Corneal Allograft Research Articles

MiR-151-5p alleviates corneal allograft rejection by activating PI3K/AKT signaling pathway and balancing Th17/Treg after corneal transplantation via targeting IL-2Rɑ.

BackgroundWorldwide, corneal transplantation (CT) is the most common type of tissue replacement and the increased rate of corneal graft rejection (CGR) after CT is a critical problem. Corneal endothelium cells (CECs) are often targets of the immune response mediated by graft-attacking effector T cells. However, the molecular mechanism underlying CGR remains poorly understood.MethodsThe differentially expressed microRNAs (miRNAs) and mRNA of graft-fail corneas were measured by transcriptome sequencing (RNA-Seq). real-time quantitative polymerase chain reaction was used to measure gene expression levels. Western blot and immunofluorescence staining were used to measure protein expression levels. Kaplan-Meier survival curves were constructed to assess corneal graft survival. Hematoxylin and eosin staining was used for histopathological examination. CCK-8 and ELISA staining were used to detect cell viability and inflammatory cytokines levels, respectively. Flow cytometry was used to detect cell apoptosis and the population of Treg and Th17. Transwell migration and wound-healing assays were used to measure cell migration.ResultsWe identified 453 miRNAs and 4,279 mRNAs aberrant expression in the corneas showing CGR. The differentially expressed miR-151-5p and its potential target gene [interleukin 2 receptor subunit alpha (IL-2Rɑ)] were selected from the RNA-Seq microarrays. The levels of miR-151-5p and IL-2Rɑ were respectively downregulated and upregulated in the CGR. The luciferase activity assay suggested that IL-2Rɑ is a target of miR-151-5p in 293 T cells. In addition, the miR-151-5p inhibitor, si-IL-2Rɑ, and oe-IL-2Rɑ transfection tests in CECs further confirmed that miR-151-5p downregulation and IL-2Rɑ overexpression promoted apoptosis of CECs and inhibited CEC migration, tight junction-related protein ZO-1 and Claudin-5 expression, and PI3K/AKT signaling pathway activity; however, downregulation of IL-2Rɑ abolished the inhibitor effect of miR-151-5p. Similarly, upregulation of miR-151-5p alleviated CGR via activation of the PI3K/AKT signaling pathway and balancing of Th17/Treg, and upregulation of IL-2Rɑ abolished the alleviating effect of miR-151-5p.ConclusionsUpregulation of miR-151-5p alleviated CGR by activating the PI3K/AKT signaling pathway and balancing Th17/Treg via targeting of IL-2Rɑ, which contributes to improving the results of CT.

Ultrastructural features of the corneal epithelium with micromolecular compound J2 in the corneal allograft of rats using atomic force microscopy

Objective: To study the ultrastructural features of the corneal epithelium in the corneal allograft of rats with micromolecular compound J2. Methods: An experimental study. Primarily cultured corneal epithelial cells of rats were used. Mononuclear cells (MNCs) by density gradient centrifugation were assigned into the experimental group [MNCs (2 ml)+corneal epithelial cells+J2], control group [MNCs (2 ml)+corneal epithelial cells] and blank group (corneal epithelial cells). Quantity of CD80 expression was obtained by flow cytometry after coculture. Amplitude and height images were obtained by tapping mode atomic force microscopy (AFM) with a scan rate of 2 Hz and an integral gain of 0.3 to 0.5. Statistical analysis of Ra, Rq, Rvm and Rt was performed using the single-factor analysis of variance, and P value was calculated. Results: There were obvious differences in the ultrastructure measured by AFM among groups. Ra was 86.75±12.60 in the experimental group, 120.23±12.11 in the control group, and 61.94±10.62 in the blank group (F=306.92, P<0.01). Rq was 102.53±9.45, 138.30±10.13, and 91.96±7.25, respectively, in the three groups (F=361.85, P<0.01). Rvm was -42.21±14.22, -67.36±10.89, and -32.18±19.01, respectively (F=72.22, P<0.01). Rt was 437.32±15.66, 495.32±13.96, and 339.92±11.22, respectively (F=1634.26, P<0.01). The one way analysis of variance showed significant differences in these parameters among groups. Conclusions: Corneal epithelial cells of rats activated by MNCs had higher CD80 expression, but the expression became decreased with micromolecular compound J2. The ultrastructure of the corneal epithelium became coarser after MNCs activation, and the number of protrusions increased significantly. The ultrastructural changes were alleviated by J2.(Chin J Ophthalmol, 2021, 57: 608-613).

