Human Corneal Transplantation Research Articles

Immunophenotypical Characterization of Limbal Mesenchymal Stromal Cell Subsets during In Vitro Expansion.

Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders.

Alginate Hydrogel Integrated with a Human Fibroblast-Derived Extracellular Matrix Supports Corneal Endothelial Cell Functionality and Suppresses Endothelial-Mesenchymal Transition.

Human corneal transplantation is still the only option to restore the function of corneal endothelial cells (CECs). Therefore, there is an urgent need for hCEC delivery systems to replace the human donor cornea. Here, we propose an alginate hydrogel (AH)-based delivery system, where a human fibroblast-derived, decellularized extracellular matrix (ECM) was physically integrated with AH. This AH securely combined with the ECM (ECM-AH) was approximately 50 μm thick, transparent, and permeable. The surface roughness and surface potential provided ECM-AH with a favorable microenvironment for CEC adhesion and growth in vitro. More importantly, ECM-AH could support the structural (ZO-1) and functional (Na+/K+-ATPase) markers of hCECs, as assessed via western blotting and quantitative polymerase chain reaction, which were comparable with those of a ferritic nitrocarburizing (FNC)-coated substrate (a positive control). The cell density per unit area was also significantly better with ECM-AH than the FNC substrate at day 7. A simulation test of cell engraftment in vitro showed that hCECs were successfully transferred into the decellularized porcine corneal tissue, where they were mostly alive. Furthermore, we found out that the endothelial-mesenchymal transition (EnMT)-inductive factors (Smad2 and vimentin) were largely declined with the hCECs grown on ECM-AH, whereas the EnMT inhibitory factor (Smad7) was significantly elevated. The difference was statistically significant compared to that of the FNC substrate. Moreover, we also observed that TGF-β1-treated hCECs showed faster recovery of cell phenotype on the ECM. Taken together, our study demonstrates that ECM-AH is a very promising material for hCEC culture and delivery, which endows an excellent microenvironment for cell function and phenotype maintenance.

Istiḥsān Method and Its Relevance to Islamic Law Reform: Content Analysis of Fatwa of Majelis Ulama Indonesia on Corneal Transplant

The progress of science in the field of medicine has brought immense advantages to human existence. Nonetheless, these advancements have both positive and negative consequences for humanity. They also give rise to deliberation and disagreement, particularly concerning Islamic jurisprudence, as not all medical technological breakthroughs can be embraced without reservation in society. This study is a descriptive, analytic, and comparative literature review. The ushuliy approach is employed to examine the methodology utilized by the two organizations in determining their stance on human corneal transplants. The primary source material used in this research is the fatwa issued by Majelis Ulama Indonesia in 2009 regarding Corneal Transplants. The data for this research was collected through a documentation method, which involves gathering information from various articles and books. The collected data was then analyzed using content analysis, which involves a descriptive and scientific examination of the key messages. The findings of this study reveal that Majelis Ulama Indonesia holds the view that corneal transplants can be performed on individuals in need, with the intention of Tabarru' (acts of charity), if there are no alternative medical options available, based on the principle of Maslahah (benefit or public interest).

Artificial cornea: Myth or reality?

AbstractSince the first human corneal transplantation was performed by Eduard Zirm over 110 years ago, the paradigm of using human donor corneas for treating blinding corneal disease has remained largely unchanged. Intense research efforts, however, have been undertaken during the past decades, to engineer an alternative to the human cornea to meet the large global demand for this specialized donor tissue. The various artificial, or engineered corneas that have reached preclinical and in several cases clinical testing during the past years will be presented, along with the advantages and limitations of each technology. Various technologies use either cell‐free or cell‐loaded tissues, de‐cellularized or re‐cellularized human or animal tissues, and aim to replace various layers of the cornea or in certain cases, the entire cornea. For the most prevalent indications for corneal transplantation, however, human donor tissue remains the method of choice due to the unparalleled visual outcomes. Nevertheless, newer emerging treatment methods and alternatives to donor cornea transplantation are being reported, that can potentially ease the global demand for donor tissue and tissue banking in the future. Here we present these developments and provide the latest update on the most promising engineered corneal replacements tested in the clinic.

