The influence of obesity, diabetes mellitus and smoking on fuchs endothelial corneal dystrophy (FECD).

To detect environmental factors, which may be possible risk factors in the disease course of Fuchs' endothelial corneal dystrophy (FECD). Evaluation of patients with FECD registered in the FECD genetics database of the Center for Ophthalmology, University Hospital Cologne. For the evaluation, disease onset, central corneal thickness, best spectacle corrected visual acuity (BSCVA, logMAR), and modified Krachmer grading (grades 1-6) were correlated with the presence of diabetes mellitus (DM), body mass index (BMI), and smoking behavior. To put the age-related increase in Krachmer grading into perspective, a correction of grading were formed. Depending on the variables studied, differences between groups were examined by Mann-Whitney U test and chi-square test. The significance level was 5%. 403 patients with FECD were included in the analysis. The mean age of the patients was 70.0 ± 10.32 (range 28-96) years. The mean age at diagnosis of those patients was 63.1 ± 13.2 years. The female-to-male ratio was 1.46:1. Patients with a BMI > 30.0 kg/m2 developed FECD significantly earlier than patients with a BMI < 30 kg/m2, p = 0.001. Patients with DM showed significantly more often an Krachmer grade of 5, p = 0.015. Smoking had a negative effect on Krachmer grading (p = 0.024). Using the mediation analysis, the presence of DM correlated Krachmer Grade 5 (p = 0.015), and the presence of DM correlated with BMI > 30.0 kg/m2 (p = 0.012). In addition to smoking and DM our study shows for the first time that obesity may have a negative impact on the development of FECD. Whether dietary interventions and hormones can influence the development or progression of the disease needs to be investigated in future studies.

[Diagnostics, clinical aspects and genetics of congenital corneal opacities]. / Diagnostik, Klinik und Genetik kongenitaler Hornhauttrübungen.

BACKGROUND: Congenital corneal opacities are comparatively rare diseases with high amblyogenic potential. PURPOSE: The present work provides an overview of the diagnostics, clinical aspects and genetics of congenital corneal opacities. METHODS: A literature search was carried out to compile an overview and illustration with own clinical case examples. RESULTS: Differentiated diagnostics are of high importance in the treatment of patients with congenital corneal opacities. A close cooperation between the medical departments involved and also the parents is absolutely essential. The structured classification of congenital corneal opacities provides the basis for a targeted treatment. DISCUSSION: The causes and the clinical symptoms of congenital corneal opacities are manifold. The correct diagnosis should be made early and in an interdisciplinary manner. Based on this, conservative and surgical treatment measures can be planned and an impending development of amblyopia can be specifically counteracted.

[Preconditioning of vascularized high-risk eyes using fine-needle diathermy and cross-linking]. / Präkonditionierung von vaskularisierten Hochrisikoaugen mittels Feinnadeldiathermie und Crosslinking.

BACKGROUND: The risk of allograft rejection following high-risk keratoplasty increases with the area of corneal neovascularization. Pharmaceutical and physical regression of corneal neovascularization before keratoplasty may offer the potential to reduce the risk of graft rejection after high-risk keratoplasty. OBJECTIVE: This article provides a review of the literature on the preconditioning of vascularized high-risk eyes using fine-needle diathermy and corneal cross-linking (preoperative preconditioning by lymphangioregression). METHODS: A literature search was carried out in PubMed and a summary of own data is presented. RESULTS: Animal experimental studies showed that both fine-needle diathermy and corneal cross-linking lead to a regression of corneal neovascularization and prolong graft survival after high-risk keratoplasty. Furthermore, studies from our institute provide first evidence that both procedures also lead to a reduction of corneal neovascularization in the clinical practice and thus potentially reduce the risk of allograft rejection after subsequent high-risk keratoplasty. DISCUSSION: Fine-needle diathermy and corneal cross-linking provide effective therapeutic approaches for angioregressive treatment and seem to prolong graft survival following high-risk keratoplasty. Larger prospective and controlled clinical trials are needed to further investigate these promising therapeutic approaches.

[Descemet membrane endothelial keratoplasty (DMEK) for graft failure after penetrating keratoplasty and in vascularized high-risk eyes]. / "Descemet membrane endothelial keratoplasty" (DMEK) bei Transplantatversagen nach perforierender Keratoplastik und in vaskularisierten Hochrisikoaugen.

