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Purpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK). Materials and Methods: pCECs were isolated, cultured up to P2 and seeded onto LinkCell (TM) bioengineered matrices of 20 mu m (LK20) or 100 mu m (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM). Results: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 +/- 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a "taco" for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. Conclusions: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications. Funding Agencies|European Unions Horizon 2020 research and innovation programme [667400]

Corneal transplantation is currently the most effective treatment to restore corneal clarity in patients with endothelial disorders. Endothelial transplantation, either by Descemet membrane endothelial keratoplasty (DMEK) or by Descemet stripping (automated) endothelial keratoplasty (DS(A)EK), is a surgical approach that replaces diseased Descemet membrane and endothelium with tissue from a healthy donor eye. Its application, however, is limited by the availability of healthy donor tissue. To increase the pool of endothelial grafts, research has focused on developing new treatment options as alternatives to conventional corneal transplantation. These treatment options can be considered as either 'surgery-based', that is tissue-efficient modifications of the current techniques (e.g. Descemet stripping only (DSO)/Descemetorhexis without endothelial keratoplasty (DWEK) and Quarter-DMEK), or 'cell-based' approaches, which rely on in vitro expansion of human corneal endothelial cells (hCEC) (i.e. cultured corneal endothelial cell sheet transplantation and cell injection). In this review, we will focus on the most recent developments in the field of the 'cell-based' approaches. Starting with the description of aspects involved in the isolation of hCEC from donor tissue, we then describe the different natural and bioengineered carriers currently used in endothelial cell sheet transplantation, and finally, we discuss the current 'state of the art' in novel therapeutic approaches such as endothelial cell injection.

Purpose To assess whether combined analysis of specular microscopy and Scheimpflug imaging improves detection of an upcoming allograft rejection following Descemet membrane endothelial keratoplasty (DMEK). Methods Retrospective analysis of 22 eyes that had developed a clinical proven allograft rejection 28 (±22) months (range: 4-84 months) after DMEK. Specular microscopy and Scheimpflug images routinely made after DMEK were retrospectively analysed for changes in endothelial cell morphology (e.g. nuclear activation), cell density (>10%) and pachymetry (>7%), and/or the presence of subclinical keratic precipitates. The same parameters were evaluated for 22 control eyes matched for age, gender and surgery indication. Results A total of 20/22 eyes (91%) showed detectable changes 0.25-75 months before allograft rejection became clinically manifest: 13/22 (59%) showed both specular microscopy and Scheimpflug imaging changes; 5/22 (23%) only had changes on Scheimpflug imaging; and 2/22 (9%) only had specular microscopy changes. In 18/22 (82%) and 14/22 (64%) eyes, subclinical keratic precipitates and endothelial cell morphology changes could be detected, respectively. A total of 11/22 (50%) eyes concurrently showed a >10% drop in endothelial cell density and 4/22 (18%) a >7% pachymetry increase. Of the control eyes, 7/22 (32%) showed changes with specular microscopy but not with Scheimpflug imaging. Conclusions Combined analysis of specular microscopy and Scheimpflug imaging may allow recognizing an upcoming allograft rejection in over 90% of eyes and up to 6 years before rejection becomes clinically manifest. Early recognition of eyes at risk may allow for targeted intensified steroid treatment to prevent endothelial cell damage associated with rejection.

Purpose: To identify the origin of corneal endothelial cells (host or donor) present on grafts at various time points after Descemet membrane endothelial keratoplasty (DMEK), using fluorescence in situ hybridization (FISH) of sex chromosomes on post mortem corneas with sex mismatch between the donor and host. Methods: Corneoscleral buttons of 6 post mortem DMEK eyes of 4 patients, operated for Fuchs endothelial dystrophy, with an average postoperative time of 2.6 (±1.8) years (range, 7 months-4.5 years), of 2.5 (±1.7) years (range, 7 months-4 years), were processed for FISH detection of XX (female) or XY (male)-labeling signals in corneal endothelial cells in the central area of the DMEK graft. Two male patients underwent bilateral DMEK with grafts from female donors, and 2 female patients underwent unilateral DMEK and received a graft from a male donor. Results: FISH consistently showed the presence of donor endothelial cells across the graft area, with signaling of sex chromosomes opposite to the sex of the host. Conclusions: Donor endothelial cells may survive up to 4.5 years after DMEK. If so, the lower incidence of allograft rejection in DMEK may not be explained by early host cell replacement. Potential host cell migration may be limited by donor/recipient cell-cell contact inhibition.

