Semiautomatic assessment of endothelial density and morphology in organ-cultured corneas - potential predictors for transplantation suitability and clinical outcome?

BACKGROUND: The quality of the endothelial cell layer is a major criterion for the approval of organ-cultured human donor-corneas for transplantation. We wanted to compare the predictive capacities of initial endothelial density and endothelium cell morphology for the approval of donor corneas for transplantation and for the clinical outcome after transplantation. METHODS: The endothelial density and endothelium morphology in organ culture were examined by semiautomatic assessment of 1031 donor corneas. We performed a statistical analysis for correlations of donor-data and cultivation parameters regarding their predictive capacities for the final approval of donor corneas for transplantation and the clinical outcome of 202 transplanted patients. RESULTS: Corneal endothelium cell density proved to be the only parameter with a certain predictive capacity with regard to the final decision, whether donor corneas are suitable for transplantation - however, the correlation was low (area under the curve [AUC] = 0.655). Endothelial cell morphology lacked any predictive power (AUC = 0.597). The clinical outcome regarding visual acuity seemed to be largely independent from both corneal endothelial cell density and morphology. Sub-analyses on transplanted patients stratified for their diagnoses vindicated these findings. CONCLUSIONS: Higher endothelial density (above a cut-off level of 2000 cells/mm2), as well as better endothelial morphology do not seem to be critical for transplant-corneal functionality in organ culture and up to 2 years after transplantation. Comparable long-term studies on graft survival are recommended to determine, whether the present endothelial density cut-off levels might be too stringent.

Effect of elevated irrigation bottle height during cataract surgery on corneal endothelial cells in porcine eyes.

BACKGROUND: Cataract surgery induces corneal endothelial cell loss (ECL). This study investigates the relationship between bottle height (BH) and ECL induced due to irrigation and aspiration (I/A) in cataract surgery and quantifies protective effects of intraoperatively used ophthalmic viscoelastic substances. METHODS: Intermittent I/A without phacoemulsification was performed in porcine eyes for 10 min with varying BHs of 100 cm (BH100), 125 cm (BH125), 150 cm (BH150) or no treatment (control, no I/A). Additionally, in one group a dispersive ophthalmic viscoelastic substance was injected into the anterior eye chamber before treatment with I/A at a BH of 150 cm (BH150 + V). After exposure of the corneal endothelium to I/A, the corneas were prepared to split corneal buttons on day 0 and cultivated for 15 days. Endothelial cell density (ECD) was analyzed blinded on days 1, 8 and 15. RESULTS: Relative ECL significantly correlated with irrigation BH (control (n = 13): -9.69 ± 6.03% (average ± standard deviation); BH100 (n = 12): -9.69 ± 4.81%-p = 1.000; BH125 (n = 14): -19.44 ± 7.30% - p < 0.001; BH150 (n = 13): -21.99 ± 6.70%-p < 0.001). I/A-induced ECL was significantly decreased by the injection of ophthalmic viscoelastic, as BH150 + V (n = 14; -10.92 ± 4.09%-p = 1.000) showed a cell loss comparable to the control group. CONCLUSIONS: ECL is altered by I/A BH and reduced when viscoelastic substances are used.

The Development and Status of Eye Banking with Special Focus on the Commitment of LIONS Clubs. / Entwicklung und Stand des Hornhautbankings unter besonderer Berücksichtigung des Engagements von LIONS Clubs.