BACKGROUND: Descemet membrane endothelial keratoplasty (DMEK) is considered the gold standard for the treatment of corneal endothelial dysfunction and generally leads to good postoperative results. Recently, studies have also analyzed the outcome of DMEK in so-called high-risk eyes. MATERIAL AND METHODS: The relevant literature and own data on DMEK for graft failure after penetrating keratoplasty and in vascularized high-risk eyes are presented and discussed. RESULTS: A DMEK for the treatment of transplant failure after penetrating keratoplasty can be considered for eyes without stromal scars and without high astigmatism. A retrospective analysis of 52 patients with failed penetrating grafts showed that DMEK leads to a significant increase in visual acuity, albeit to a lesser extent than after primary DMEK. Rejection and transplant failure rates seem to be similar those seen after penetrating re-keratoplasty and are thus higher than after primary DMEK. A DMEK might also be a feasible option for eyes with corneal neovascularization and stromal edema without stromal scars. A retrospective analysis of 24 eyes with at least 2 vascularized corneal quadrants demonstrated that DMEK leads to a significant improvement in visual acuity and regression of corneal neovascularization. The rejection rate in this cohort was 4.2% and is therefore slightly higher than after low-risk DMEK in eyes without corneal neovascularization but still much better compared to penetrating keratoplasty. CONCLUSION: Indications for DMEK are expanding and it can be a therapeutic option for transplant failure after penetrating keratoplasty with acceptable outcomes. Furthermore, DMEK seems to be a good option for the treatment of endothelial dysfunction in vascularized high-risk eyes without stromal scars.

[Treatment of vascularized high-risk eyes with a Boston keratoprosthesis]. / Versorgung von vaskularisierten Hochrisikoaugen mittels Boston-Keratoprothese.

BACKGROUND: In severely compromised and vascularized high-risk eyes, the Boston keratoprosthesis procedure is often the last treatment option. The transparency of the optics is not impaired by endothelial immune reactions. METHODS: This review article discusses relevant literature as well as our own data and experiences with the Boston keratoprosthesis in high-risk eyes. The relevant complications as well as the postoperative management are discussed. RESULTS: In more than 60% of the high-risk eyes a long-term increase in visual acuity can be achieved. Keratoprosthesis retention rates show a variable span with reported mean 5­year retention rates of 75%. The most common postoperative complications include the formation of a retroprosthetic membrane and keratolysis in up to 50% each. More than 70% of the eyes already suffer from (secondary) glaucoma preoperatively, so that glaucoma surgery is performed simultaneously in at least 20% of cases and in the postoperative course further antiglaucomatous surgery is necessary in up to 31%. Vitreoretinal complications include, in particular, sterile vitritis and infectious endophthalmitis but persistent hypotonia is also described in one third of patients. CONCLUSION: The Boston keratoprosthesis is an alternative to conventional corneal replacement if the prognosis for allogeneic transplants is poor. Postoperative complications are common; therefore, postoperative management plays an important role. For vascularized high-risk eyes, however, it is often the only remaining option for visual rehabilitation.

[Boston keratoprosthesis: 73 eyes from Germany : An overview of experiences from two centers]. / Boston-Keratoprothese: 73 Augen aus Deutschland : Übersicht der Erfahrungen aus zwei Zentren.

