Helga Spelsberg

Influencing factors on chronic endothelial cell loss characterised in a homogeneous group of patients.

Background/aim: Advanced donor age, long death to excision time interval, and factors related to organ culture can trigger unfavourable intracellular processes in the graft endothelium and contribute to chronic endothelial cell loss after penetrating keratoplasty. The aim of this study was to investigate factors influencing chronic endothelial cell loss in a homogeneous group of patients. Methods: 177 patients after first normal risk keratoplasties for keratoconus were retrospectively selected from the quality control database of our clinic. For 71 of them at least four central endothelial cell density values were documented in follow up. From these patients, only those 53 without any further intraocular procedures, without glaucoma, and without graft rejection were considered. A scatter plot of logarithmised endothelial cell density values against postoperative time was drawn for each patient. The slope of the regression line then equals the constant of decay in central endothelial cell density. The influence of donor age and storage time in organ culture on this index value of cell loss was investigated by means of linear regression analysis. Results: Mean loss of central endothelial cell density was 16.7% per year. Regression analysis revealed a statistically significant negative linear effect of both postmortem time (β = –0.324; p = 0.014) and donor age (β = –0.282; p = 0.036) and a trend for storage time in organ culture (β = –0.195; p = 0.142) in a combined linear regression model. Conclusion: Increased postmortem time and advanced donor age exert a significant negative effect on chronic endothelial cell loss. Storage time in organ culture seems to be third influencing factor. These negative influences may be reduced by compensating advanced donor age with minimised postmortem and storage time.

Mycophenolate mofetil versus cyclosporin A in high risk keratoplasty patients: a prospectively randomised clinical trial

BACKGROUND/AIMS The requirement for an effective, minimally toxic immunosuppressive agent remains a major obstacle to performing high risk corneal transplantation. Although therapy with cyclosporin A (CSA) allows superior graft survival, its use is limited because of a wide range of side effects. Mycophenolate mofetil (MMF) has been shown to be a safe and effective immunosuppressive agent following renal transplantation. This prospective, randomised clinical trial was carried out to investigate the efficacy and safety of MMF in preventing corneal allograft rejection. METHODS Recipients of corneal transplants who were at high risk for graft failure were randomly assigned to either CSA or MMF immunosuppressive therapy. CSA was given in doses to achieve whole blood trough levels of 120–150 ng/ml. MMF was given in a daily dose of 2 g. Both therapy groups additionally received oral corticosteroids (fluocortolone 1 mg/kg) which were tapered and discontinued within the first 3 postoperative weeks. Patients were monitored closely for evidence of corneal graft rejection and adverse side effects. Drug efficacy was measured, primarily, by the number of patients who experienced at least one episode of clinical graft rejection. Safety analysis focused on reported adverse side effects and laboratory measurements. RESULTS 41 patients were enrolled in the study. There was no statistically significant difference between the two groups. 20 patients received CSA and 21 patients received MMF. Two patients in each group showed evidence of acute graft rejection which could be treated effectively by corticosteroids. All corneal grafts remained clear throughout the follow up. CONCLUSIONS In this study it was shown that MMF is just as effective as CSA in preventing acute rejection following high risk corneal transplantation. Mycophenolate mofetil represents a promising alternative therapeutic option in patients who are at high risk for corneal graft failure.

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.

Organ-cultured corneal grafts from septic donors: a retrospective study

Purpose To evaluate the quality of corneal grafts from donors, who have died from septic multi-organ failure and who are called septic donors in the following.Methods One hundred and eighty-two corneal grafts from septic donors were stored in organ culture for 10–14 days. Graft evaluation was performed according to the criteria of the European Eye Bank Association. Only donor corneas with cell density values above 2000 cells/mm2 were transplanted. Ninety-one patients who received these transplanted corneas were examined retrospectively with special emphasis on endophthalmitis, graft failure and incidence of immune reactions.Results Ninety-one of 182 donor corneas (50%) from septic donors were discarded mainly due to endothelial damage (61; 67%). Only seven (8%) were discarded due to medium contamination. In contrast, 452 of 1261 donor corneas (36%) from non-septic donors during the same period were discarded, again mainly due to endothelial damage (264; 58%). In this group, 48 donor corneas (11%) were discarded due to medium contamination. No patient who had received a graft from a septic donor has experienced endophthalmitis. The rate of immune reactions and graft failure was in the same range when compared to a larger group who received grafts from non-septic donors.Conclusion Our data reveal no contraindication against the use of corneal grafts derived from septic donors, critical graft assessment in organ culture provided.