Corneal Allograft Rejection Associated With Herpes Zoster Recombinant Adjuvanted Vaccine.

The purpose of this article is to report 3 cases of corneal allograft rejection that occurred in temporal proximity to administration of the zoster subunit vaccine (RZV). Three cases of corneal transplant rejection that developed after RZV administration were identified. Clinical history, including existence of other risk factors, timing of rejection, corticosteroid therapy at the time of onset of rejection, and course were reviewed. The onset of symptoms occurred 5 weeks after the first RZV dose in 1 patient and 1 and 6 weeks after the second dose in the other 2 patients. Coexisting risk factors included history of endothelial keratoplasty in the fellow eye in 1 patient and previous failure of a penetrating keratoplasty because of rejection in a second patient. The third patient had a history of 1 episode of rejection in a previous graft that resolved and then experienced graft failure over several years. In 2 patients, rejection developed despite relatively high levels of topical steroid therapy: prednisolone acetate 1%, 4 × per day in 1 patient and difluprednate 0.05%, 3 × per day in a second patient. RZV, which elicits a more robust immune reaction than the zoster live-attenuated vaccine, ZVL, may increase the risk of allograft rejection in immunocompetent patients with preexisting corneal endothelial or penetrating transplants. Based on the available data, it may be reasonable to increase the topical corticosteroid regimen before the first dose, until approximately 3 months after the second dose of ZVL.

Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

PurposeTo investigate the effects of ex vivo–induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation.MethodsBone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription–quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo–induced BM-MDSCs. IFN-γ, IL-2, IL-10, and TGF-β1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo–induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry.ResultsThe combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo–induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo–induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-β1 production was observed in coculture of ex vivo–induced BM-MDSCs. Injected ex vivo–induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis.ConclusionsThe ex vivo–induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.

Kaempferol alleviates corneal transplantation rejection by inhibiting NLRP3 inflammasome activation and macrophage M1 polarization via promoting autophagy

Corneal transplantation rejection remains a major threat to the success rate of high-risk patients. Given the many side effects presented by traditional immunosuppressants, there is an urgency to clarify the mechanism of corneal transplantation rejection and to identify new therapeutic targets. Kaempferol is a natural flavonoid that has been proven in various studies to possess anti-inflammatory, antioxidant, anticancer, and neuroprotective properties. However, the effect of Ka on corneal transplantation remains largely unexplored. To address this, both at the in vivo and in vitro levels, we established a model of corneal allograft transplantation in Wistar rats and an LPS-induced inflammatory model using human THP-1-derived macrophages. In the transplantation experiments, we observed an enhancement of mRNA and protein level in the NLRP3/IL-1 β axis and in M1 macrophage polarization post-operation. In groups to which kaempferol intraperitoneal injections were administered, this response was effectively reduced. However, the effect of kaempferol was reversed after the application of autophagy inhibitors. Similarly, in the inflammatory model, we found that different concentrations of kaempferol reduced the LPS-induced M1 polarization and NLRP3 inflammasome activation. Moreover, we confirmed that kaempferol induced autophagy and that autophagy inhibitors reversed this effect in macrophages. In conclusion, we found that kaempferol can inhibit the activation of NLRP3 inflammasomes by inducing autophagy, thus inhibiting macrophage polarization, and ultimately alleviating corneal transplantation rejection. Thus, our study suggests that kaempferol is a potential therapeutic agent in the treatment of allograft rejection.

Characteristics of endothelial corneal transplant rejection following immunisation with SARS-CoV-2 messenger RNA vaccine

AimWe report two cases of endothelial corneal allograft rejection following immunisation with SARS-CoV-2 messenger RNA (mRNA) vaccine BNT162b2 and describe the implications for management of transplant recipients postvaccination for COVID-19.MethodsA...

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.