BACKGROUND: The Boston Keratoprosthesis (B-KPro) is a therapeutic option for corneal replacement for visual rehabilitation in eyes with poor a prognosis for penetrating keratoplasty (high-risk eyes). Since 2009, this approach has been used in the MVZ Prof. Neuhann, Munich, and since 2013 also at the Department of Ophthalmology, University of Cologne. An overview of results from the first 73 eyes in these departments is presented. METHODS: All recipient eyes of both centers which received a B-KPro type I (BI-KPro) between November 2009 and March 2017 were included. Indications, preoperative findings, intraoperative procedures, and clinical results, as well as postoperative complication and retention rates were documented and analyzed. RESULTS: A total of 73 eyes from 68 patients (mean age 55 ± 21.1 years, range 7 months-93 years; 26 women, 42 men) were treated with a BI-KPro. In 53 eyes (72.6%) the BI-KPro was implanted due to corneal graft failure, whereas BI-KPro was used as the primary corneal replacement in 20 eyes (27.4%). In 46 eyes (63.0%) a long-term increase in visual acuity was achieved and in 21.9% visual acuity was stabilized, while a decrease in preoperative visual acuity occurred in 15.1% of cases. During an average follow-up of 24.7 ± 23.0 months (range 1-78 months), 1.7 ± 2.4 re-interventions (range 0-9) were required. Only 4 keratoprostheses (5.5%) could not be retained. In 3 eyes (4.2%), a change of the BI-KPro device was necessary. CONCLUSION: There is a wide range of indications for BI-KPro in eyes with a poor prognosis for conventional keratoplasty. The surgical technique is safe but with a notable postoperative complication rate, which does not seem to decrease in the medium or long term. The retention rate is excellent for eyes without stem cell deficiency. Nevertheless, the BI-KPro is the only therapeutic option for high-risk eyes and leads to a gain in visual acuity in most cases.

[Deep Anterior Lamellar Keratoplasty : Experiences and results of the first 100 consecutive DALK from the University Eye Hospital of Cologne]. / Tiefe anteriore lamelläre Keratoplastik : Erfahrungen und Ergebnisse der ersten 100 konsekutiven DALK aus der Kölner Universitätsaugenklinik.

BACKGROUND: Deep anterior lamellar keratoplasty (DALK) is a treatment option for patients with diseases of the corneal stroma without endothelial involvement and has been used at the Department of Ophthalmology of the University of Cologne since July 2011. Experiences and results of the first 100 consecutive cases from Cologne with a minimum follow-up of 1 year are summarized. MATERIALS AND METHODS: We included the first 100 consecutive DALKs. Indications for anterior lamellar keratoplasty, the feasibility of the surgery, the postoperative course, in addition to the complications and clinical outcome 3 months after complete suture removal, were examined. The rate of conversion to penetrating keratoplasty in all planned DALKs during the observation period was also recorded. Mean follow-up was 25.6 ± 13 months. RESULTS: In 99 eyes of 97 patients (72 men, 25 women; aged 43.5 ± 16.9 years), the first 100 DALKs were performed in the period between 26 July 2011 and 27 January 2015 (87 cases electively, 13 cases as DALK à chaud, 1 of which was a second DALK). The most common indications were keratoconus (68%), corneal ulcers (13%), and corneal scarring (13%). On average, visual acuity increased from 0.83 ± 0.4 to 0.09 ± 0.1 logMAR. In 6 eyes (6%) grafts could not be preserved because of a corneal ulcer (n = 5, n = 3 of which were recurrent episodes), and in 1 case because of postoperative bulbus trauma. Rate of conversion to penetrating keratoplasty during the observation period was 11.5%. In eyes with keratoconus, visual acuity increased from 0.89 ± 0.4 before DALK to 0.06 ± 0.1 logMAR (best-corrected with contact lens) and the conversion rate was 17.6%. CONCLUSION: DALK represents a reliable technique for corneal transplantation in different pathological conditions of the cornea and the risk of endothelial immune responses can be avoided completely. In keratoconus and corneal scarring in particular, DALK can be used as a safe treatment option with very good results.

[Update Minimally Invasive Lamellar Keratoplasty: DMEK, DSAEK and DALK]. / Update minimalinvasive lamelläre Keratoplastik: DMEK, DSAEK und DALK.

BACKGROUND: The spectrum of operative interventions in corneal disorders requiring keratoplasty has been expanded considerably in recent years. In addition to the standard technique with full-thickness replacement of the cornea (perforating keratoplasty), lamellar techniques have been introduced. The aim of this review is to highlight current opportunities, indications and complications, as well as possible strategies to standardise lamellar keratoplasties. MATERIALS AND METHODS: Our own data and a review of the literature in PubMed are summarised. RESULTS: Performing "Descemet Membrane Endothelial Keratoplasty" (DMEK), "Descemt Stripping Automated Endothelial Keratoplasty" (DSAEK) and "Deep Anterior Lamellar Keratoplasty" (DALK) can provide patients with disorders of the corneal endothelium or the anterior corneal stroma with minimally invasive corneal grafts at a reduced risk of complications. CONCLUSION: DMEK and DSAEK are now internationally the surgical methods of choice in patients with endothelial corneal pathologies, i.e. in Fuchs' Endothelial Dystrophy. The DALK technique for lamellar replacement of the anterior stroma, i.e. in keratoconus, needs further standardisation, so that DALK can in future be performed more often even in less specialised departments. Major advantages of lamellar keratoplasties are the very good results in visual outcome and fewer immune reactions or graft rejections. Even intraoperative complications are rare. In the long term, further strategies are desirable for the standardisation of lamellar keratoplasties and its establishment as the primary standard procedure.

