Levels of Foxp3 in regulatory T cells reflect their functional status in transplantation.

Foxp3 expressing CD4(+)CD25(+) regulatory T cells (Tregs) have been shown to prevent allograft rejection in clinical and animal models of transplantation. However, the role of Foxp3 in regulating Treg function, and the kinetics and mechanism of action of Tregs in inducing allograft tolerance in transplantation, are still not fully understood. Thus, we investigated the kinetics and function of Tregs in a mouse model of orthotopic corneal transplantation, the most common form of tissue grafting worldwide. In this study, using in vitro functional assays and in vivo Treg adoptive transfer assays, we show that far more relevant than Treg frequency is their level of Foxp3 expression, which is directly associated with the potential of Tregs to prevent allograft rejection by producing regulatory cytokines and suppressing effector T cell activation. In addition, our data clearly demonstrate that Tregs primarily suppress the induction of alloimmunity in regional draining lymph nodes rather than suppressing the effector phase of the immune response in the periphery. These findings provide new insights on Treg dynamics in transplantation, which are crucial for designing therapeutic strategies to modulate Treg function and to optimize Treg-based cell therapies for clinical translation.

Histopathologic examination of failed grafts in descemet's stripping with automated endothelial keratoplasty.

PURPOSE: To study the histopathologic features of 19 corneal posterior lamellar grafts in eyes for which Descemet's stripping with automated endothelial keratoplasty (DSAEK) has failed. DESIGN: Retrospective case series with clinicopathologic correlation. PARTICIPANTS: Nineteen cases of DSAEK failures undergoing repeat DSAEK or penetrating keratoplasty. METHODS: The histopathologic results of posterior lamellar grafts (also termed DSAEK grafts), recipient corneas, or both from 19 cases of failed DSAEK were examined. MAIN OUTCOME MEASURES: Abnormalities in the DSAEK graft and in the interface between the recipient cornea and the DSAEK graft were assessed. RESULTS: Histopathologic features in 19 failed DSAEK grafts revealed attenuation of endothelial cells (16 cases) and presence in the graft-host interface of fibrocellular tissue (11 cases), retained Descemet's membrane (5 cases), epithelial ingrowth (4 cases), or a combination thereof. Four DSAEK grafts had full-thickness corneal layers at 1 edge. CONCLUSIONS: Presence of interface material, such as fibrocellular tissue, retained Descemet's membrane, and epithelial ingrowth, are potential causes of dislocation. Endothelial attenuation was the most common finding in failed grafts. Decentered DSAEK grafts with full-thickness corneal layers at 1 edge are a potential cause for epithelial ingrowth.

A clinicopathologic series of primary graft failure after Descemet's stripping and automated endothelial keratoplasty.

PURPOSE: To characterize the clinical and histologic features of primary graft failure after Descemet's stripping and automated endothelial keratoplasty (DSAEK). DESIGN: Retrospective observational case series. PARTICIPANTS: Sixteen cases of DSAEK graft failure from 15 patients, all with detailed histologic examination of failed graft tissue. METHODS: Hematoxylin-eosin, periodic acid-Schiff staining, and light microscopy were used to examine the failed DSAEK graft tissue from all patients. MAIN OUTCOME MEASURES: Examination of specimens for corneal endothelial cell viability and host-donor interface characteristics. RESULTS: Clinical history revealed that 88% (14/16) of studied DSAEK grafts detached before failure, and pathologic examination found that 75% (12/16) of failed grafts had atrophic corneal endothelium. Examples of residual host Descemet's membrane in the graft site and improper donor trephination were also identified. CONCLUSIONS: Marked loss of the corneal endothelium is the prominent feature of primary DSAEK graft failure. Examples of surgical features, such as incomplete Descemet's stripping and residual full-thickness cornea with a DSAEK graft, are shown.

DNA microarray-based gene expression profiling in porcine keratocytes and corneal endothelial cells and comparative analysis associated with xeno-related rejection.

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model.

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.

Corneal imaging with anterior segment optical coherence tomography for lamellar keratoplasty procedures.