BACKGROUND: Ever since the first successful keratoplasty in 1905, there has been a need to store corneas for transplantation. R. Townley Paton founded the first eye bank in New York in 1944. With Helen Keller's call in 1925 for LIONS to "constitute themselves Knights of the Blind in the crusade against darkness", LIONS Clubs International has become involved in the establishment of eye banks worldwide. This paper presents the development of eye banking in general and with special attention to the support offered by LIONS Clubs. METHODS: Selective literature search through PubMed, Google Scholar and Google in close cooperation with the LIONS Eye Banks already established in Germany, LIONS Clubs International (USA) and the Julius Hirschberg Society (Austria). Analysis focused on the founding processes of 6 German eye banks and their current services. RESULTS: Filatov was the first to keep donor eyes in a cool, moist container for a few days. In 1973, Summerlin et al described the technique of organ culture for donor corneas, and McCarey & Kaufman described a liquid storage medium in 1974. LIONS Clubs International and their organisational structure first supported an eye bank in the US in 1952, outside America in Hong Kong in 1962 and in Germany in 1969. Funding is provided across all levels of LIONS as network support and material resources. In general, staff funding is not provided. Of the 88 eye banks operating worldwide today, 44 are called LIONS Eye Banks. 6 of the current 26 eye banks in Germany are operating under LIONS sponsorship and run by departments of ophthalmology at university medical centres. Although the number of transplants has increased in recent years due to new surgical techniques, the number of patients waiting for donor tissue is also growing as a result of the broadening indication. CONCLUSIONS: Even today, the availability of donor corneas limits patient care. Eye banks help to meet the need for donor corneas. However, the techniques and technical equipment of eye banks must undergo continuous improvement. The local, national and international network of LIONS Clubs can assist in establishing these in order to facilitate legal requirements and structural developments. This support frequently lasts for many years, often triggers additional public commitment and is thus also a supporting element for the future development of eye banking in Germany.

A new storage solution for the hypothermic preservation of corneal grafts: an experimental study.

In this experimental study we used for the first time Tiprotec® as a solution for corneal preservation and cold storage. We compared the resultant endothelial cell morphology and viability with this obtained after preservation of the ex-vivo corneas with both usual standard techniques: conventional cold storage (using Eusol-C) and organ culture. This prospective, in vitro, 3-armed parallel study was performed with the use of 90 porcine corneas (examined for their endothelial quality and transparency) randomly selected for preservation in three storage methods (each 30 corneas): organ culture, standard cold storage (Eusol-C) and experimental cold storage (Tiprotec®) Endothelium cell quantity and quality as well as corneal opacification were assessed. The degree of endothelial transparency was significantly reduced over time with all preservation media, without any significant difference among the three groups at any point of time. A reduction in endothelial cell density was also observed with all three preservation media after 30 days of storage without statistically significant differences between groups. The number of hexagonal and pentagonal endothelium cells was significantly reduced overtime in all media with significantly more hexagonal and pentagonal in the organ culture group compared to the cold storage groups. We could show that the cryopreservation medium Tiprotec®, used until now for the preservation of vascular grafts, was of similar quality compared to the medium Eusol-C for the hypothermic storage of corneal tissue for an extended period of time up to 30 days. In comparison to organic culture with culture medium KII, both Tiprotec® and Eusol-C were found less effective in preserving endothelial cell quality, as assessed by the morphometric analysis, and viability, as assessed by the degree of vacuolization at least up to the 30th day of storage. However, both, Tiprotec®- and Eusol-C-preserved corneas demonstrated a certain capacity to recover after their submission in organ culture.

Establishment of a porcine corneal endothelial organ culture model for research purposes.

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas-displaying endothelial cell death rates comparable to those of cultured human corneas-would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called "split corneal buttons" (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.

Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm.