To describe the clinical outcome and histopathology of Descemet membrane endothelial keratoplasty (DMEK) performed for secondary graft failure after penetrating keratoplasty (PK).A total of 11 eyes from 10 patients who underwent DMEK for secondary PK graft failure at a tertiary referral center were included in this retrospective study. Best-corrected visual acuity, endothelial cell density, and central pachymetry were evaluated before and at regular time intervals up to 36 months after DMEK and complications were recorded; 1 post mortem cornea was available for light microscopy.At their last follow-up visit (on average, 16 months after DMEK), 7 of 11 transplanted corneas were clear. In the 7 eyes with clear grafts, 5 had a best-corrected visual acuity of ≥20/25 (≥0.8), central pachymetry averaged 535 (±70) μm, and endothelial cell density averaged 1045 (±500) cells/mm. Of the 11 eyes, 4 required rebubbling in the early postoperative phase; 1 eye was left with a small (1/3) detachment. Light microscopy of the pathology specimen showed complete attachment of the DMEK graft onto the preexisting PK posterior stroma, with interface scarring over DMEK graft folds and underneath the graft area that had initially been detached.DMEK may be a viable option to manage secondary PK graft failure with acceptable outcomes in many cases. Rebubbling for graft detachment may be anticipated, especially because of preexisting glaucoma conditions (severe decompensation, hypotony, and tubes from glaucoma-draining devices). Graft reattachment may occur through interface scarring.

Purpose To describe the use of 360-degree Scheimpflug imaging as a diagnostic tool for detection and documentation of subtle corneal changes preceding upcoming allograft rejection after Descemet membrane endothelial keratoplasty (DMEK). Methods A total of 17 eyes (16 patients) were diagnosed with clinically manifest allograft rejection 2 to 42 months after DMEK. 360-degree Scheimpflug images of consecutive follow-up examinations (from 3-60 mo) of "asymptomatic" eyes before, during, and after rejection were retrospectively analyzed, to determine which abnormalities could be detected before allograft rejection became clinically manifest. The images were compared with DMEK control eyes (without rejection episode). Results Scheimpflug images at the time of rejection showed keratic precipitates as distinct retrocorneal nodular elevations and/or a significant increase in pachymetry of ≥7%. More subtle changes could be identified retrospectively in 9/17 eyes (53%) on an average at 8 (±5) months before rejection became clinically manifest; in all eyes, these subtle changes were not recognized at routine slit-lamp examinations by various ophthalmologists as inflammatory changes heralding allograft rejection. Secondary graft failure occurred in 4/17 eyes (24%). None of the control eyes showed relevant abnormalities with Scheimpflug imaging. Conclusions By screening the posterior corneal surface with 360-degree Scheimpflug imaging, subtle inflammatory retrocorneal deposits can be detected and recorded during consecutive follow-up visits. Hence, Scheimpflug imaging may have the potential to become a diagnostic tool for early detection of upcoming allograft rejection in asymptomatic DMEK eyes, that is, before the immune response becomes clinically manifest and before substantial endothelial cell damage occurs.

Purpose To investigate in vitro central and peripheral corneal endothelial cell (EC) migration from Quarter–Descemet membrane endothelial keratoplasty (Quarter‐DMEK) grafts. Methods Quarter‐DMEK grafts were obtained from 10 corneas ineligible for transplantation but with intact and viable ECs. Ten Quarter‐DMEK grafts were ‘sandwiched’ between two glass slides and cultured over 1 week in a humidified atmosphere at 37 °C and 5% CO2. Cell migration was evaluated by light microscopy at standardized time intervals. In addition, immunohistochemistry analyses were performed to assess the detailed structural organization of ECs in the corneal centre and far periphery. Results Endothelial cell (EC) migration occurred from the radial cut graft edges, but not from the far peripheral area. Cell migration followed three different migration patterns: (1) individual cell migration, (2) uncoordinated cell migration of cell clusters and (3) collective migration in which ECs moved as a sheet. Immunostaining showed the presence of ECs up to the far periphery but with different expression patterns of phenotypical markers ZO‐1, Na+/K+ ‐ATPase and vimentin compared to central ECs. Conclusion In vitro EC migration from Quarter‐DMEK grafts occurs along the radial cut edges with a decrease in migration activity towards the corneal far periphery. No migration occurred along the outer peripheral corneal edge possibly due to a different anatomical matrix in the far periphery. Hence, ECs from the far periphery may not contribute to corneal clearance of the adjacent bare area after Quarter‐DMEK surgery, but these cells may constitute a valuable cellular reserve on the graft.