Subconjunctival injections of dimethyl fumarate inhibit lymphangiogenesis and allograft rejection in the rat cornea

Corneal lymphangiogenesis induced by macrophages played a critical role in corneal allograft rejection (CGR). However, there are few Food and Drug Administration (FDA)-approved drugs that target lymphangiogenesis. The aim of our study is to evaluate the effects of dimethyl fumarate (DMF) on corneal allograft survival in rats. Penetrating corneal transplantation was performed in rats. Subconjunctival injections of dimethyl fumarate (20 µg) were administered at the end of the operation and postoperative day 3 to day 11. The clinical signs of corneal allografts were evaluated. Immunohistochemistry, quantitative real-time PCR (qPCR), flow cytometry and western blot were performed respectively. The effects and mechanism of DMF on RAW264.7 cells were determined by qPCR, enzyme-linked immunosorbent assay (ELISA), and western blot in vitro. The results showed that subconjunctival injections of DMF could significantly inhibit corneal lymphangiogenesis and CGR with decreased corneal macrophage infiltration compared with the vehicle group. Moreover, DMF could reduce the mRNA expression of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and vascular endothelial growth factor-C (VEGF-C) in the corneal grafts and RAW264.7 macrophages by inhibiting NF-κB activation. Furthermore, compared with the vehicle group, the number of dendritic cells in the ipsilateral cervical lymph nodes of the DMF-treated group was decreased significantly. Collectively, our findings showed that DMF could suppress CGR by inhibiting the macrophage-induced corneal lymphoangiogenesis.

Secondary Endothelial Keratoplasty-A Narrative Review of the Outcomes of Secondary Corneal Endothelial Allografts.

We review the literature on the efficacy and safety outcomes of secondary Descemet stripping endothelial keratoplasty (DSEK) and Descemet membrane endothelial keratoplasty (DMEK). Literature search of English-written publications up to September 27, 2020 in PubMed database, using the terms "endothelial keratoplasty" in combination with keywords "secondary" or "repeat." In addition, we manually searched the references of the primary articles. Twenty-seven studies (n = 651 eyes) were retained and reviewed, including 10 studies on repeat DSEK, 8 studies on repeat DMEK, 6 studies of DMEK following DSEK, and 3 studies of DSEK after failed DMEK. All studies reported significant improvement in visual acuity after secondary endothelial keratoplasty (EK). Twelve studies compared visual outcomes between primary and secondary EK, reporting conflicting findings. Sixteen studies reported endothelial cell loss rates after secondary EK, and only 1 study reported significantly increased endothelial cell loss rates compared with primary EK. Allograft rejection episodes occurred in 1.8% of eyes (range, 0%-50%). Six studies compared complication rates between primary and secondary EK eyes, and only 1 study found a higher median number of complications. However, 2 studies reported higher regraft failure rates compared with primary EK eyes. Secondary EK is surgically feasible and renders significant visual improvement after failed primary EK, although it is not clear whether visual outcomes and allograft survival are comparable with primary EK, raising the question of whether secondary EK eyes are "low risk" as primary EK eyes. Further larger, prospective studies are encouraged to obtain additional quality data on secondary corneal endothelial allotransplantation.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after (“post-transplant,” PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular “immune privilege.” PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

Sterile Excimer Laser Shaped Allograft Corneal Inlay for Hyperopia: One-year Clinical Results in 28 Eyes

ABSTRACT Purpose: This study aimed to evaluate the one-year clinical results of an allograft corneal inlay (ACI) implantation in a case series of 28 hyperopic eyes of 16 patients. Methods: Patients with manifest refraction spherical equivalent (MRSE) between +1.00 and +6.00 D and having a cylindrical refraction of less than 1 D were included in this prospective study. The refractive powers of excimer laser-shaped ACIs were determined based on the refractive error of the individual subject’s eyes. After the creation of a femtosecond flap, the inlays were centered on the pupillary axis. Visual acuities, refractive results, and other clinical findings were reported for the 6- and 12-month follow-up exams. Results: The mean age of the patients included in the study was 36.2 ± 12.4 years (range 22–65 years). The mean pre-operative MSRE of 3.6 ± 1.51 D decreased to 0.21 ± 0.56 D (P < .001). The uncorrected distance and near visual acuity increased from 0.33 ± 0.22 and 0.17 ± 0.13 to 0.75 ± 0.22 (P < .001) and 0.72 ± 0.19 (P < .001), respectively. The corrected distance visual acuity remained unchanged (pre-OP: 0.79 ± 0.22; post-OP: 0.80 ± 0.21; P = .916), and the corrected near visual acuity increased from 0.78 ± 0.22 to 0.84 ± 0.20 (P = .003). The mean K-value and central corneal thickness increased from 42.57 ± 0.81 D and 557.5 ± 43.0 µm to 44.8 ± 1.4 D (P < .001) and 597.1 ± 58.1 µm (P < .001), respectively. No significant postoperative complications such as diffuse lamellar keratitis, epithelial ingrowth, or decentralization were observed. Conclusion: Excimer laser-shaped ACI offers an alternative treatment modality for patients with hyperopia. Acceptable visual results and similar regression rates were observed with ACI implantation compared with other laser refractive procedures.