Dextran is added to corneal culture medium for at least 8 h prior to transplantation to ensure that the cornea is osmotically dehydrated. It is presumed that dextran has a certain toxic effect on corneal endothelium but the degree and the kinetics of this effect have not been quantified so far. We consider that such data regarding the toxicity of dextran on the corneal endothelium could have an impact on scheduling and logistics of corneal preparation in eye banking. In retrospective statistic analyses, we compared the progress of corneal endothelium (endothelium cell loss per day) of 1334 organ-cultured corneal explants in media with and without dextran. Also, the influence of donor-age, sex and cause of death on the observed dextran-mediated effect on endothelial cell counts was studied. Corneas cultured in dextran-free medium showed a mean endothelium cell count decrease of 0.7% per day. Dextran supplementation led to a mean endothelium cell loss of 2.01% per day; this reflects an increase by the factor of 2.9. The toxic impact of dextran was found to be time dependent; while the prevailing part of the effect was observed within the first 24 h after dextran-addition. Donor age, sex and cause of death did not seem to have an influence on the dextran-mediated toxicity. Based on these findings, we could design an algorithm which approximately describes the kinetics of dextran-toxicity. We reproduced the previously reported toxic effect of dextran on the corneal endothelium in vitro. Additionally, this is the first work that provides an algorithmic instrument for the semi-quantitative calculation of the putative endothelium cell count decrease in dextran containing medium for a given incubation time and could thus influence the time management and planning of corneal transplantations.

Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer.

Using an Affymetrix 10K SNP array to screen for gene copy number changes in breast cancer, we detected a single-gene amplification of the ESR1 gene, which encodes estrogen receptor alpha, at 6q25. A subsequent tissue microarray analysis of more than 2,000 clinical breast cancer samples showed ESR1 amplification in 20.6% of breast cancers. Ninety-nine percent of tumors with ESR1 amplification showed estrogen receptor protein overexpression, compared with 66.6% cancers without ESR1 amplification (P < 0.0001). In 175 women who had received adjuvant tamoxifen monotherapy, survival was significantly longer for women with cancer with ESR1 amplification than for women with estrogen receptor-expressing cancers without ESR1 amplification (P = 0.023). Notably, we also found ESR1 amplification in benign and precancerous breast diseases, suggesting that ESR1 amplification may be a common mechanism in proliferative breast disease and a very early genetic alteration in a large subset of breast cancers.

A Cancer-Indicative microRNA Pattern in Normal Prostate Tissue.

We analyzed the levels of selected micro-RNAs in normal prostate tissue to assess their potential to indicate tumor foci elsewhere in the prostate. Histologically normal prostate tissue samples from 31 prostate cancer patients and two cancer negative control groups with either unsuspicious or elevated prostate specific antigen (PSA) levels (14 and 17 individuals, respectively) were analyzed. Based on the expression analysis of 157 microRNAs in a pool of prostate tissue samples and information from data bases/literature, we selected eight microRNAs for quantification by real-time polymerase chain reactions (RT-PCRs). Selected miRNAs were analyzed in histologically tumor-free biopsy samples from patients and healthy controls. We identified seven microRNAs (miR-124a, miR-146a & b, miR-185, miR-16 and let-7a & b), which displayed significant differential expression in normal prostate tissue from men with prostate cancer compared to both cancer negative control groups. Four microRNAs (miR-185, miR-16 and let-7a and let-7b) remained to significantly discriminate normal tissues from prostate cancer patients from those of the cancer negative control group with elevated PSA levels. The transcript levels of these microRNAs were highly indicative for the presence of cancer in the prostates, independently of the PSA level. Our results suggest a microRNA-pattern in histologically normal prostate tissue, indicating prostate cancer elsewhere in the organ.

Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase.

BACKGROUND: The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide-dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. METHODS AND RESULTS: Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography-mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg(-1) · min(-1)) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 µmol · kg(-1) · d(-1)) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO(-/-) mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. CONCLUSIONS: ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.

Contamination of multi dose eyedrops in the intra and perioperative context.

In this study, we examined the rate of contamination of multi-dose ophthalmic solutions in the operating theatre and the underlying risk for infection by examining the microbiological load on the tips of the dispenser bottles. A total of 245 samples of eye drop bottles were collected and analysed between June 2018 and January 2019. All were collected in the operating theatre of the University Eye Hospital Hamburg-Eppendorf. Contamination of the dropper tip occurred in 2% of the samples. Although the prevalence of contamination was low, the results of this study reveal the possibility of contamination of multi-dose eyedrops even when used by health care professionals in the controlled environment of an operating theatre. Following these results, we recommend the use of single-dose eyedrops in the pre- and intraoperative context.