Clinical outcomes in a large multi‐Centre prospective corneal transplant study

AbstractThe output from Work package 2 (WP2) of the VISICORT project included four deliverables: Establishment of secure multi‐site clinical database of human corneal transplant recipients. Report of 1‐year outcomes from multi‐site prospective biological sampling and clinical follow‐up of full thickness (FT)‐ and posterior lamellar (PL)‐CT recipients Report of 3‐year prospective outcomes of FT‐ and PL‐CT recipients Report of clinical factors associated with adverse immunological outcomes from FT‐ and PL‐CT within the overall transplant practice of a network of 5 leading EU centres. In more general terms, the main mission of WP2 was to develop a cloud‐based system to collect relevant, well‐defined clinical information and track biological samples, to recruit patients from the five clinical centers, to collect biological samples and clinical data and to follow these patients clinically over several years.The current speech focus on the clinical outcomes 928 prospectively enrolled patients followed up to 3 years.Beyond confirming known factors associated with an adverse immunological outcome after FT‐ and PL‐CT such as the influence of primary diagnosis and type of surgery as risk factors, the clinical factors study revealed factors which needs to be explored further on. Identification of a re‐bubbling procedure (repeated injection of air to keep a PL graft in place) as an important risk factor for development of a later acute rejection episode is important as re‐bubbling is necessary in a fair large number of CT‐PL procedures (15–25%). The finding that presence of loose sutures after CT‐FT is a significant risk factor for AGR is not surprising, but further confirms that patients undergoing CT‐FT needs to be followed closely.Concerning graft failure, it is worth to note that presence of diabetes 2 was identified as a significant risk factor for graft failure after CT‐FT. This may be due to a partially neurotrophic disease state in many patients with diabetes 2 and this has to be taken into account when deciding to perform CT‐FT and in the clinical follow‐up, which need to be meticulous.The current clinical variables study is unique, and it would seem compelling to continue the clinical follow‐up of the recruited patients beyond the funding period for VISICORT. This would of course require new grant applications and obtaining new ethical approvals from the ethics committees at the five clinical centres.

Quantitative analysis of cornea endothelial cell damage from enucleation, corneal buttoning, and storage in donor corneas using trypan blue dye staining.

We aimed to quantitatively analyze the corneal endothelial cell damage by measuring the area stained with trypan blue dye, and to confirm the degree of corneal endothelial cell damage resulting from enucleation, corneal buttoning, and storage in donor corneas intended for use in human corneal transplantation. This study was a retrospective analysis of medical records and videos recorded during keratoplasty. Twenty-one corneal buttons of 21 donors that underwent endothelial cell staining using trypan blue for the donor preparation during DALK or DMEK were included in the study. The percentage of stained area in entire corneal endothelia and the percentage of the stained area in the 8-mm diameter circle were quantitatively analyzed using Adobe Photoshop. The mean percentage of the stained area in the entire corneal endothelia in 13 corneas was 8.1 ± 13.3% (range, 0.0-56.1%), and the mean percentage of the stained area in a circle with a diameter of 8 mm in 21 corneas was 3.4 ± 5.2% (range, 0.0-18.9%). The correlations between the death-to-preservation time, the training duration of the residents who performed donor corneal buttoning, and the percentage of the stained area in the 8-mm diameter circle were not significant(P = .441, P = .495, respectively). Cornea thickness and endothelial cell density did not differ between 10 eyes in the group with the percentage of the stained area in a circle with a diameter of 8 mm <5% and 5 eyes in the group with the percentage more than 5% damage (P = .854, P = .358). The corneal endothelial cell damage could be quantitatively analyzed using trypan blue staining before keratoplasty in donor cornea. The amount of corneal endothelial cell damage in the central 8-mm circle was mostly acceptable, but some cases showed significantly severe endothelial cell damage. The corneal thickness and endothelial cell density did not differ between 10 eyes in the group with the percentage of the stained area in a circle with a diameter of 8 mm <5% and 5 eyes in the group with the percentage more than 5% damage. Therefore, pachymetry and specular microscopy are not sufficient for evaluating donor corneas before keratoplasty.

Recent Advances in Natural Materials for Corneal Tissue Engineering.

Given the incidence of corneal dysfunctions and diseases worldwide and the limited availability of healthy, human donors, investigators are working to generate engineered cellular and acellular therapeutic approaches as alternatives to corneal transplants from human cadavers. These engineered strategies aim to address existing complications with human corneal transplants, including graft rejection, infection, and complications resulting from surgical methodologies. The main goals of these research endeavors are to (1) determine ideal mechanical properties, (2) devise methodologies to improve the efficacy of engineered corneal grafts and cell-based therapies, and (3) optimize transplantation of engineered tissue structures in the eye. Thus, recent innovations have sought to address these challenges through both in vitro and in vivo studies. This review covers recent work aimed at evaluating engineered materials, potential therapeutic cells, and the resulting cell-material interactions that lead to optimal corneal graft properties. Furthermore, we discuss promising strategies in corneal tissue engineering techniques and in vivo studies in animal models.