PURPOSE: To assess and describe the uses of anterior segment optical coherence tomography (OCT) in the evaluation of the cornea before and after lamellar corneal transplantation procedures. DESIGN: Prospective, noncomparative, observational case series. METHODS: Seven eyes of seven patients undergoing anterior and posterior lamellar corneal transplantation procedures at the Singapore National Eye Centre were included in the study. High-resolution anterior segment OCT scans of the cornea and anterior segment were performed both before and after lamellar transplantation procedures on the cornea with the Visante anterior segment OCT system (Visante OCT; Carl Zeiss Meditec, Inc, Dublin, California, USA), and the imaging findings were correlated with the clinical picture. Measurements of lamella thickness were performed with the software provided. RESULTS: Anterior segment OCT images were able to provide valuable information on donor apposition, Descemet membrane detachment after deep anterior lamellar keratoplasty (DALK), posterior lamellar dislocation, primary graft failure, and anterior chamber crowding with consequent chamber angle encroachment and pupillary block after Descemet stripping automated endothelial keratoplasty (DSAEK). CONCLUSIONS: Anterior segment OCT is a valuable imaging tool for assessing the feasibility of lamellar transplantation surgery in the diseased cornea and in the management of surgical complications after such procedures.

Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.

Survival of cultured allogeneic limbal epithelial cells following corneal repair.

In this study a technique for determining donor cell fate following corneal grafting was evaluated. Patients treated for limbal deficiency with allogeneic cultured corneal epithelial cells were studied to determine the fate of the grafted cells. The technique was evaluated initially through the use of donor eyes and then applied to the clinical analysis of 7 patients who had received a cultured corneal epithelial allograft. Cells removed from the cornea and any retrieved tissue were analyzed via polymerase chain reaction (PCR) genotyping to determine the origin of the cells populating the patients' healed cornea. A mixture of genotypes was detected in a cornea retrieved from a patient following a fully penetrating keratoplasty who had received a mixture of allogeneic tissue. Donor cells were no longer detected on the corneal surface of all 7 cases beyond 28 weeks postgraft. At these later time points, only patient genotype could be detected. These results demonstrate that PCR genotyping can be used to determine the origin of cells populating the surface of the cornea following the grafting of cultured allogeneic cells and demonstrates that transplanted cultured limbal epithelial cells do not persist on the surface of the host cornea for more than 28 weeks.

Corneal hysteresis, resistance factor, topography, and pachymetry after corneal lamellar flap.

PURPOSE: To measure prospectively the early changes in corneal hysteresis, topography, and pachymetry after the creation of a stromal flap cut without laser photoablation. METHODS: A 37-year-old man was referred for a bioptic procedure to correct for compound myopic astigmatism in the left eye. A 159-microm-thick 8x8.5-mm superior hinged flap was created with a mechanical microkeratome in the left cornea. Changes in the corneal hysteresis, corneal resistance factor, Goldmann correlated intraocular pressure (lOP), corneal compensated IOP, anterior and posterior topography, and optical and ultrasound pachymetry were monitored prospectively before and at 1 hour, 1 day, 5 days, and 25 days after flap creation. The right eye served as a control. RESULTS: In the left eye, corneal hysteresis and corneal resistance factor decreased immediately after the flap cut and remained lower than preoperatively at 1 hour, 1 day, 5 days, and 25 days. Corneal compensated IOP varied significantly less than Goldmann correlated IOP in both eyes. Central flattening of the horizontal meridians was observed on the difference topography maps. The values of the left eye posterior best fit sphere increased after the flap cut. Increased central corneal thickness occurred immediately after the flap cut and decreased over time without returning to its preoperative value. CONCLUSIONS: The creation of a stromal flap can modify the biomechanical properties of the cornea, including a reduction in corneal hysteresis. The topographic changes were consistent with previously reported cases of flap cut in normal corneas.

Corneal wound healing from the perspective of keratoplasty specimens with special reference to the function of the Bowman layer and Descemet membrane.

PURPOSE: To review corneal wound healing with special reference to the function of the Bowman layer and Descemet membrane. METHODS: Corneal specimens were obtained from keratoplasties, including regrafted cases. Recipient corneal buttons were evaluated histopathologically with attention to 5 layers of corneal structure: 3 cellular layers consisting of epithelial cells, keratocytes, and endothelial cells and 2 acellular layers consisting of the Bowman layer and Descemet membrane. RESULTS: Subepithelial fibrosis was found in advanced bullous keratopathy. The possible source of subepithelial fibrosis was either conjunctival stroma or corneal stroma through disruption of the Bowman layer. Subepithelial fibrosis was observed in the area of the Bowman layer disruption at the host-graft junction in regrafted cases. The Bowman layer was disrupted in eyes with not only keratoconus but also corneal dystrophy such as macular dystrophy and gelatinous drop-like dystrophy. Newly formed, thin Descemet membrane was found in keratoconic eyes of patients with acute hydrops. Retrocorneal membranes were observed in eyes with advanced bullous keratopathy and graft failure. Abnormal wound healing of Descemet membrane such as override and separation was found in the host-graft interface of regrafted eyes, causing stromal overgrowth. CONCLUSIONS: The Bowman layer and Descemet membrane seem to serve as barriers to separate 3 cellular layers of epithelium, stroma, and endothelium. Disruption of the Bowman layer forms a new epithelial-stromal interaction and may cause cellular proliferative response. Separation of Descemet membrane can provide the trigger for emanating stromal tissue from the wound edge.