PURPOSE To evaluate endothelial cell density (ECD) in the first 6 months after Descemet membrane endothelial keratoplasty (DMEK) by eliminating method error as a confounding variable. METHODS From 24 DMEK eyes operated for Fuchs endothelial corneal dystrophy, from which specular microscopy images could be taken at 1 day and 6 months postoperatively, ECD values were compared between these 2 time points. RESULTS Using the 1-day ECD measurement as baseline, mean ECD decreased from 1913 (±326) cells/mm to 1524 (±393) cells/mm at 6 months, a decline of -18 (±19)%. With the 1-week ECD as baseline [1658 (±395) cells/mm], the decline at 6 months was -6 (±19)% and when using preoperative ECD as baseline [2521 (±122) cells/mm], the decline was -39 (±16)% at 6 months. CONCLUSIONS After DMEK, ECD shows an in vivo decline of 18% from 1 day to 6 months postoperatively, with a sharp 13% drop in the first week, and a slower decrease thereafter. The remaining difference of 20% from preoperative ECD values may be attributed to a measurement error in the eye bank with an overestimation of the graft's viable endothelial cell population and/or intraoperative trauma to the graft.

Fuchs endothelial corneal dystrophy is a common disorder characterized by the progressive thickening of Descemet membrane (DM), manifesting as guttae and leading to a decrease in endothelial cells and corneal clearance. Numerous studies have tried to better characterize the genetics of Fuchs endothelial corneal dystrophy, and suggest that it is a complex heterogenic disorder with an array of variants in severity and disease progression. Currently the most effective treatment for replacing diseased endothelium is endothelial keratoplasty (EK). In the last decade, EK has evolved into selective transplantation of an isolated DM and its endothelium, referred to as Descemet membrane endothelial keratoplasty (DMEK), which enables near normal anatomical and visual outcomes after surgery. Unexpected observations after DMEK, however, may bring new insights on endothelial cell wound healing, potentially allowing novel ‘non-keratoplasty’ surgical approaches like Descemet membrane endothelial transfer, endothelial ce...

Anterior donor grafts (including scleral rim, without Descemet membrane) increase in thickness and become hazy upon storage in organ culture (OC) medium. Transfer of these grafts to standard dehydration media just before transplantation does not reduce their thickness to normal. Therefore, we assessed the efficacy of different media enriched with polyethylene glycol (PEG) as dehydrating agents for organ-cultured anterior donor grafts. Grafts were harvested and stored in the commercial OC medium ‘Max’ (without dextran) for 1 week, and subsequently dehydrated in the standard commercial dehydration medium ‘Jet’ (with dextran) supplemented with 4–20 % PEG3350, or ‘Max’ supplemented with 20 % PEG6000 and PEG20.000, or 5–20 % PEG35.000. Central corneal thickness (CCT), as assessed by anterior segment-optical coherence tomography, and transparency were evaluated before, and at 1, 4 and 7 days of dehydration. Transfer of grafts after 1 week of OC (average 1,200 µm) to ‘Jet’ supplemented with PEG3350 revealed a concentration-dependent effect of dehydration; CCT was restored to normal (500–600 µm) when 10 % PEG3350 was added. However, transparency was only temporarily restored; after 1 day, the grafts turned hazy. In contrast, grafts transferred to ‘Max’ supplemented with 20 % PEG35.000 were transparent throughout the evaluation period, but were dehydrated to beyond normal levels (average 300 µm). ‘Max’ supplemented with 5 % PEG35.000 dehydrated grafts to normal values and restored transparency throughout. Thus, dehydration of anterior donor grafts prior to surgery in dextran-free OC medium supplemented with 5 % PEG35.000 reduces graft thickness to normal and may facilitate anterior keratoplasty procedures.