Endothelial Safety and Efficacy of Ex Vivo Collagen Cross-linking of Human Corneal Transplants

To assess endothelial safety and efficacy of exvivo corneal collagen cross-linking (CXL) in human corneal transplants stored in 2 different culture media. Fellow-eye controlled laboratory study of exvivo human donor corneas. Three sets of paired human donor corneas, 5 pairs each, were stored in organ culture medium before deswelling either at 31 C or at room temperature. One eye of each pair was cross-linked by 0.1% riboflavin in hydroxylpropyl methylcellulose (HPMC) instillation for 10minutes followed by 10minutes of ultraviolet-A (9 mW/cm2) irradiation while contralateral eyes served as controls. In Set 1, endothelial cell densities were determined. In Set 2, paired samples were assigned to the 2 deswelling media and CXL efficacy was assessed comparing to untreated controls using collagenase-A-assisted enzymatic digestion. In Set 3, biomechanical testing was performed in the eye pairs (treated vs control) by stress/strain measurements. There was no difference in endothelial cell counts between CXL samples and controls (P= .21). No statistically significant difference in digestion dynamics was found between tissues stored in the 2 different culture media. Complete enzymatic digestion was slowed down by 3 hours in the cross-linked samples (P= .036). Stress needed for a 12% strain was increased by 34% in the treatment group compared to control (P= .04). Exvivo CXL of human donor tissue is an effective and safe procedure with no difference regarding efficacy between 2 commercially available deswelling media. Biochemical and biomechanical resistance were significantly increased after CXL. Patients requiring keratoplasty owing to corneal melting might benefit from the strengthening effect of preoperative CXL of donor tissue.

High-risk Corneal Transplantation: Recent Developments and Future Possibilities.

Human corneal transplantation (keratoplasty) is typically considered to have superior short- and long-term outcomes and lower requirement for immunosuppression compared to solid organ transplants because of the inherent immune privilege and tolerogenic mechanisms associated with the anterior segment of the eye. However, in a substantial proportion of corneal transplants, the rates of acute rejection and/or graft failure are comparable to or greater than those of the commonly transplanted solid organs. Critically, while registry data and observational studies have helped to identify factors that are associated with increased risk of corneal transplant failure, the extent to which these risk factors operate through enhancing immune-mediated rejection is less clear. In this overview, we summarize a range of important recent clinical and basic insights related to high-risk corneal transplantation, the factors associated with graft failure, and the immunological basis of corneal allograft rejection. We highlight critical research areas from which continued progress is likely to drive improvements in the long-term survival of high-risk corneal transplants. These include further development and clinical testing of predictive risk scores and assays; greater use of multicenter clinical trials to optimize immunosuppressive therapy in high-risk recipients and robust clinical translation of novel, mechanistically-targeted immunomodulatory and regenerative therapies that are emerging from basic science laboratories. We also emphasize the relative lack of knowledge regarding transplant outcomes for infection-related corneal diseases that are common in the developing world and the potential for greater cross-pollination and synergy between corneal and solid organ transplant research communities.

Lymphatic vessels identified in failed corneal transplants with neovascularisation

BackgroundCorneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human...

Activation of human macrophages by human corneal allogen in vitro.

PurposeTo study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection.MethodsHuman monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively.ResultsCorneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1β, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation.ConclusionsHuman macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.

Da Vinci Xi Robot-Assisted Penetrating Keratoplasty.

PurposeThis study aims (1) to investigate the feasibility of robot-assisted penetrating keratoplasty (PK) using the new Da Vinci Xi Surgical System and (2) to report what we believe to be the first use of this system in experimental eye surgery.MethodsRobot-assisted PK procedures were performed on human corneal transplants using the Da Vinci Xi Surgical System. After an 8-mm corneal trephination, four interrupted sutures and one 10.0 monofilament running suture were made. For each procedure, duration and successful completion of the surgery as well as any unexpected events were assessed. The depth of the corneal sutures was checked postoperatively using spectral-domain optical coherence tomography (SD-OCT).ResultsRobot-assisted PK was successfully performed on 12 corneas. The Da Vinci Xi Surgical System provided the necessary dexterity to perform the different steps of surgery. The mean duration of the procedures was 43.4 ± 8.9 minutes (range: 28.5–61.1 minutes). There were no unexpected intraoperative events. SD-OCT confirmed that the sutures were placed at the appropriate depth.ConclusionsWe confirm the feasibility of robot-assisted PK with the new Da Vinci Surgical System and report the first use of the Xi model in experimental eye surgery. Operative time of robot-assisted PK surgery is now close to that of conventional manual surgery due to both improvement of the optical system and the presence of microsurgical instruments.Translational RelevanceExperimentations will allow the advantages of robot-assisted microsurgery to be identified while underlining the improvements and innovations necessary for clinical use.