Descemet-stripping automated endothelial keratoplasty: effect of inserting forceps on DSAEK donor tissue viability by using an in vitro delivery model and vital dye assay.

PURPOSE: To qualitatively assess the extent and pattern of endothelial trauma on corneal donor Descemet-stripping automated endothelial keratoplasty (DSAEK) buttons resulting from DSAEK insertion forceps. METHODS: An in vitro model was used with corneoscleral rims, DSAEK quality corneal donor tissue, and DSAEK insertion forceps. After insertion of the donor button through the corneoscleral rim, a vital dye assay was used to identify devitalized and necrotic endothelial cells (with alizarin red S and typan blue). RESULTS: Corneal buttons evaluated with the forceps delivery model showed that, for each arm of the forceps, there were 2 parallel bands of purple/red staining. In addition, orthogonal wrinkles of scattered blue devitalized nuclei were seen in a parallel arrangement. CONCLUSIONS: The DSAEK insertion forceps resulted in a reproducible pattern of endothelial damage. A thorough understanding of iatrogenic endothelial trauma could result in improved forceps design and perhaps help mitigate the high rate of donor dislocation and graft failure in the future.

Deep lamellar endothelial keratoplasty: histopathology of complications in initial cases.

PURPOSE: To report complications encountered after 2 initial cases of deep lamellar endothelial keratoplasty. METHODS: Review of clinical findings of 2 initial cases in which intraoperative and postoperative complications were encountered. Corneal buttons from the 2 cases were evaluated by light microscopy and transmission electron microscopy. RESULTS: Complications encountered were related to the graft-recipient interface and the lamellar dissection of the recipient and donor corneas. CONCLUSION: Deep lamellar endothelial keratoplasty is an evolving procedure that shows much promise. These 2 cases show that the procedure entails a separate skill set from penetrating keratoplasty and requires significant surgeon practice before being introduced into the individual's transplant practice.

Major endothelial loss from corneas in organ culture: importance of second endothelial count.

PURPOSE: The aim of this study was to show that major losses can still occur on corneas judged suitable for grafting at the first count. In addition, we studied the frequency of these losses on 1992 corneas over a period of 4 years to evaluate the risk incurred. METHODS: We evaluated the incidence of these major losses and the associated risk factors. An Ishigawa diagram was created with the Cornea Bank team and the ophthalmologists involved in organ retrieval. Endothelial losses caused by bacterial or fungicidal contamination were excluded from the study. For the 29 corneas that suffered major losses, we analyzed the donor files for donor age, clinical file, geographical origins of the corneas, the person who did the retrieval, the length of time the cornea was stored, the data resulting from examining the endothelium at the bank by optical microscope, and the method used for sterilizing the material used. Specific analyses in cases of major loss of endothelial content: anatomopathologic examination of the corneas and search for the herpes simplex virus (HSV; type 1 or 2) by polymerase chain reaction (PCR). We carried out a statistical analysis using a chi(2) test on the 1992 corneas studied to see if the presence of diabetes (type 1 or 2) in the donor led to reduction levels different from those of corneas originating from nondiabetic donors. RESULTS: The incidence was evaluated at between 0.4% and 3% of corneas sampled, and the associated risk factor was between 0.8% and 6% of grafted corneas. The occurrence of major losses was independent of donor age and was independent of the person who did the retrieval. The occurrence of major losses was independent of geographical origin. We tested our media for endotoxin before use and found levels from 0.22 to 3.9 UI/mL. We verified the absence of a chronological relationship between the batches of media used in the bank and the number of major losses observed, showing that the pyrogenicity limit was independent of cytotoxicity limits. Data analysis showed no difference in reduction levels between diabetic and nondiabetic donors (P < 0.05). Results on the detection of HSV-1 by PCR on the storage media were all negative, and these results agree with the anatomopathologic examinations that showed no signs of viral infection. CONCLUSION: Total endothelial losses amounted to 1.4%/yr. Without the double endothelial counts, we would have had 29 primary graft rejections over that period. During storage, this loss has not been linked to a specific cause, but risk factors such as traumatic death, herpes infections, and badly controlled endotoxin levels should be considered when taking preventative actions. For the moment, a second endothelial count before grafting should be carried out, because all these problem grafts conformed to grafting criteria after the first count. The possibility of carrying out this second count is one of the recognized advantages of storage in organ culture.