To report the failure rate of 2 graft preparation techniques for Descemet membrane endothelial keratoplasty (DMEK) and to evaluate how to minimize graft preparation failure.Retrospective, nonrandomized study at an eye bank specialized in graft preparation for lamellar keratoplasty. For 1416 donor corneas, the DMEK graft preparation failure rate was evaluated for 2 different techniques, technique I: "Standardized traditional technique" (n = 341) and technique II: "Standardized no-touch technique" (n = 933), and for grafts that were converted from technique II to technique I during preparation (n = 142).The overall failure rate averaged 3.9% (55/1416): 7.0% (24/341) for technique I and 2.9% (31/1075) for technique II (P0.05). Tissue preparations which were converted from technique II to technique I failed in 13.4% (19/142), whereas for grafts that were entirely prepared by technique II, the failure rate was only 1.3% (12/933). The endothelial cell density decrease (before compared with after preparation) did not differ for both techniques (1.1% vs. 0.2%, P0.05).Various DMEK graft preparation techniques may provide failure rates of4%. A "no-touch preparation" approach (technique II) may combine good graft quality (completely intact endothelial cell layer, ie, negligible preparation-induced endothelial cell density decrease) with low risk of dissection failure, leaving the possibility of conversion to "traditional preparation" (technique I) as a backup method.

Purpose: The aim of this study was to describe the ultrastructure of the host-donor interface in the eye of a recently deceased patient, who had undergone Descemet membrane endothelial keratoplasty. Methods: The eye was enucleated postmortem, and after standard decontamination, the corneoscleral button was excised, cut into 4 quadrants, and processed for light and transmission electron microscopy evaluation. Results: Transmission electron microscopy revealed close attachment of the donor's Descemet membrane to the host's stroma and projection of stromal collagen fibers into the interfacial matrix, resembling a normal "virgin" corneal architecture. Conclusions: Ultrastructurally, an attached Descemet membrane endothelial keratoplasty graft closely resembles that of an unoperated, healthy eye with no appreciable adventitious or missing structures.

As epiretinal membranes (ERMs), the internal limiting membrane (ILM) and the vitreous cortex are essentially transparent tissues, or translucent structures, nontraumatic removal may be challenging in various types of macular surgery. Vital dyes stain these thin tissues, thus allowing for better visualization of these structures during vitrectomy and selective ‘membrane peeling' from the underlying retina. To avoid swirling of the dye within the fluid-filled vitreous cavity, and to better target the dye onto the macula, a fluid-air exchange is commonly performed. However, this may jeopardize visualization of the macula during peeling due to clouding of the posterior lens capsule, and may lead to postoperative visual field defects. Recently, a new dye solution for staining the ERM and ILM simultaneously has been developed that circumvents the need for fluid-air exchange, i.e. MembraneBlue-Dual™. This paper will focus on the hydrodynamics and biocompatibility of this ‘heavy' dual dye and its efficacy for staining of the ILM and/or ERMs during posterior segment surgery in a multicenter clinical setting.

PURPOSE To describe the presence of "salt and pepper endothelium", that is, typical cellular inclusion bodies in a patient with Fuchs endothelial corneal dystrophy (FECD), that recurred in the donor corneal endothelial cells after Descemet membrane endothelial keratoplasty (DMEK). METHODS A 76-year-old man underwent DMEK for FECD in his left eye. Routine specular microscopy imaging, best-corrected visual acuity measurements, and pachymetry measurements were performed before and after surgery. RESULTS Besides large guttae indicating FECD, preoperative specular microscopy images showed variable-sized dark cellular inclusion bodies in the endothelial cells. One month after DMEK, donor endothelial cells appeared normal; however, at 3 months, the typical inclusion bodies reappeared and progressed slowly within a 4-year follow-up period. Both best-corrected visual acuity and pachymetry were stable throughout the study period. CONCLUSIONS "Salt and pepper endothelium" recurred after the host tissue was exchanged by donor Descemet membrane, that is, a DMEK graft. These changes may indicate that either the donor corneal endothelial cell morphology is modified by adjacent tissue structures or that it is completely replaced by recipient endothelium within the first months after surgery.