[Corneal donation : Dilemma between growing demand and declining donor rate]. / Hornhautspende : Ein Dilemma zwischen wachsender Nachfrage und sinkender Spenderrate.

BACKGROUND: One of the most important requirements for successful corneal transplantation is the availability of donor tissue and thus the approval for postmortem corneal tissue donation. The aim of this study was to investigate donor willingness compared to the continuously increasing demand in recent years. MATERIAL AND METHODS: Archives of the local eye bank at the Department of Ophthalmology, University of Cologne, Germany in the time period between 1 July 2011 and 31 December 2015 were examined regarding the willingness for corneal donations in deceased patients from the University Hospital of Cologne. Absolute numbers of deceased, exclusion criteria for donation as well as the rate of negative and affirmative decisions were evaluated. RESULTS: In 235 (5.1 %) out of 4593 deceased at the University Hospital of Cologne, corneal donation was accomplished during the observation period. Of the patients 2923 (63.6 %) were excluded because of absolute contraindications for corneal donation and the rate of absolute contraindications increased from 46.6 % in 2011 to 68.9 % in 2015. Willingness for corneal donation in potentially suitable deceased patients diminished from 34.9 % in 2011, to 34.3 % in 2012, 35.5 % in 2013, 28.4 % in 2014 and to 24.1 % in 2015. In relation to the total number of deceased, the number of corneal tissue donations decreased from 11.5 % in 2011 to 3.5 % in 2015. CONCLUSION: Despite a rising demand, data from Cologne seem to indicate that the number of corneal donations has declined to some extent. In order to increase the number of corneal donors in the future, further educational work as well as standardization and optimization of the tissue donation process seem to be urgently needed.

[Boston-keratoprosthesis : Preliminary experiences in 13 high-risk eyes from the Department of Ophthalmology of the University of Cologne]. / Boston-Keratoprothese : Erste Erfahrungen bei 13 Hochrisikoaugen aus der Kölner Universitätsaugenklinik.

BACKGROUND: Corneal transplantation in high-risk eyes remains a challenge. The Boston keratoprosthesis (B-KPro) is a final option for patients with end-stage corneal disease and a poor prognosis with conventional penetrating keratoplasty. In this article the results of the first 13 eyes that received a B-KPro type I at the Department of Ophthalmology, University of Cologne, Germany are reported and the usefulness of postoperative slit-lamp optical coherence tomography (SL-OCT) for control purposes is evaluated. MATERIAL AND METHODS: All recipients of a B-KPro type I between September 2013 and May 2015 were included in the study. The feasibility of the operation, clinical outcomes, complications and revision surgery were investigated. The visualization of wound healing by SL-OCT was analyzed. RESULTS: The age of the patients ranged from 26 to 92 years (mean 57.3 ± 20.9 years). In all 13 eyes from 12 patients (6 males and 6 females) dense corneal opacification with vascularization and sometimes also conjunctivalization was present. Preoperative visual acuity was reduced and ranged from mere light perception up to a maximum of 1/35 eye chart. All 13 eyes could be supplied with a B-KPro type I without any intraoperative complications, in 6 eyes no significant postoperative complications occurred, whereas in 7 eyes various additional surgical interventions were required and 1 B-KPro could not be preserved. Postoperative visual acuity ranged from light perception to 20/32 and was significantly improved in 85 % of the treated eyes. The use of SL-OCT reproducibly allowed the postoperative assessment of stromal thinning. CONCLUSION: The B-KPro provides the possibility of visual rehabilitation in high-risk eyes that could never be achieved without artificial cornea replacement. Despite higher complication rates this technique represents a significant progress in the surgical treatment of complex corneal pathologies. Regular and intensive postoperative controls are necessary to achieve good long-term results.

Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.

The chemokine receptor CCR7 is essential for migration of mature dendritic cells (DCs) to the regional lymph nodes, and it has been shown that blocking of CCR7 improves graft survival after high-risk corneal transplantation in vascularized recipient corneas. However, it is so far unknown whether blocking of CCR7 reduces migration of DCs from the avascular cornea to the draining lymph nodes and whether this leads to improved graft survival also in the low-risk setting of corneal transplantation, which accounts for the majority of perforating transplantations performed. Therefore, in this study, pellets containing Freund's adjuvant and bovine serum albumin (BSA) conjugated to Alexa488 fluorescent dye were implanted into the corneal stroma of BALB/c mice to analyze antigen uptake by corneal DCs and their migration to the regional lymph nodes. After pellet implantation, mice were either treated by local administration of a CCR7 blocking fusion protein that consisted of CCL19 fused to the Fc part of human IgG1 or a control-IgG. In vivo fluorescence microscopy showed uptake of Alexa488-conjugated BSA by corneal DCs within 8 h. Furthermore, analysis of single cell suspensions of draining lymph nodes prepared after 48 h revealed that 2.1 ± 0.3% of CD11c(+) cells were also Alexa488(+). Importantly, DC migration was significantly reduced after topical administration of CCL19-IgG (1.2 ± 0.2%; p < 0.05). To test the effect of CCR7 blockade on graft rejection after allogeneic low-risk keratoplasty, corneal transplantations were performed using C57BL/6-mice as donors and BALB/c-mice as recipients. Treatment mice received two intraperitoneal loading doses of CCL19-IgG prior to transplantation, followed by local treatment with CCL19-IgG containing eye drops for the first two weeks after transplantation. Control mice received same amounts of control-IgG. Kaplan-Meier survival analysis showed that in the CCL19-IgG treated group, 76% of the grafts survived through the end of the 8 week observation period, whereas 38% of the grafts survived in the control group (p < 0.05). Taken together, our study shows that blockade of CCR7 reduces the migration of mature corneal DCs to the draining lymph nodes and leads to improved graft survival in low-risk corneal transplantation.

[Influence of 20 % sulfur hexafluoride (SF6) on human corneal endothelial cells : An in vitro study]. / Einfluss von 20 % Schwefelhexafluorid (SF6) auf humane korneale Endothelzellen : Eine In-vitro-Studie.

BACKGROUND: In posterior lamellar keratoplasties, such as Descemet membrane endothelial keratoplasty (DMEK) and Descemet's stripping automated endothelial keratoplasty (DSAEK) an air bubble is left inside the anterior chamber to promote graft attachment during the early postoperative period. In the case of insufficient graft adhesion a renewed intracameral air injection is often necessary. The use of sulfur hexafluoride diluted with air (SF6 20 %) as an alternative to pure air may further enhance graft attachment and reduce the rebubbling rate. The effect of SF6 20 % on corneal endothelium is currently unclear and was therefore examined in vitro. MATERIAL AND METHODS: For this study 12 human corneoscleral discs were mounted in artificial anterior chambers, the systems were continuously filled with culture medium and the anterior chambers with air (n = 5) or SF6 20 % (n = 7) as tamponade. After 6 days of storage in the incubator endothelial cell density, toxicity on endothelial cells and corneal thickness were evaluated. RESULTS: There were no significant differences in endothelial cell loss (p = 1.000), endothelial cell count (p = 0.648), toxicity on endothelial cells (p = 0.048) and central corneal thickness (p = 0.905) between the two groups after 1 week. The level of significance was defined as p ≤ 0.05 and adjusted to p ≤ 0.0056 according to the Bonferroni correction for multiple testing. CONCLUSION: The use of SF6 20 % as tamponade in the anterior chamber for posterior lamellar keratoplasty can be proposed as a safe alternative to pure air filling related to endothelial cell loss. Increased toxic effects on the corneal endothelium by SF6 20 % were not detected in this study; however, further prospective clinical trials are needed to examine the long-term effects in humans.

Transient postoperative vascular endothelial growth factor (VEGF)-neutralisation improves graft survival in corneas with partly regressed inflammatory neovascularisation.