SARS-CoV-2: Impact on, Risk Assessment and Countermeasures in German Eye Banks.

INTRODUCTION: Since the beginning of the COVID-19 pandemic there has been some debate regarding the risk of transmission through tissue transplantation and tissue banking processes. AIM OF THE STUDY: To analyze the changes that SARS-CoV-2 has caused regarding the harvesting of corneal donor tissue and eye bank activities in Germany. METHODS: A questionnaire was provided to 26 eye banks in Germany, consisting of questions about adaptations made in the screening of potential donors and the harvesting of corneal tissue following the pandemic spread of SARS-CoV-2. RESULTS: Eighteen eye banks actively reduced recruitment of donors and two banks ceased all activity. Additional diagnostic screening was performed in eight banks, using conjunctival swabs and/or nasopharyngeal swabs. In six eye banks, additional protective measures, such as FFP2 masks and/or facial shields, were implemented. Overall, a mean reduction in the number of obtained donor tissues of 17% was observed. DISCUSSION: Conjunctival and/or nasopharyngeal swabs of donors have been implemented by a minority. Reasons for not performing additional tests may be moderate sensitivity and lack of validation for postmortem use of RT-PCR testing. Also, the hazard of SARS-CoV-2 entering the corneal donor pool with subsequent transmission might be perceived as theoretical. Face shields provide a sufficient barrier against splash and splatter contamination but may be insufficient against aerosols. Additional face masks would provide support against aerosols, but it remains debatable if corneal harvesting can be considered an aerosol-producing procedure. In the future we expect to see changes in current guidelines because of a surge in scientific activities to improve our understanding of the risks involved with cornea donation in the COVID-19 pandemic, and because current practice may reduce the availability of donor corneas due to new exclusion criteria while the demand remains unchanged.

Effects of Perfluorobutylpentane (F4H5) on Corneal Endothelial Cells.

Purpose: To evaluate the effects of perfluorobutylpentane (F4H5) on corneal endothelial cell density (ECD) and morphology using a porcine corneal endothelial organ culture model. Materials and methods: "Split corneal buttons" were cultivated for 15 days (d) after incubation in F4H5 (15, 30, 60, and 120 min) or BSS (controls). ECD was assessed manually on d1, d8, and d15. After histological staining (trypan blue, alizarin red S) on d15 morphological changes (reformation figures, rosette formations, and alizarin red cells) were evaluated. Results: ECD was significantly reduced after incubation in F4H5 for 120 min (median ± 25%/75%-quartile; 3281 ± 43/222 cells/mm2; p = 0.046) on d15 compared to controls (3658 ± 129/296 cells/mm2), but not after shorter incubation times (15, 30, and 60 min). Morphological assessment supports these findings as reformation figures (F4H5 120 min: 10.5 ± 9.3/13.9/mm2 vs. controls: 5.2 ± 2.8/7.2/mm2; p = 0.010), rosette formations (F4H5 120 min 25.566 ± 17.044/36.219/mm2 vs. controls: 8.333 ± 0.000/15.667/mm2; p = 0.002), and alizarin red cells (F4H5 120 min: 38.350 ± 29.827/51.333/mm2 vs. controls: 20.833 ± 10.417/25.000/mm2; p = 0.049) were significantly more prevalent after incubation in F4H5 for 120 min compared to controls. Also, F4H5 60 min showed significantly more rosette formations (25.452 ± 16.968/36.057/mm2; p = 0.006) and alizarin red cells (46.662 ± 42.420/50.903/mm2; p = 0.007), but not reformation figures (7.0 ± 2.2/1.6 %; p = 0.953). Conclusion: Short exposure (≤30 min) of porcine corneal endothelial cells to F4H5 does not have significant effects on ECD or morphological characteristics. Longer exposure times (≥60-120 min) may cause ECD decline and/or induce morphological changes.

Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer.