Expression of p63 in conjunctival intraepithelial neoplasia and squamous cell carcinoma

Backgroundp63 is a homologue of the tumour suppressor gene p53, which is expressed in human basal squamous epithelium. Some investigators maintain that p63 plays a role in the development of squamous epithelium and, despite its homology to p53, it is considered to act as an oncogene. This study investigated the expression of p63 in conjunctival intraepithelial neoplasia of different grades, and conjunctival squamous cell carcinoma and its correlation to the proliferation marker MIB-1.Material and methodsSeventeen conjunctival specimens excised with the suspicion of either conjunctival intraepithelial neoplasia (CIN) or squamous cell carcinoma were diagnosed histologically as follows: 2 squamous cell carcinomas of the conjunctiva, 2 CIN grade I, 3 CIN grade II, 7 CIN grade III, 2 CIN with beginning invasion and 1 normal conjunctiva with no dysplasia. Sixteen microscopically-normal postmortem conjunctival specimens and normal conjunctiva, CIN and carcinoma specimens were stained immunohistochemically with antibodies against p63 and MIB-1. At least 500 cells per specimen were counted and the percentage of positively-stained cells of each antibody was calculated.ResultsA mean of 80% (57–89%) of the dysplastic cells from the CIN specimens stained positively with antibodies against p63, especially in the lower two-thirds of the epithelium, statistically significantly more compared with the normal specimens (9–55%, mean 36%, p<0.001). Nevertheless, we did not find a correlation between the percentage of p63-positive cells and the differentiation grade of the malignant specimens. MIB-1 positivity was shown by 0–1% of the cells in the normal postmortem controls, by 3–30% (mean 12%) of the cells in the basal and occasionally in the middle layer of the CIN specimens, and 16–61% (mean 23%) in the carcinoma specimens.ConclusionIn conjunctival intraepithelial neoplasia and squamous cell carcinoma of the conjunctiva, p63 is preferentially expressed in the immature dysplastic epithelial cells. Its staining does not correlate with MIB-1-expression, and therefore does not appear to be linked to cell proliferation.

In situ corneal excision – Experience of the Lions Cornea Bank of Nordrhein-Westfalen in 1995 and 1996

SummaryBackground: Donor corneas are normally obtained by whole globe enucleation – a procedure often refused by the bereaved. To increase the acceptance of cornea donation, we have exclusively obtained donor corneas by in situ excision since the end of 1994. There have been reports of increased endothelial damage and higher contamination rates. We report our experience in 1995 and 1996. Methods: The in situ excision was performed by staff trained in microsurgical techniques. Only donor corneas with negative end-storage cultures after at least 10 days and an endothelial cell count of more than 2500 cells/mm2 were used for transplantation. Results: In all, 705 corneoscleral buttons were excised from 1/95 to 12/96. The bereaved consented in 34 % in 1996. A total of 30.5 % of the corneas were ineligible for transplantation which corresponds to the discard figures from all cornea banks with culture methods. We did not observe any primary transplant failure nor endophthalmitis after 444 perforating keratoplasties. Conclusion: In situ corneal excision is safe, and helps to reduce the shortage in donor corneas.Hintergrund: Spenderhornhäute werden meist durch Enukleation gewonnen, was bei Angehörigen oft auf Ablehnung stößt. Um die Akzeptanz der Hornhautspende bei dem herrschenden Transplantatmangel zu erhöhen, entnehmen wir seit Ende 1994 Spenderhornhäute nur noch korneoskleral. Bedenken gegen diese Entnahmetechnik bestehen wegen möglicher Endothelschädigung und mikrobieller Kontamination. Wir berichten über unsere Erfahrungen in den Jahren 1995 und 1996. Methode: Die korneosklerale Entnahme an der Leiche erfolgte ausschließlich durch mikrochirurgisch geschulte Mitarbeiter. Es wurden nur Korneoskleralscheiben mit mikrobiologisch unauffälligem Organkulturmedium nach mindestens 10 Tagen Kultur und mit einer Endothelzelldichte von über 2500 Zellen/mm2 zur Transplantation verwendet. Ergebnisse: Von Januar 1995 bis Dezember 1996 haben wir 705 Korneoskleralscheiben entnommen. Die Zustimmungsquote (Einverständnisse der Angehörigen/Anzahl der Gespräche) betrug 1996 34 %. Der Anteil zur Transplantation nicht geeigneter Hornhäute betrug 30,5 % und lag damit im Rahmen aller Hornhautbanken mit Kulturverfahren. Bei 444 perforierenden Keratoplastiken wurde weder ein primäres Transplantatversagen noch eine Endophthalmitis beobachtet. Schlußfolgerung: Die korneosklerale Transplantatentnahme ist eine sichere Methode, die durch eine hohe Akzeptanz bei Spenderangehörigen den Transplantatengpaß zu beseitigen hilft.

Homologous penetrating central limbokeratoplasty in granular and lattice corneal dystrophy.