Self-assembled collagen-like-peptide implants as alternatives to human donor corneal transplantation

PEG-conjugated collagen-like peptides promote corneal regeneration in a pig cornea.

Boston Keratoprosthesis: Outcomes and Complications: A Report by the American Academy of Ophthalmology

To review the published literature on safety and outcomes of the Boston type I keratoprosthesis (BI-KPro) for the surgical treatment of corneal opacification not amenable to human cadaveric corneal transplantation. Searches of peer-reviewed literature were conducted in PubMed and the Cochrane Library in December 2012, July 2013, and January 2014 without date restrictions. The searches were limited to studies published in English and yielded 587 citations. The abstracts of these articles were reviewed, 48 articles were selected for possible clinical relevance, and 22 were determined to be relevant for the assessment objectives. Nine studies were rated as level II evidence and 13 studies were rated as level III evidence. Excluded were level III evidence, case reports, review articles, letters, editorials, and case series with fewer than 25 eyes. In 9 articles, a best-corrected Snellen visual acuity (BCSVA) of 20/200 or better occurred in 45% to 89% of eyes. Five articles described a BCSVA of 20/50 or better in 43% to 69% of eyes, and 4 articles found a BCSVA of 20/40 or better in 11% to 39% of eyes. Retention rates of the BI-KPro ranged from 65% to 100%. Reasons for loss of vision after BI-KPro implantation most commonly included corneal melts resulting from exposure keratopathy, endophthalmitis, and infectious keratitis or corneal ulceration. The 2 most common complications after surgery were retroprosthetic membrane formation (range, 1.0%-65.0%; mean ± standard deviation [SD], 30.0±19.0%) and elevated intraocular pressure (range, 2.4%-64.0%; mean ± SD, 27.5±18.1%). The 2 most common posterior segment complications were endophthalmitis (range, 0%-12.5%; mean ± SD, 4.6±4.6%) and vitritis (range, 0%-14.5%; mean ± SD, 5.6±4.7%). The reviewed articles on BI-KPro use suggest that the device improves vision in cases of severe corneal opacification that were not amenable to corneal transplantation using human cadaveric keratoplasty techniques. A number of severe anterior and posterior segment complications can develop as follow-up continues, making ongoing close observation paramount for patients undergoing this surgery. These complications include infection, device extrusion, and permanent vision loss.

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.

A brief history of corneal transplantation: From ancient to modern.

This review highlights many of the fundamental concepts and events in the development of corneal transplantation – from ancient times to modern. Tales of eye, limb, and even heart transplantation appear in ancient and medieval texts; however, in the scientific sense, the original concepts of corneal surgery date back to the Greek physician Galen (130-200 AD). Although proposals to provide improved corneal clarity by surgical interventions, including keratoprostheses, were better developed by the 17th and 18th centuries, true scientific and surgical experimentation in this field did not begin until the 19th century. Indeed, the success of contemporary corneal transplantation is largely the result of a culmination of pivotal ideas, experimentation, and perseverance by inspired individuals over the last 200 years. Franz Reisinger initiated experimental animal corneal transplantation in 1818, coining the term “keratoplasty”. Subsequently, Wilhelmus Thorne created the term corneal transplant and 3 years later Samuel Bigger, 1837, reported successful corneal transplantation in a gazelle. The first recorded therapeutic corneal xenograft on a human was reported shortly thereafter in 1838—unsurprisingly this was unsuccessful. Further progress in corneal transplantation was significantly hindered by limited understanding of antiseptic principles, anesthesiology, surgical technique, and immunology. There ensued an extremely prolonged period of debate and experimentation upon the utility of animal compared to human tissue, and lamellar versus penetrating keratoplasty. Indeed, the first successful human corneal transplant was not performed by Eduard Zirm until 1905. Since that first successful corneal transplant, innumerable ophthalmologists have contributed to the development and refinement of corneal transplantation aided by the development of surgical microscopes, refined suture materials, the development of eye banks, and the introduction of corticosteroids. Recent developments, including the replacement of selected corneal layers rather than full-thickness keratoplasty, have the potential to improve or transform corneal transplant surgery in the future.

Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model

To evaluate the potential use of decellularized porcine stromal matrix (PSM) for reconstruction of corneal stroma in a rabbit model. Ten chinchilla bastard rabbit corneas were exposed to a circular half-thickness keratotomy with a 3.0 mm diameter at the central cornea. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 K IU/ml) and 0.3% sodium dodecyl sulphate (SDS). The 3.0 mm in diameter decellularized corneal stromal xenograft was inserted into the pocket, and the incision was closed with four 10.0 nylon sutures. Clinical photographs were taken at day 1, day 7, day 30 and on a monthly basis for up to 6 months after transplantation. Six months after surgery, the rabbits were killed and eyes were enucleated. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm the complete removal of the corneal cells after decellularization of porcine corneas and repopulation with rabbit cells. Alcian blue staining was performed for analysing the structure of the extracellular matrix (ECM). Efficient elimination of porcine cells was achieved by our decellularization protocol and confirmed via HE and DAPI stainings. Moreover, the major histoarchitectural ECM structure had been maintained as visualized by the alcian blue stain. Finally, the PSM was biocompatible with the host's epithelium evidenced as a regrowth covering the exposed xenograft. This novel technique of tissue engineering may provide one of many solutions to addressing anterior corneal pathological conditions in the face of a shortage of human corneal material.

Experience With Preventive Genetic Testing of Corneal Donors in Slovakia

The purpose of this study was (1) to detect asymptomatic carriers of the prion protein gene mutation E200K, which is associated with Creutzfeldt-Jakob disease (CJD), in corneal donors and in the general population of Slovakia and (2) to assess the genetic testing of corneal donors as an effective preventive measure against iatrogenic infection in a country with an unusually high incidence of genetic CJD. The prion protein gene (PRNP) was analyzed in 1133 corneal donors and 970 control subjects to search for E200K mutation and to determine the genotype at codon 129. Mutation E200K was found in 2 of the 1133 donors and in 4 of the 970 control subjects. The most frequent genotype at the codon 129 polymorphic region was methionine homozygous (48% of donors and controls). An E200K mutation, which confers a risk of developing genetic CJD, was detected in corneal donors and in the general population. The majority of subjects were codon 129 methionine homozygous that increases susceptibility to CJD. Genetic testing of corneal donors in Slovakia is a useful and effective preventive measure against iatrogenic CJD through human corneal transplantation in the investigated population.

Survival of Donor-Derived Cells in Human Corneal Transplants

To determine the fate of donor epithelial, stromal, and endothelial cells after corneal transplantation in humans. Fifty-two transplanted corneal buttons were explanted over a 2-year period from patients who required regrafting and had received corneas from donors of opposite sex. Fluorescence in situ hybridization of the sex chromosomes of the epithelial, stromal, and endothelial cells was performed in histologic sections prepared from each freshly explanted graft. Fluorescence microscopy was subsequently used to determine the origin of cells in the graft (donor or recipient) and to quantify the relative proportion of donor and recipient cells of each corneal cell type. As early as 3 months after transplantation, donor epithelial cells were completely replaced by recipient epithelium in all corneal buttons examined. Donor stromal and endothelial cells, however, were found in all 52 buttons, with 4% to 95% of stromal cells and 6% to 95% of endothelial cells being of donor origin. No significant correlation between donor cell proportion and the age of the graft could be found. Donor-derived cells were found in significant numbers up to 32 years after transplantation. Eight corneas in this study were transparent, compensated grafts, and a similar long-term survival of donor stromal and endothelial cells was found in these cases. Although donor epithelial cells are promptly replaced, a high proportion of donor stromal and endothelial cells can survive within the corneal transplant in the long-term. The proportion of surviving donor cells is highly variable; however, the source of this variability remains unknown.

Increased Rac2 mRNA expression in peripheral blood during human corneal graft rejection

Allograft rejection is the main cause of graft failure in human corneal transplantation, for which underlying pathomechanism is not yet clear. We compared gene expression in the peripheral blood of patients who after undergoing corneal transplantation experienced graft rejection with those patients who accepted grafts. Sixty-six patients who underwent corneal transplantation were studied including 18 patients who suffered subsequent graft rejection. cDNA array technology was used to survey and quantify transcript expression. A semiquantitative reverse transcriptase-PCR (RT-PCR) was used to confirm the gene expression pattern measured by a cDNA array of selected genes. Among 265 genes present on the array, eight genes were found to be differentially expressed. Four genes (Rac 2, RhoA, paxillin, and CD18) were further analysed by semiquantitative RT-PCR, and significant differences in mRNA expression levels in the rejection group were confirmed. Our study demonstrated that the expression of Rac2 mRNA was upregulated in the peripheral blood of patients experiencing corneal transplantation rejection compared to those patients who had no rejection episodes. In addition, three genes, RhoA, paxillin, and CD18, showed decreased expression in rejecting patients. cDNA array technology provides a potentially useful approach to identify novel genes that might participate in pathogenic pathways during corneal graft rejection.