Based on recent clinical observations made after DMEK, where the cornea cleared even when an endothelial graft was almost completely detached [1], a new surgical concept was presented at the Netherlands Institute for Innovative Ocular Surgery in 2012: Descemet membrane endothelial transfer (DMET) [2]. Going forward from DMEK, DMET consists of the implantation of a Descemet roll that is mostly “free floating” in the anterior chamber being attached only to the corneal incision [2]. In this chapter, we will discuss the observations that led into the development of DMET and the possible mechanism(s) behind (spontaneous) corneal clearance.

Purpose: To describe a standardized ‘no-touch’ harvesting technique of anterior and Descemet membrane (DM) grafts for use in deep anterior lamellar keratoplasty (DALK) and Descemet membrane endothelial keratoplasty (DMEK), which provides undamaged anterior and posterior corneal grafts. Methods: A retrospective evaluation was performed of our standard method for harvesting DM grafts and DALK grafts (Technique I; n = 31) versus a newly designed ‘no-touch’ technique (Technique II; n = 31), in which a peripheral ring of trabecular meshwork tissue is left in-situ, and the DM graft is trephined on an underlying soft contact lens. Endothelial cell density (ECD) before and immediately after DM stripping was used as the main outcome parameter. Results: Endothelial cell density did not differ within Techniques I and II (before versus after DM stripping) (p = 0.75 and p = 0.54, respectively) or among Techniques I and II (p = 0.61). With the latter technique, anterior corneal grafts and posterior DM grafts could be harvested with negligible damage to the endothelial cell layer or the posterior stromal bed. All 93 grafts (62 DM grafts) were eligible for transplantation, and six months post-operatively all transplants used were functional. Conclusion: The new technique offers the following advantages: (i) production of ‘undamaged’ grafts for DALK and DMEK, (ii) better controlled tissue handling of the thin DM graft during DM stripping and (iii) an increase in availability of corneal grafts obtained from the same donor tissue pool.

The aim of this study was to report the efficacy of adding chlorhexidine to the protocol for decontamination of human donor globes prior to excision of corneo-scleral rims for future keratoplasty procedures. In 2005, chlorhexidine was introduced by our eye bank as an additional step in the protocol for decontaminating human donor globes. After 5 years, we prospectively evaluated the number of contaminations. Out of 2,891 globes included in our study, 2,663 globes were processed, of which 36 (1.4%) were considered contaminated. Seventeen contaminations (0.6%) were detected by culturing limbal swabs, directly after decontamination, eight (0.3%) by visible discoloration of the culture medium carrying a corneo-scleral rim, and eleven (0.4%) after inoculation of the culture medium on blood agar plates. Importantly, after 4 weeks of incubation, none of the aerobic and anaerobic cultures taken from the secondary 'transport medium' (dextran containing medium used to transport corneal tissue to the transplantation centre) showed microbiological growth. In conclusion, the combined use of 0.02% chlorhexidine and 0.5% povidone-iodine may allow decontamination of donor globes to a level at which the risk of tissue contamination at the time of transplantation is minimized, while corneal viability is preserved.

Objective The adoptive transfer of regulatory T cells (Treg) in murine models has been shown to ameliorate graft-versus-host disease while it may preserve the graft-versus-leukemia effect. However, the impact of Treg on infectious immunity after bone marrow transplantation (BMT) is still unclear. Immunocompetence against opportunistic viral infections depends on the kinetics of T cell recovery after BMT through two distinctive processes, i.e. lymphopenia-induced proliferation (LIP) of mature T cells and generation of T cells through thymopoiesis. Methods In this study, we set out to assess the effects of adoptively transferred Treg on T cell regeneration in a homeostatic peripheral T cell expansion model and a thymopoiesis-dependent BMT model, and on murine cytomegalovirus (mCMV) clearance and mortality following mCMV challenge. Results Using lymphopenic Rag-2 −/− γc −/− mice that received a limited number of congenic T cells, we demonstrate that adoptively transferred Treg abrogate LIP of T cells. mCMV challenge resulted in a rapid increase of viral load and death in mice that received Treg, but not in controls. In contrast, following syngeneic T cell-depleted BMT in Rag-2 −/− γc −/− mice, adoptively transferred Treg did not delay T cell reconstitution nor suppressed thymic output and had no effect on viral clearance and survival following mCMV-challenge. Conclusion The effect of Treg on T cell-mediated immunocompetence against mCMV early after BMT depends on the relative contribution of peripheral expansion and thymopoiesis to T cell regeneration.

