inhibits the Ox-62 expression in grafts after high-risk PKP.

Corneal Transplantation and Immune Privilege

Corneal transplants have been successfully performed in human subjects for over 100 years and enjoy an immune privilege that is unrivaled in the field of transplantation. Immune privilege is defined as the reduced incidence and tempo in the immune rejection of corneal allografts compared to other categories of organ allografts performed under the same conditions. Skin allografts transplanted across various MHC or minor histocompatibility barriers undergo rejection in approximately 100% of the hosts. By contrast, orthotopic corneal allografts experience long-term survival in 50% to >90% of the hosts, depending on the histocompatibility barriers that confront the host. The capacity of corneal allografts to evade immune rejection is attributable to multiple anatomical, physiological and immunoregulatory conditions that conspire to prevent the induction and expression of alloimmunity.

Expression of cytokine in mouse graft of corneal allograft rejection

Background Cytokines play a crucial role in mediating immune tolerance or immune rejection of corneal transplantation.However,the study on the expression of cytokines in corneal graft is seldom.ObjectiveThe purpose of the study is to investigate the change of cytokine expression in allografts at different time points after corneal transplantation.Methods BALB/c and C57BL/6 mice aged 6 to 8-week old were used to establish autologous and allografts keratoplasty models.BALB/c mouse was use as donor and receipt in autologous group,and the cornea of C57BL/6 mouse was used to graft on the BALB/c mouse in the allografts group.The graft inflammation was clinically scored,and graft inflammatory scores of ≥5 or opaciflcation scores of ≥2 were identified as rejection.BALB/c mice were randomized into normal control group(3 mice)and allografts group(15 mice).Reverse transcriptPCR (RT-PCR) was used to detect the expression of interleukin-4 (IL-4) mRNA,interferon-γ (IFN-γ) mRNA,IL-10mRNA and tumor necrosis factor(TNF-α) mRNA in graft 6 hours and 1 day,3,7,14 days after operation.Results The corneal graft opacification score was ＜2 and inflammatory score was ＜5 in the 10 mice with autologous keratoplasty until 60 days with the survival rate 100％.The edema,opacification and new blood vessel were seen in the BALB/c mice received allografts keratoplasty.The inflammatory score was ≥ 5 and the opacification score was ≥2 24 days after surgery with the rejection rate 100％,in the allografts group,and the graft survival time was (17.80±4.66)days.RT-PCR showed that IL-4 and IFN-γ were positively expressed,and IL-10 and TNF-α were absently expressed in normal mouse cornea.In the allografts keratoplasty mice,positive responses for IL-4,IFN-γ and IL-10 were found in 6hours after operation,but TNF-α was absent.From 1 day through 3 days after operation,the expressions of IL-4,IFN-γand TNF-α were enhanced but IL-10 was disappeared in the graft.IL-10,IFN-γ and TNF-α were expressed till the 7th day,but on the 14th day,only IL-10 was detected in graft in the allografts keratoplasty mice.Conclusions TNF-αis a main factor among the variety of cytokines that may influent corneal allograft rejection locally. Key words: Corneal transplantation; Immunity; Rejection; Tolerance; Cytokine

Inhibitory effect of neutralizing interleukin-17 antibody on corneal allograft rejection

Background Interleukin-17 (IL-17)is a potent pro-inflammatory cytokine and plays a pathogenic role in autoimmune disease.It was confirmed that IL-17 is implicated in allograft rejection of many transplanted organs.Recent studies have foensed on the effect of IL-17 antagonists on allograft rejection.Objective This study aimed to investigate the inhibitory effect of anti-mouse IL-17 monoclonal antibody (mAb) on corneal allograft rejection.Methods Twenty-five 8 to 10-week-old C57BL/6 mice and 50 BALB/c mice were collected.Donor cornea grafts with 2 mm diameter from 25 C57BL/6 mice was transplanted to 50 eye of BALB/c mice to establish a model of corneal transplantation.The recipients were randomized into 2 groups,and neutralizing mouse IL-17antibody or isotype control antibody was intraperitoneally injected immediately after transplantation for experimental treatment,respectively.Allografts were scored clinically at appropriate time points after treatment based on Plskova criteria,and ≥5 was confirmed as rejection.Infiltrating cells in corneal graft were detected qualitatively and quantitatively by immunohistochemistry and reverse transcription-PCR separately.The cytokine levels of T helper type 1 (Th1),Th2,and Th17 in recipients' spleen wer(c) analyzcd by ELISA.The use of the animals followed the Statement of ARVO.Results Compared with the isotype control antibody group,the survival of grafts was improved in the IL-17mAb group(P＜0.05).The levels of neutrophile granulocyte mRNA,CD4+ and CD8+ T lymphotes mRNA were 2.22±0.10,1.64±0.04 and 1.32±0.10 in the IL-17 mAb group,showing a significant decline in comparison with those of the isotype control antibody group(3.61 ±0.08,2.69±0.06 and 2.17±0.04) (P=0.000,0.000,0.000).Interferon-γ(IFN-γ),IL-12 p40 and IL-17 concentrations in recipients ' splenocytes were (529.80 ± 13.83) ng/L,(539.58 ±10.74) ng/L and(173.70±8.11)ng/L in the IL-17 mAb group,and thosc in the isotype control antibody group were (741.48± 10.51) ng/L,(1156.90 ± 69.93) ng/L and (366.13± 7.93) ng/L,with significant differences between them (P=0.000,0.001,0.000).Conclusions Neutralization IL-17 bioactivity inhibits mouse corneal allograft rejection to a certain extent. Key words: Corneal transplantation; Interleukin-17; Neutralization antibody; Immune rejection

Effect of Donor and Recipient Factors on Corneal Graft Rejection

To assess the relationship between donor and recipient factors and corneal allograft rejection in eyes that underwent penetrating keratoplasty in the Cornea Donor Study. Overall, 1090 subjects undergoing corneal transplantation for a moderate risk condition (principally Fuchs dystrophy or pseudophakic corneal edema) were followed for up to 5 years. Associations of baseline recipient and donor factors with the occurrence of a probable or definite rejection event were assessed in univariate and multivariate proportional hazards models. Eyes with pseudophakic or aphakic corneal edema (n = 369) were more likely to experience a rejection event than eyes with Fuchs dystrophy (n = 676) [34% ± 6% vs. 22% ± 4%; hazard ratio = 1.56; 95% confidence interval (CI), 1.21-2.03]. Among eyes with Fuchs dystrophy, a higher probability of a rejection event was observed in phakic posttransplant eyes compared with those that underwent cataract extraction with or without intraocular lens implantation during penetrating keratoplasty (29% vs. 19%; hazard ratio = 0.54; 95% CI, 0.36-0.82). Female recipients had a higher probability of a rejection event than male recipients (29% vs. 21%; hazard ratio = 1.42; 95% CI, 1.08-1.87) after controlling for the effect of preoperative diagnosis and lens status. Donor age and donor recipient ABO compatibility were not associated with rejection. There was a substantially higher graft rejection rate in eyes with pseudophakic or aphakic corneal edema compared to that in eyes with Fuchs dystrophy. Female recipients were more likely to have a rejection event than male recipients. Graft rejection was not associated with donor age.