Blastoschizomyces capitatus keratitis and melting in a corneal graft.

CASE REPORT: We present the first case, to our knowledge, of Blastoschizomyces capitatus keratitis and melting in a corneal graft. An 80-year-old man was treated for a large, severe corneal abscess and melting in the corneal graft of his right eye. Medical history was negative for hematologic disorders. Urgent penetrating keratoplasty was performed. COMMENTS: Corneal cultures grew B. capitatus. Systemic fluconazole and systemic and topical amphotericin B treatment were started. Three months after the last operation, no recurrence was observed and the graft remained clear. B. capitatus can rarely cause severe keratitis in patients even in the absence of hematologic disorders.

Corneal graft rejection occurs despite Fas ligand expression and apoptosis of infiltrating cells.

BACKGROUND/AIMS: Constitutive expression of Fas ligand (CD95L) protects the eye against cell mediated immune responses by inducing apoptosis in infiltrating Fas bearing T cells. This study was designed to examine Fas ligand expression on acutely rejecting rat corneal grafts and to investigate the kinetics of induction of apoptosis in infiltrating leucocytes. METHODS: Orthotopic penetrating corneal transplantation was performed between genetically disparate inbred rats. Fas ligand expression and the phenotype of infiltrating leucocytes were examined by immunohistochemistry. Apoptotic nuclei were visualised in sections of normal rat cornea, rejecting allografts, and time matched isografts by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling (TUNEL) and quantified by video image analysis. Staining with Hoechst dye 33258 was used to confirm the presence of apoptotic nuclei. RESULTS: Fas ligand was expressed on corneal endothelial and epithelial cells during acute corneal graft rejection. At all time points examined, including as early as the fifth postoperative day, the cells infiltrating both corneal isografts and allografts were TUNEL positive. By the 15th postoperative day, over 90% of all nuclei, many of which were T cells, were apoptotic. CONCLUSION: Expression of Fas ligand is not downregulated on the cornea during allograft rejection and infiltrating leucocytes in both isografts and allografts die rapidly in situ. Despite the death of the cells believed to be responsible for rejection, isografts survive indefinitely whereas allografts are irreparably damaged.

CD4+ T cells are critical for corneal, but not skin, allograft rejection.

BACKGROUND: The relative contribution of CD4+ or CD8+ T cells in allograft rejection remains to be fully characterized. Some reports indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas others report that CD4-depleted mice are capable of rejecting certain types of allografts. METHODS: We compared the ability of CD4- knockout (KO), CD8- KO, and normal CD4+/CD8+ mice to reject allogeneic corneal or skin grafts. We also examined delayed-type hypersensitivity and CTL responses to donor alloantigens. RESULTS: Engraftment of C57BL/6 corneas to C.B6-(n5-7) CD4-KO mice resulted in significantly higher rates of acceptance (>85%) than either C.B6-(n5-7) CD8- KO (30%) or normal BALB/c mice (40%). Likewise, mean survival times for B6 skin grafts placed on C.B6-(n5-7) CD4- KO mice (29.2 +/- 3.5 days) were significantly increased over those of normal BALB/c mice (13.2 +/- 1 days), although most CD4- KO mice (70%) eventually reject their grafts. C.B6-(n5-7) CD4- KO mice that reject allogeneic grafts fail to develop a delayed-type hypersensitivity response, but they did demonstrate significantly greater cytotoxic T lymphocyte precursor (CTLp) frequencies than did CD4- KO mice that accepted such grafts or that were not grafted. CONCLUSIONS: This study indicates that mice lacking CD4+ T cells have a significantly impaired ability to reject corneal allografts, but are able, in most cases, to reject allogeneic skin grafts. Thus, in the absence of CD4+ T cells, the likely mechanism for rejection appears to involve the generation of CD8+ CTLs.

Coadministration of the new macrolide immunosuppressant RAD and mycophenolate mofetil in experimental corneal transplantation.