BACKGROUND: We previously identified a novel exogenous gammaretrovirus (xenotropic murine leukemia virus-related gammaretrovirus (XMRV)) using a pan-viral microarray. XMRV is the first MLV-related virus found in human infection. Forty percent (8/20) of familial prostate cancer patients homozygous for a mutation in RNase L (R462Q) were positive for XMRV, while the virus was rarely (1/66) detected in familial prostate cancer patients heterozygous for R462Q or carrying the wild type allele. OBJECTIVES: To determine the presence of XMRV in non-familial prostate cancer samples. STUDY DESIGN: RNA from prostate tissue was analyzed for XMRV using nested RT-PCR. In all samples, RNase L (R462Q) genotyping was performed using an allele-specific PCR. RESULTS: XMRV-specific sequences were detected in one of 105 tissue samples from non-familial prostate cancer patients and from one of 70 tissue samples from men without prostate cancer. The two XMRV-positive patients were wild type or heterozygous for the R462Q mutation and thus carried at least one fully functional RNase L allele. CONCLUSIONS: XMRV was rarely detected in non-familial prostate cancer samples from Northern European patients. The homozygous mutation R462Q (QQ) was significantly underrepresented (<6%) in this cohort when compared to other studies (11-17%).

Methylation of the TPEF- and PAX6-promoters is increased in early bladder cancer and in normal mucosa adjacent to pTa tumours.

OBJECTIVE: To evaluate CpG island methylation patterns of cancer-associated genes for their applicability as molecular biomarkers for the detection of superficial bladder cancer and for the discrimination of invasive from noninvasive tumours. PATIENTS AND METHODS: We analysed the methylation status of CpG islands in the promoter region of the cancer-associated genes GSTP1, DAPK, MDR1, TPEF, PAX6, and TSLC1 in primary papillary bladder cancer specimens from 39 patients (pT1 10, pTis one, pTa 20, pT2 five). Tumour-adjacent normal mucosa served as the control. The DNAs were bisulphite-treated and submitted to methylation-specific real-time polymerase chain reactions. RESULTS: Only TPEF and PAX6 had substantial CpG island methylation percentages. The TPEF- and PAX6-promoters also had significantly higher methylation rates in tumour tissue compared with the normal tumour-adjacent tissue. Interestingly, the methylation rates of the TPEF- and the PAX6-promoter were higher in adjacent normal tissues from bladders with pTa then in those with pT1 tumours. CONCLUSION: Our results shed a critical light on the hypothesis that CpG island hypermethylation of the GSTP1-, DAPK-, MDR1- and TSLC1-promoter could represent molecular biomarkers for bladder cancer diagnosis and detection. However, methylated PAX6- or TPEF-promoters could represent biomarkers for this disease. Additional studies are needed to evaluate whether methylation rates of these genes in normal bladder tissues are applicable as accessory markers for the tumour state or its invasive behaviour.

A comprehensive analysis of transcript signatures of the phosphatidylinositol-3 kinase/protein kinase B signal-transduction pathway in prostate cancer.

OBJECTIVE: To assess the gene activities of various important members of the phosphatidylinositol 3 kinase (PIK3)/protein kinase B (PKB/Akt) pathway (involved in the promotion and regulation of cellular metabolism, proliferation and apoptosis) for alterations in prostate carcinoma. PATIENTS, SUBJECTS AND METHODS: Using quantitative real-time reverse-transcription polymerase chain reaction, we analysed the transcript levels of 12 genes involved in the PIK3/PKB pathway in microdissected tumour tissues from 20 patients with varying stages of prostate cancer, assessing differences from adjacent normal tissues and from a pool of prostate tissues from healthy controls. RESULTS: In cancer samples with a high Gleason grade, the PIK3/PKB pathway was principally affected by marked decreases in expression over almost all the investigated stages of the pathway. These changes were in effectors of the pathway, especially PIK3 p85 alpha (PIK3R1) and integrin-linked kinase, and the pathway target fork-head box protein (FOXO)-1A, while the transcript quantities of regulators, e.g. phosphatase/tensin homologue (PTEN), were decreased in a smaller proportion of the patients. Transcript amounts of FOXO-1A and FOXO-3A were significantly higher in normal tumour-adjacent tissues than in the healthy controls. CONCLUSIONS: Down-regulation of the PIK3/PKB pathway by repression of involved effector and regulator genes at all stages of the molecular pathway could represent a marker for the formation of highly de-differentiated prostate cancers from low-grade tumour foci. Also, parts of the pathway are deviant in normal tumour-adjacent tissue; this might represent a reaction to neighbouring tumours or be a sign of pre-cancerous biological alterations.