The role of soluble programmed death protein-1 (sPD-1) and soluble programmed death ligand-1 (sPD-L1) in rat corneal transplantation rejection.

Immunological rejection is one of the problems in corneal transplantation. Recently, some research found out that soluble programmed death protein-1 (sPD-1) and soluble programmed death ligand protein-1 (sPD-L1) play a significant role in immunologic suppression. To explore expression of sPD-1 and sPD-L1 in a penetrative corneal transplantation model and its relationship with transplant rejection. Autologous corneal transplantation rat models and allogeneic corneal transplantation rat models were used as the control group and the experimental group, respectively. Changes of the transplanted grafts were observed under a slit-lamp microscope. Hematoxylin-eosin (H&E) staining was applied to examine the histopathological features of the corneal grafts. Flow cytometry was used to analyze CD4+CD25+Treg in the serum and spleen. The sPD-1, sPD-L1, interleukin 10 (IL-10) and interleukin 4 (IL-4) levels in serum and the aqueous humor of the rats were detected using enzyme-linked immunosorbent assay (ELISA). After the operation, no transplant rejection occurred in the control group. Flow cytometry results showed that expressions of CD4+CD25+Treg in serum in the experimental group were lower than those in the control group (p < 0.05). The ELISA results showed that after the operation, sPD-1 and sPD-L1 expression levels in serum in the experimental group were higher than in the control group (all p < 0.05). After the operation, lL-10 and IL-4 content in serum in the experimental group was lower than in the control group (all p < 0.05). The sPD-1/sPD-L1 ratio in the experimental group was higher than in the control group. Increases of sPD-1 content and decreases of CD4+CD25+Treg, IL-10 and IL-4 levels may be involved in corneal allograft rejection. Dynamic detection of the content of sPD-1 and sPD-L1 in serum and aqueous humor after the operation would help in understanding the local immune response in a clinical setting and predicting the occurrence of corneal graft 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.

Incidence of corneal transplant rejection following BNT162b2 SARS-CoV-2 messenger RNA vaccination

Purpose: We report seven cases of corneal allograft rejection following immunization with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccine BNT162b2, and describe the implications for the management of transplant recipients post-vaccination for coronavirus disease 2019 (COVID-19). Methods: This retrospective observational study included 66 patients post-keratoplasty between 98 and 2054 days following transplantation. Data on the incidence of rejection, time series of the onset of rejection, corneal transplantation, and vaccination with the SARS-CoV-2 mRNA vaccine BNT162b2 were collected. In addition, the use of ophthalmic solutions including topical steroids and immunosuppressants at the time of rejection, and background including systemic diseases, such as autoimmune or allergic disease, were collected. Results: The incidence of rejection following BNT162b2 vaccination was observed in seven cases (observational period following vaccination was 74.0 ± 28.4 days), while no vaccination was observed in 2 cases (observational period following keratoplasty was 938.3 ± 1501.5 days). The odds ratio of rejection related to vaccination was 5.57 (95% confidence interval [CI], 1.18–25.57; p =0.036; Fisher’s exact test). The relative risk of rejection related to vaccination is 4.47 (95% CI, 1.15–18.34). Conclusion: A temporal association between corneal transplant rejection following immunization against COVID-19 was suspected. A possible mechanism could be the allogeneic response initiated by the host antibody following vaccination.

Mesenchymal Stem Cell Derived Exosomes Promote Corneal Allograft Survival in a Rat Model

Mesenchymal Stem Cell Derived Exosomes Promote Corneal Allograft Survival in a Rat Model

Corneal graft rejection after COVID-19 vaccination.