INTRODUCTION: The effect of RAD, a new macrolide immunosuppressant, was examined as mono- and combination therapy with mycophenolate mofetil (MMF) in prevention of acute allograft rejection in murine corneal transplantation. METHODS: Both drugs were administered orally for 18 days beginning at the day of transplantation. The inbred strains Fisher and Lewis were used as donors and recipients, respectively. Five groups were involved: syngeneic control, allogeneic control, 2.5 mg/kg RAD, 40 mg/kg MMF, and double drug therapy with 1.5 mg/kg RAD and 20 mg/kg MMF. RESULTS: The median transplant survival time in the allogeneic combination was 12 (+/-0.3) days. Monotherapy with 2.5 mg/kg RAD and 40 mg/kg MMF led to a statistically significant prolongation of transplant survival to 25.5 (+/-12.5, P=0.0001) days and 19.5 (+/-13.9, P=0.0053) days, respectively. Combination therapy was superior to both monotherapies (100+/-15.8 days, P=0.03). There was a significant reduction in the number of CD4+, CD8+, as well as CD45RA+ cells in the RAD- and double drug-treated animals when compared with the allogeneic control. This significant reduction in graft-infiltrating lymphocytes has not been found in the MMF monotherapy. CONCLUSIONS: The unique finding of this first study on the combination of RAD and MMF in murine corneal transplantation is that double drug therapy produces a highly synergistic effect in prevention of acute allograft rejection without a higher incidence of complications related to drug toxicity or overimmunosuppression.

Roles of tumor necrosis factor-related apoptosis-inducing ligand in corneal transplantation.

BACKGROUND: Allograft rejection is still a main cause of graft failure in high-risk keratoplasty. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis of tumor cells without significant toxicity and inhibit autoimmune disease. Therefore, we attempted to determine the roles of TRAIL in graft survival. METHODS: Thirty-two BALB/C mice were recipients of corneal grafts from C57BL/6 mice. The 32 eyes were equally divided into four groups receiving graft without incubation with viral preparations, graft immersed in Dulbecco's minimum essential medium containing soluble human death receptor 5 (sDR5) before transplantation, graft carrying the recombinant adenovirus with TRAIL gene (Ad-TRAIL), and graft carrying the recombinant adenovirus with green fluorescent protein (Ad-GFP), respectively. Pathologic and immunohistochemical examinations were performed, and apoptotic cells were detected. RESULTS: High levels of TRAIL expression were detected in the Ad-TRAIL group, which lasted more than 2 weeks. The mean graft survival time was 17.7, 12.3, 22.0, and 17.4 days for control group, sDR5 group, Ad-TRAIL group, and Ad-GFP group, respectively. The degrees of inflammation in tissue sections taken from all groups after the onset of rejection were similar. There were more apoptotic cells in the graft of the Ad-TRAIL group than in other groups. CONCLUSION: TRAIL appears to play an important role in corneal-allograft rejection and may be used to prolong the survival of allografts.

Cytokine profiles of aqueous humor and graft in orthotopic mouse corneal transplantation.

BACKGROUND: Cytokine profile is a key in understanding the mechanisms of allograft rejection. Cytokine expression in the aqueous humor and the correlation between the aqueous humor cells and corneal infiltrating cells are not fully understood in corneal transplantation. METHODS: Orthotopic mouse corneal transplantation was performed using BALB/c (H2d) mice as recipients, and C3H/He (H2k) and BALB/c mice as donors for allografts and isografts, respectively. Immunocytochemistry was performed on aqueous humor cells. Corneal graft was studied immunohistochemically. Cytokine gene expressions of the cells infiltrating the aqueous humor and corneal grafts were determined by the semiquantitative reverse transcription and polymerase chain reaction method. RESULTS: Interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 were detected in the cells infiltrating the aqueous humor and corneal grafts at both the protein and gene expression levels. T helper 1 (Th1) cytokine expressions at the protein level, however, were consistently predominant in the rejected allografts compared to those of Th2 cytokines. The cytokine and surface marker profiles of the cells in the aqueous humor corresponded well to those of the cells infiltrating the corneal grafts. Cytokine protein and mRNA expression levels in the aqueous humor decreased rapidly. CONCLUSIONS: Allorejection in corneal transplantation is Th1 cytokine-predominant. Infiltrating cells do not express Th2 cytokine so much in allograft rejection, as compared with Th1 cytokine. The cell infiltration patterns of the aqueous humor were well correlated with those of the cornea.

Reciprocal corneal transplantation fails to correct mucopolysaccharidosis VI corneal storage.

This report contains the results of studies designed to evaluate corneal clearing in mucopolysaccharidosis VI (MPS VI)-affected cats. Corneal buttons from affected cats were transplanted into normal cat corneas and, as controls, normal-to-normal and normal-to-affected transplants also were done. No clearing of the MPS VI graft or host beds occurred, nor was there any clouding of the normal donor or recipient corneal tissues. This assessment was made by serial clinical examinations over a 14-30 mo period and by light and electron microscopic examination of the corneal tissues at the end of the study. Lack of corneal clearing under conditions that would maximize such a process in this animal model indicates that corneal clearing is not an appropriate index for measuring the success of systemic therapy in MPS VI.