Estradiol modulates stress reactions in female rats. Several studies showed anxiolytic effects of estradiol in behavioral tests, but the underlying mechanisms are still unclear. The aim of the current study was to explore how estradiol-treated rats respond to acute and chronic stress compared to ovariectomized rats. Ovariectomized rats received vehicle or 17 beta-estradiol injections (10 mu g/250 g) once every 4 days, which induced alternating high and low plasma 17 beta-estradiol levels. Stress was presented by daily exposure to an adverse environment in which the animals received five footshocks for either 3 or 22 days. Under control conditions no differences were observed, but as soon as stress was applied, reactions of ovariectomized and estradiol-treated rats diverged. Both acute and chronic stress increased the c-Fos protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Cyclic estradiol treatment reduced this stress-induced activation of the PVN, an effect that seems to be dependent on the plasma estradiol levels. No differences in stress-induced corticosterone responses were revealed between the treatment groups. An increase in the number of ER beta-expressing cells in the PVN of ovariectomized and estradiol-treated rats during chronic stress implied increased ER beta-mediated mechanisms during these conditions. The dampening effect of estradiol on the excessive stress-induced activity in the PVN may be beneficial for the animal in its response to chronic recurrent stress by reducing the output of the PVN. (c) 2005 Elsevier Inc. All rights reserved.

Purpose: To evaluate the feasibility of two novel ‘heavy’ dye solutions for staining the internal limiting membrane (ILM) and epiretinal membranes (ERMs), without the need for a prior fluid-air exchange, during macular surgery. Methods: In this prospective nonrandomized multicenter cohort study, the high molecular weight dyes ILM-Blue™ [0.025% brilliant blue G, 4% polyethylene glycol (PEG)] and MembraneBlue-Dual™ (0.15% trypan blue, 0.025% brilliant blue G, 4% PEG) were randomly used in vitrectomy surgeries for macular disease in 127 eyes of 127 patients. Dye enhanced membrane visualization of the ILM and ERMs, ‘ease of membrane peeling’, visually detectable perioperative retinal damage, postoperative best-corrected visual acuity (BCVA), dye remnants and other unexpected clinical events were documented by 21 surgeons. Results: All surgeries were uneventful, and a clear bluish staining, facilitating the identification, delineation and removal of the ILM and ERMs, was reported in all but five cases. None of the surgeries required a fluid-air exchange to assist the dye application. BCVA at 1 month after surgery improved in 83% of the eyes in the MembraneBlue-Dual™ group and in 88% in the ILM-Blue™ group. No dye remnants were detected by ophthalmoscopy, and no retinal adverse effects related to the surgery or use of the dyes were observed. Conclusion: The ‘heavy’ dye solutions ILM-Blue™ and MembraneBlue-Dual™ can be injected into a fluid-filled vitreous cavity and may facilitate staining and removal of the ILM and/or ERMs in macular surgery without an additional fluid-air exchange.

Purpose To report early, specific changes in donor endothelial cell morphology as a predictor of an upcoming allograft rejection after Descemet membrane endothelial keratoplasty (DMEK). Design Retrospective, observational case series. Methods Out of a cohort of 500 eyes that underwent DMEK at a tertiary referral center, 7 eyes developed typical clinical signs of an allograft rejection. Specular microscopy images before, during, and after the rejection episode were analyzed and compared with a case-control group of 49 asymptomatic DMEK eyes that matched baseline characteristics of the rejection group. Endothelial cell morphology was evaluated by subjective scoring (range 1–5) in a masked fashion as well as by an objective comparison of endothelial cell density, cell size, coefficient of variation, and hexagonality in rejection vs control eyes. Results Subjective scores (median) were higher before and after rejection (2.5 and 5, respectively) than in the DMEK control group (2.0 and 2.5, respectively) at comparable time points ( P = .0230 and P = .0005, respectively). Endothelial cell density also differed before ( P = .0106) and after rejection ( P = .0240), while hexagonality differed before ( P = .0499) but not after rejection ( P = .1767). Conclusion Our study suggests that allograft rejection may not be an acute event, but rather a slow-onset immune response. Early, specific changes in endothelial cell morphology were found to "announce" an upcoming allograft rejection. If so, monitoring donor endothelium after DMEK or other forms of keratoplasty may be used to anticipate a rejection episode and/or to prevent an allograft rejection from clinically manifesting itself.