A 62-year-old man presented with corneal graft rejection after COVID-19 vaccination. He had undergone penetrating keratoplasty for a childhood corneal scar in his right eye, 2 years ago. The aphakic and amblyopic eye had regained corrected vision of 6/36, N12 and was on topical steroid once a day. He presented with congestion and diminution of vision of 5 days duration, having taken his first shot of COVID-19 vector vaccine (ChAdO × 1 nCoV-19 Corona Virus Vaccine Recombinant COVISHIELD™), 3 weeks prior. There were no loose sutures, neovascularization, iris synechiae, or any other stimulus for rejection. There was an advancing horizontal Khodadoust’s rejection line in the center of the endothelium with graft edema at its wake inferiorly [Fig. 1] and grade 2 anterior chamber reaction. The graft superior to the rejection line was clear [Fig. 2]. Treatment for corneal allograft rejection was started. This may be the first report of graft rejection after COVID-19 vector vaccination.Figure 1: It shows graft rejection with edema inferiorly and clear graft superiorlyFigure 2: It shows an advancing Khodadoust’s endothelial rejection lineDiscussion The immune privilege of the cornea is due to lack of blood and lymph vessels in the host bed and absence of major histocompatibility complex (MHC) class II antigen presenting cells, making the donor cornea invisible to the host immune system.[1] Vaccination incites immune response that can induce Class II MHC complex antigens in all layers of the grafted cornea and could trigger allograft rejection like the influenza vaccine.[23] The recent rampant vaccination against COVID-19 has triggered a debate about similar such effect on endothelial or full thickness keratoplasty.[4] Now, it has become common practice to hike up topical steroids prior to any type of COVID-19 vaccination.[5] Our case highlights the possibility of immune corneal graft rejection after COVID-19 vaccination which corneal surgeons should be aware of. It also raises the dilemma regarding the timing of the second dose of vaccination, in patients presenting with rejection. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

First experience of α1,3-galactosyltransferase gene-knockout (GTKO) transgenic pig to nonhuman primate lamellar corneal xenotransplantation

Background: Corneal allotransplantation is a well-known technique to treat corneal blindness. However, the problem of organ scarcity in transplantology has become profound. One promising alternative could be xenograft using pig as an organ donor. Full thickness corneal xenotransplantation needs total immunosuppression. This study is an investigation of the efficacy of α1,3-ga lactosyltransferase gene-knockout (GTKO) transgenic pig-to-nonhuman primate lamellar corneal transplantation with minimal immunosuppression. Methods: We conducted 10 lamellae corneal xenotransplantation between 2016 and 2019. Clinically acceptable graft size (diam eter 7.5 mm, thickness 500 um). The dexamethasone subconjunctival injection (1.5 mg/0.3 mL) was administered for minimal immunosuppression immediately after surgery and eye drops of 0.5% levofloxacin and 1% prednisolone acetate were applied four times a day for 1 week, gradually tapered. No eye drops were added after two months. Three cases are alive without graft rejection. We examined the remaining seven cases for the histopathological features and Immunologic profiles. Results: As a result, three of the ten xenografts survival is significantly longer 1,239, 589, and 316 days. Corneal opacity result ed in graft failure, and terminated in seven cases. Rejected grafts showed extensive polymorphic cellular infiltration, different degrees of epithelial layer irregular attenuation, stromal neovascularization, and inflammatory cell infiltrations including lym phocytes, plasma cells, eosinophils, neutrophils. Stromal irregularity, fibrosis and edema are observed in two of seven cases resulting in a single case of sub epithelial detachment. Immunologic profiles of the recipients with rejected grafts shows minimal increase in anti-Gal antibody IgG and IgM but increase in anti-Gal IgG is seen in one case. Four cases have different systemic in flammatory conditions with regards to plasma C3a and D-dimer levels. The anterior stromal surface of the graft showed epitheli al nests and fibrous proliferation. Conclusions: The GTKO transgenic pig to NHP lamella xeno corneal transplantation could be a promising substitute for human corneal allograft. Lamella xeno corneal transplantation may be a feasible option with minimal immunosuppression.

Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival

Corneal allograft survival is mediated by the variety of immunological reactions and wound healing process. Our aim was to explore the effects of topical administration of ripasudil, a selective Rho-associated coiled-coil protein kinase inhibitor, on corneal allograft survival. Ripasudil was administered to mice thrice a day after allogeneic corneal transplantation. Corneal graft survival, opacity, neovascularization, re-epithelization, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in the grafted cornea and draining lymph nodes (dLNs) were evaluated with slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. Graft survival was significantly prolonged with lower graft opacity and neovascularization scores in 0.4% and 2.0% ripasudil-treated groups, and mRNA levels of angiogenic and pro-inflammatory factors in ripasudil-treated grafted corneas were reduced. Moreover, 0.4% and 2.0% ripasudil reduced CD45+-infiltrated leukocyte frequency, Cd11b and Cd11c mRNA levels, and the frequencies of mature dendritic cells, IFNγ-, and IL-17- producing CD4+T cells in the dLNs of recipients. Re-epithelization rate of the grafted cornea was significantly higher in the 0.4% and 2.0% ripasudil groups than in the control. Topically applied ripasudil prolonged graft survival by downregulating neovascularization and inflammation factors, while promoting corneal re-epithelization, suggesting that ripasudil may be useful for suppressing immunological rejection in corneal transplantation.

Myeloid-derived suppressor cells improve corneal graft survival through suppressing angiogenesis and lymphangiogenesis.

Myeloid-derived suppressor cells (MDSC) are one of the major negative regulators of immune responses during many pathological conditions such as cancer and transplantation. Emerging evidence indicates that MDSC also contribute to tumor progression through their pro-angiogenic activity in addition to immunosuppressive function. However, virtually nothing is known about the role of MDSC in the regulation of neovascularization after transplantation. Here we showed that antibody-mediated depletion of MDSC in mice led to robust growth of blood and lymphatic neovessels and rapid allograft rejection after corneal penetrating keratoplasty. In contrast, adoptive transfer of ex vivo generated MDSC from cytokine-treated bone marrow cells (evMDSC) suppressed neovascularization and prolonged corneal allograft survival in an inducible nitric oxide synthase (iNOS)-dependent manner. Mechanistically, compared to naïve MDSC control, evMDSC have increased expression of an anti-angiogenic factor thrombospondin 1 (Tsp-1) and decreased expression of two critical pro-angiogenic factors, vascular endothelial growth factor A (VEGF-A), and VEGF-C. These findings demonstrate MDSC as a critical anti-angiogenic regulator during transplantation. Our study also indicates that evMDSC are a valuable candidate agent for development of novel cell therapy to improve allograft survival after transplantation.

Blockade of costimulatory CD27/CD70 pathway promotes corneal allograft survival

PurposeTo determine whether the CD27/CD70 pathway plays a significant role in corneal allograft rejection by investigating the effect of blocking the CD27/CD70 pathway by anti-CD70 antibody on corneal allograft survival. MethodsOrthotopic penetrating keratoplasty was performed using C57BL/6 donor grafts and BALB/c recipients. Expression of CD27 and CD70 on rejected cornea was examined by immunohistochemistry. Corneal transplant recipients received intraperitoneal injection of anti-CD70 antibody (FR70) or control rat IgG. Alloreactivity was measured by mixed lymphoid reaction (MLR) in recipients administered control rat IgG and those administered anti-CD70 antibody. Corneal expression of IFN-γ and IL-12 was also examined in both groups. Graft opacity was assessed over an 8-week period and graft survival was evaluated using Kaplan-Meier survival curves. Proportion of CD4+CD44+ memory T cells in lymph nodes was measured by flow cytometry. ResultsCD4+CD27+ cells and CD11c+CD70+ cells were present in rejected cornea. Anti-CD70 antibody administration suppressed alloreactivity in corneal allograft recipients, and inhibited IFN-γ expression in recipient cornea (p < 0.05). Anti-CD70 antibody suppressed opacity score of recipient cornea and prolonged corneal allograft survival (p < 0.05). Proportion of CD4+CD44+ memory T cells in recipient lymph nodes was reduced by anti-CD70 antibody treatment. ConclusionThe CD27/CD70 pathway plays a significant role in corneal allograft rejection by initiating alloreactive Th1 cells and preserving memory T cells. Anti-CD70 antibody administration prolongs corneal allograft survival indicating the potential therapeutic effect of CD27/CD70 pathway blockade on corneal allograft rejection.