PURPOSE Subepithelial recurrences in the graft are well known after conventional penetrating keratoplasties in granular and lattice dystrophies. In contrast to what is still written in most textbooks, both dystrophies have their origin in the epithelium, which originates from the limbal stem cells. The aim of this study, therefore, is to investigate whether recurrences can be avoided or minimized by simultaneous transplantation of healthy donor limbus by means of a homologous central limbokeratoplasty. METHODS Since October 1995, five patients with granular and four patients with lattice dystrophies were treated with homologous penetrating central limbokeratoplasties. Approximately 40% of the grafts circumference contained limbal donor stem cells. After surgery, cyclosporin A was administered for at least 6 months to protect the limbal stem cells from immunologic destruction. RESULTS During a follow-up period of 12-35 months, one recurrence was observed, probably because of an immune reaction against the transplanted limbal stem cells after tapering off the immune prophylaxis. CONCLUSION These first results are in agreement with our expectations. More patients, however, must be operated on in this way. Follow-up must occur for many years before we will definitely know under which conditions long-term survival of donor stem cells occurs to such an extent that subepithelial recurrences in granular and lattice dystrophies stop being a major problem after keratoplasty in these patients.

Expression of p16 in Conjunctival Intraepithelial Neoplasia Does Not Correlate with HPV-Infection

The aim of our study was to identify the frequency of expression of p16INK4a (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN). Twelve specimens including CIN I (2), II (3), III (5), and CIN with beginning invasion (2), as well as 15 control specimens, were stained with antibodies against p16INK4a and MIB1. The presence of HPV was examined by PCR. p16 as well as MIB1 were significantly elevated in CIN compared to control specimens (p<0.01) without correlation with the differentiation grade. Only two cases with CIN grade 3 contained HPV 16. As few control specimens also showed increased p16INK4a expression, p16INK4a seems not to be a very reliable marker for the exact determination of CIN. It could serve as an additional diagnostic tool besides the morphological characterization. Our study suggested that p16INK4a elevation is not associated with HPV infection.

Endotoxin in storage medium of human corneal grafts and clinical course after penetrating normal-risk keratoplasty

AbstractPurpose It is well known that endotoxins in storage medium may stimulate cytokine production and expression of adhesion molecules as well as endothelial damage in human corneal grafts. It has been supposed that endotoxin exposure of corneal grafts may, therefore, cause immune reactions and lead to reduced endothelial cell count after penetrating keratoplasty. It was the purpose of this prospective study to evaluate if this hypothesis is true.Methods A consecutive series of 274 samples of sterile organ culture storage medium from 274 human corneal grafts was collected between August 1998 and February 1999 and tested for endotoxin using Limulus amebocyte-lysate assay (LAL)after 7 days of organ culture. Threshold endotoxin level was set at 1.0 U/ml. A total of 161 grafts were transplanted and 113 were discarded. Within the 161 corneas transplanted, 62 were grafted to normal-risk patients and 99 to high-risk patients. Only normal-risk keratoplasty patients were included in the study and followed for at least 10 months. Immune reactions, graft failures, and postoperative endothelial cell counts were recorded.Results The mean endotoxin level in organ culture medium of all transplanted grafts was 1.07±2.96. Mean endotoxin level in organ culture medium of discarded grafts was 1.68±5.76, with 71 samples being below and 42 above the threshold of 1.0 U/ml called endotoxin-negative and endotoxin-positive, respectively. In all 36 culture medium samples from the 62 grafts transplanted to the group of normal-risk keratoplasty patients were endotoxin-negative and 26 endotoxin-positive. An influence of endotoxin levels on incidence of immune reactions, graft failure, and postoperative endothelial cell counts could not be revealed in patients with normal-risk keratoplasty.Conclusion Low endotoxin levels in storage medium neither seem to promote immune reactions nor to contribute to postoperative chronic endothelial cell loss in normal-risk keratoplasty patients.

Beneficial effect of preoperative mycophenolate mofetil in murine corneal transplantation

To investigate the effect of preoperative mycophenoiate mofetil (MMF) on allograft survival in a murine corneal transplantation model. Corneal grafting was performed from Brown Norway to Lewis rats. Groups were divided as follows: Rats that received syngeneic or allogeneic grafts without therapy served as controls. MMF treatment was either started 7 days prior to transplantation and continued for 14 postoperative days (POD) or started at the day of corneal grafting until POD 14. MMF (20 mg/kg) administered postoperatively had no significant beneficial effect on corneal graft survival when compared with controls. However, the group receiving 40 mg/kg MMF postoperatively showed a statistically significant prolonged graft survival. A 1‐week preoperative administration of 20 mg/kg MMF allowed superior graft survival. Priming the immune system of corneal transplant recipients preoperatively with MMF proved to be a beneficial therapeutic regimen for prolonging corneal allograft survival in rats.