BACKGROUND: High-risk keratoplasties are usually performed after an uninflamed and quiescent interval in corneas with partly regressed blood and lymphatic vessels. We analysed whether the inhibition of post-keratoplasty revascularisation in mice with partly regressed corneal vessels ("intermediate-risk") improves graft survival. METHODS: Three interrupted stromal sutures (11-0) in corneas of Balb/c mice (6-8 weeks old) were placed for 6 weeks. Six months after suture removal, penetrating keratoplasty was performed with C57BL/6 donors. The treatment group received a vascular endothelial growth factor-A specific cytokine trap (VEGF Trap) intraperitoneally at days 0, 4, 7 and 14 after keratoplasty (25 mg/kg per mouse; controls received equal amounts of Fc protein). Pathological haemangiogenesis and lymphangiogenesis prior to as well as 3 days or 8 weeks after keratoplasty and graft survival were analysed. RESULTS: Three days after keratoplasty corneal revascularisation was sufficiently reduced by VEGF Trap (haem-vascularised areas 42.7% reduction; lymph-vascularised areas 54.7% reduction). Survival proportions 8 weeks after keratoplasty were 36% in the treatment group compared with 9% in the control group (n = 11; p<0.05). At that time no differences in haemangiogenesis or lymphangiogenesis were observed between the two groups. CONCLUSION: Early transient postoperative induction of haemangiogenesis and lymphangiogenesis and reformation of regressed corneal blood and lymphatic vessels are important for transplant rejections after "intermediate-risk" corneal transplantation.

Changes in donor corneal lenticule thickness after Descemet's stripping automated endothelial keratoplasty (DSAEK) with organ-cultured corneas.

AIMS: The aim of the study was to quantify changes in donor and host corneal tissue after Descemet's stripping automated endothelial keratoplasty (DSAEK) using organ-cultured corneas and to correlate these findings with the visual acuity during intermediate-term follow-up. METHODS: Fifteen eyes of 15 consecutive patients, with Fuchs' endothelial dystrophy treated with DSAEK using organ-cultured corneas, underwent ophthalmological examination, including slit lamp-adapted optical coherence tomography, at 1, 3 and 7 days, and 4 weeks, 8 weeks and 6 months after the surgery. RESULTS: The mean best spectacle-corrected visual acuity (BSCVA) improved from 20/100 pre-operatively to 20/40 at 6-months post-operatively (p<0.0001). A continuous decrease of thickness of the grafted lenticule was observed during the follow-up (mean thickness immediately after surgery 191(SD 56) mum, compared with 100 (SD 38) mum 6 months after surgery, p<0.001). The central corneal thickness decreased from 1057 (SD 86) mum at the first post-operative day to 661 (SD 74) mum after 6 months. Both central corneal thickness and the thickness of the posterior donor lamella correlated with the 6-month BSCVA (Pearson correlations -0.745 and -0.589, respectively, p<0.05). CONCLUSIONS: Organ-cultured corneas can be used successfully for DSAEK. The thickness of the grafted corneal lenticule correlated with the BSCVA 6 months after the surgery. It decreased continuously during the follow-up period.

[Patient satisfaction after posterior lamellar keratoplasty (DSAEK)]. / Patientenzufriedenheit nach posteriorer lamellärer Keratoplastik (DSAEK).

BACKGROUND: The advantages of posterior lamellar keratoplasty (DSAEK) compared to conventional full thickness keratoplasty are faster visual rehabilitation, little change in astigmatism and the absence of suture-associated complications. The current study evaluated the satisfaction of the first 15 patients who underwent DSAEK at our clinic. MATERIALS AND METHODS: Evaluation of patient satisfaction was conducted via a telephone survey and by means of a written patient questionnaire. The patients were asked to answer questions on a scale from 1 (very bad) to 10 (very good) about their overall satisfaction with this procedure, their satisfaction with the achieved postoperative visual acuity, their satisfaction with the progress of the healing process and the extent of the perceived burden. The patients were also asked whether they would opt again for the operation. Pre- and postoperative visual acuity, IOP, topographic astigmatism and corneal pachymetry were analysed. RESULTS: 15 eyes of 15 patients were analysed (12 women and 3 men). Average age at the time of operation was 75 +/- 7 years. Visual acuity increased and corneal thickness decreased primarily within the first three months. During that time there was an increase in overall patient satisfaction which finally reached 6.9 +/- 1.8 on the scale. Postoperative visual acuity was awarded with 7.2 +/- 2.0, progress of the healing process with 7.4 +/- 2.3. The procedure was not perceived as a big burden (8.0 +/- 1.9) and most patients would have decided again for the operation (8.2 +/- 2.0). There was no pre- and postoperative difference in IOP and corneal astigmatism. CONCLUSIONS: Like in our group patients with diseases of the corneal endothelium are of higher age and therefore benefit from a fast restitution of visual acuity and a low burden of operation. DSAEK represents a secure and practical procedure to rehabilitate elderly patients with diseases of the corneal endothelium.

Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine.

The enzyme beta-lactam synthetase (beta-LS) catalyzes the formation of the beta-lactam ring in clavulanic acid, a clinically important beta-lactamase inhibitor. Whereas the penicillin beta-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, beta-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, beta-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a beta-LS. The 1.95 A resolution structure of Streptomyces clavuligerus beta-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in beta-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate beta-lactam formation. The structural data provide the opportunity to alter the synthetic potential of beta-LS, perhaps leading to the creation of new beta-lactamase inhibitors and beta-lactam antibiotics.

Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional alpha-KG-dependent non-heme iron enzyme: correlation with mechanisms and reactivities.

Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O(2) and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, alpha-ketoglutarate (alpha-KG). CS2 can also catalyze an uncoupled decarboxylation of alpha-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/Fe(II) has a six-coordinate Fe(II) site, and alpha-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and alpha-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordinate ferrous species in the substrate complexes, which correlate to the uncoupled reaction. Five-coordinate ferrous species with similar geometric and electronic structures are present for all three substrate/alpha-KG complexes. Coordinative unsaturation of the Fe(II) in the presence of both cosubstrate and substrate appears to be critical for the coupling of the oxidative decarboxylation of alpha-KG to the different substrate oxidation reactions. In addition to the substrate orientation relative to the open coordination position on the iron site, it is hypothesized that the enzyme can affect the nature of the reactivity by further regulating the binding energy of the water to the ferrous species in the enzyme/succinate/product complex.

Kinetic mechanism of the beta-lactam synthetase of Streptomyces clavuligerus.

Streptomyces clavuligerus beta-lactam synthetase (beta-LS) was recently demonstrated to catalyze an early step in clavulanic acid biosynthesis, the ATP/Mg(2+)-dependent intramolecular closure of the beta-amino acid N(2)-(carboxyethyl)-L-arginine (CEA) to the monocyclic beta-lactam deoxyguanidinoproclavaminic acid (DGPC). Here we investigate the steady-state kinetic mechanism of the beta-LS-catalyzed reaction to better understand this unprecedented secondary metabolic enzyme. Initial velocity patterns were consistent with a sequential ordered bi-ter kinetic mechanism. Product inhibition studies with PP(i) and DGPC demonstrated competitive inhibition versus their cognate substrates ATP and CEA, respectively, and noncompetitive inhibition against their noncognate substrates. To clarify the order of substrate binding, the truncated substrate analogue N(2)-(carboxymethyl)-L-arginine was synthesized and demonstrated uncompetitive inhibition versus ATP and competitive patterns versus CEA. These data are consistent with ordered substrate binding, with ATP binding first, an abortive enzyme-DGPC complex, and PP(i) released as the last product. The pH dependence of V and V/K was determined and suggests that residues with a pK of 6.5 and 9.3 must be ionized for optimal activity. These observations were considered in the context of investigations of the homologous primary metabolic enzyme asparagine synthetase B, and a chemical mechanism is proposed that is consistent with the kinetic mechanism.

beta-Lactam synthetase: a new biosynthetic enzyme.

The principal cause of bacterial resistance to penicillin and other beta-lactam antibiotics is the acquisition of plasmid-encoded beta-lactamases, enzymes that catalyze hydrolysis of the beta-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of beta-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical beta-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg2+-dependent enzyme, a beta-lactam synthetase (beta-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N2-(carboxyethyl)-L-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the beta-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the beta-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg2+. Primary amino acid sequence comparisons suggest that this mode of beta-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.