Importance After retrospectively evaluating the clinical outcome of 500 consecutive cases after Descemet membrane endothelial keratoplasty (DMEK), we extended the analysis in this study by assessing the effect of donor-related parameters on endothelial cell density (ECD) decline and detachment rate in this group. Observations This retrospective case series included 500 cases who had undergone DMEK from October 2007 to September 2012 at the Netherlands Institute for Innovative Ocular Surgery (NIIOS), Rotterdam, the Netherlands. Logistic regression analysis (n = 332 eyes) showed that donor age might be associated with a 3% increase in the risk for a detachment (odds ratio, 0.97; 95% CI, 0.94-1.00; P = .049) (ie, higher donor age seems to be associated with lower chances of a detachment). In addition, linear regression analysis indicated that graft storage time in medium was associated with ECD decrease (ie, the longer the storage time, the larger the decrease at 6 months after DMEK) ( P = .01). Conclusions and Relevance We showed an association between graft storage time and ECD decline after DMEK and possibly between donor age and graft detachment. Therefore, donor storage times should be kept as short as possible to improve short-term ECDs. More research is needed to draw definite conclusions on the possible effect of donor age on the chance of a detachment after DMEK.

Purpose To study the validity of endothelial polymegethism, pleomorphism, and "poor swelling" as tissue discard parameters in the immediate postmortem evaluation of human donor corneal endothelium. Methods We retrospectively evaluated the quality of the endothelium at first and second evaluations for all processed corneas exhibiting moderate polymegethism, pleomorphism, or "poor swelling" in our eye bank over a 5-year period. Results Out of 2008 eyes qualifying for our study, 422 corneas (21%) showed polymegethism, pleomorphism, or poor swelling at the first tissue evaluation immediately after excision of the corneoscleral button. In 363 (86%) of these corneas, a normal endothelial mosaic was observed at the second tissue evaluation after 7 to 21 days of organ culture, whereas only 59 (14%) still showed persistent polymegethism, pleomorphism, or "poor swelling" at that time point. Conclusions A recovery of normal endothelial cell mosaic and "normal swelling" at the second evaluation suggests that cellular contour parameters do not relate to tissue viability, but rather to a cellular stress reaction. If so, the validity of endothelial cellular contour morphology as an early parameter in assessing the suitability of a donor cornea for transplantation may be reconsidered.

Keratinocyte growth factor (KGF) protects mice from acute graft-vs-host disease and graft rejection by cytoprotective and yet incompletely understood immunological mechanisms. Recently, we showed that administration of KGF induces selective peripheral expansion of CD4(+)Foxp3(+) regulatory T cells (Treg). In this study, we set out to assess whether the peripheral expansion of Treg accounts for the immunomodulatory effects of KGF after bone marrow (BM) transplantation. To exclude potentially confounding cytoprotective and thymopoietic effects of KGF, we applied KGF to congenic wild-type mice that served as T cell provider mice for T and B cell-deficient RAG-1(-/-) mice that were subsequently transplanted with allogeneic BM. Treatment of congenic T cell provider mice with KGF significantly improved engraftment and reduced graft rejection in BMT recipients. CD4(+)Foxp3(+) Treg remained increased for 4 wk, while expansion of congenic CD3(+) T cells was inhibited. To assess a causal relationship between expansion of Treg and improved BM engraftment, congenic Scurfy mice, which lack Foxp3(+) Treg, served as T cell provider mice and were treated with KGF. KGF-treatment of Scurfy mice did not affect engraftment nor did it inhibit the expansion of congenic T cells. These data demonstrate that administration of KGF to the T cell provider mice improves engraftment of allogeneic BM through a CD4(+)Foxp3(+) Treg-dependent mechanism. The Journal of Immunology, 2009, 182: 7364-7369.

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4+Foxp3+ Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4+Foxp3+ Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4+Foxp3- T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4+ T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4+Foxp3+ Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.

BACKGROUND AND OBJECTIVES: Interleukin-7 (IL-7) has been studied for its possible immunorestorative capacities following stem cell transplantation and has been shown to enhance post-transplant immune recovery predominantly by peripheral T-cell expansion. A major concern of IL-7 is its possible aggravating effect on graft-versus-host and host-versus-graft reactivity. DESIGN AND METHODS: To study the effect of IL-7 on host-versus-graft reactivity, we applied IL-7 in an experimental transplantation model using RAG-1-/- mice supplied with B6 CD45.1 congenic T cells as recipients of T-cell depleted allogeneic bone marrow grafts. RESULTS: Rejection of minor antigen-mismatched bone marrow was significantly reduced in IL-7 treated recipients compared with PBS treated control mice. Rejection was observed in 2 out of 18 IL-7 treated mice compared with 9 out of 17 PBS treated mice (11% vs. 53%; p=0.012). IL-7 administration resulted in enhanced recovery of peripheral blood CD4+CD25+ regulatory T cells (Treg) with a concomitant increase in peripheral blood Foxp3 mRNA expression. IL-7Ra (CD127) was expressed by the vast majority of CD4+Foxp3+ T cells. The incidence of graft rejection following fully MHC mismatched bone marrow transplantation was not reduced nor enhanced by IL-7 administration. INTERPRETATION AND CONCLUSIONS: Post-transplant IL-7 administration protects against minor antigen-mismatched bone marrow rejection, which may be due to enhanced Treg recovery

Administration of keratinocyte growth factor (KGF) before and shortly after experimental allogeneic bone marrow transplantation (allo-BMT) has been shown to reduce the incidence and severity of graft versus host disease (GVHD) and to inhibit graft rejection. As naturally occurring CD4+Foxp3+ regulatory T cells (Treg) play an important role in the prevention of GVHD and graft rejection, we evaluated the effect of KGF on peripheral and thymic CD4+Foxp3+ regulatory T cell numbers in normal mice and BMT recipients. KGF (5mg/kg/day) was administered subcutaneously to adult B6 mice for 3 consecutive days. KGF enhanced the frequency of CD4+Foxp3+ Treg in peripheral blood and spleen twofold, to more than 15 % of CD4+ T cells, resulting in an increase in absolute numbers of CD4+Foxp3+ Treg within one week. From 2 weeks onwards, the frequency of CD4+Foxp3+ Treg gradually normalized, but the absolute numbers of Treg remained elevated (> 10 weeks) due to an increase of both CD4 and CD8 T cell numbers. In order to assess the relative contribution of thymic output versus peripherally expanded Treg, we next determined the frequency of T-cell receptor excision circles (TREC) in peripheral blood T cells. Analysis of sorted CD4+Foxp3+ Treg and CD4+Foxp3− conventional T cells showed a decline in TREC frequency in both subsets 1 week after KGF administration, suggesting peripheral expansion. In contrast, the TREC frequency in peripheral T cells was significantly higher at 5 and 10 weeks after KGF, indicating a late thymic dependent effect. Normal B6 mice that were adoptively transferred with congenic T cells showed an enhanced, but transient, increase of the frequency of CD4+Foxp3+ Treg within one week, also suggesting that the early effect of KGF on peripheral Treg is independent of thymic output. In addition, we assessed the effects of KGF on Foxp3+ thymocytes. Thymic weight and thymic cellularity of all subsets, including CD4+Foxp3+ thymocytes, were 2 to 3-fold increased one week after KGF administration as compared to control mice. In addition, we found that KGF transiently affected the thymic architecture. The medullary epithelial compartment was virtually absent one week after KGF administration. Normal thymic architecture gradually reappeared after 2–3 weeks. To determine whether KGF-enhanced Treg numbers in BMT recipients would inhibit graft rejection, KGF was administered from day -3 until day -1 to Rag-1−/− mice. Subsequently, the mice were 3Gy irradiated, supplied with 105 B6 CD45.1 congenic T cells and transplanted with a MHC-matched minor-Ag mismatched T cell depleted 129Sv bone marrow graft. As in normal B6 mice, KGF-treatment of BMT-recipients enhanced peripheral CD45.1+CD4+Foxp3+ Treg numbers. This was associated with a reduced rate of bone marrow graft rejection (2 out of 8 KGF-treated mice as compared to 5 out of 7 PBS-treated mice). Taken together, our data show that KGF enhances the peripheral Treg pool by an early, transient and selective thymus-independent mechanism and thereafter by a non-selective thymus-dependent mechanism. The KGF-mediated expansion of the pool of peripheral Treg may contribute to the inhibitory effects of KGF on graft rejection.