Endothelial Cell Count in Eye Bank Corneal Grafts: Impact of Death Cause and Donor Diseases.

PURPOSE: The purpose of this study is to analyze the impact of death causes and documented donor diseases on initial endothelial cell counts (after retrieval) and the development of corneal graft endothelia during organ culture. METHODS: The retrospective statistic analyses was conducted on a data set of 10,185 human corneas prepared at the Hamburg Eye Bank. RESULTS: Although we observed that death by gunshot trauma or alcoholism seems to be associated with marginally higher endothelium cell counts (independently from donor age), we could prove that only donor age is a relevant predictive parameter for the initial cell-density of the endothelium and its development in vitro. CONCLUSION: We conclude that an extension of prospective quality parameters for donor selection additional to donor age (such as individual causes of death) is not necessary.

Donor Cornea Harvest Techniques: Comparison Between Globe Enucleation and In Situ Corneoscleral Disc Excision.

PURPOSE: To compare whole eye enucleation and corneoscleral disc (CD) excision as donor cornea harvesting techniques for possible effects on corneal cultivation and the clinical outcome of the corneal grafts after transplantation in 2929 cases. METHODS: A retrospective analysis was performed on the Hamburg Eye Bank database using comparative statistics. The standard method for donor cornea recovery at the Hamburg Eye Bank changed from enucleation of the whole eye to CD in situ excision in 2008. Corneas recovered between 2003 and 2013 were included in this study. We compared the contamination rate, the endothelial density after retrieval, endothelial cell loss during cultivation, and the clinical outcome (visual acuity, astigmatism, and refraction) of transplanted corneas. RESULTS: Once the retrieval method was changed from whole globe enucleation to in situ CD excision, the donation numbers increased (after several years of constant decrease). Furthermore, we observed slightly lower endothelial cell density after retrieval in corneas obtained by in situ CD excision compared with those from enucleated eyes, whereas endothelial cell loss during cultivation was similar. After changing the recovery procedure to in situ excision, initially a higher rate of contamination was observed, but but it eventually decreased. Finally, the corneas of both groups had a similar clinical outcome. CONCLUSIONS: After a transient technical learning period, in situ CD excision proved to be a method of donor cornea recovery with similar cultivation performance and clinical results compared with whole eye enucleation. It also may have led to higher willingness to donate, possibly because of better acceptance by the relatives of the deceased.

Extraction and processing of high quality RNA from impalpable and macroscopically invisible prostate cancer for microarray gene expression analysis.

Molecular analyses of early-stage prostate cancers are necessary to assess their potential clinical significance based on established and/or novel biomarkers for tailored clinical management. A prerequisite for the application of RNA-based analyses of such, mostly macroscopically-undetectable, small prostate carcinomas is the recovery and preservation of sufficient RNA quantities and quality. Furthermore, in prostate cancer, heterogeneity is a common phenomenon that includes a juxtaposition of different tissue compositions and variable histological grades within the same tumor focus. To better understand the molecular mechanisms of prostate cancer, it is essential to correlate molecular data with a specific cell type. Here, we present a tissue collecting protocol which is aligned with the preoperative evaluation of tumor localization. In combination with the technique of laser microdissection and pressure catapulting, we are able to preserve RNA of high quality from homogeneous cell populations of macroscopically-undetectable small prostate carcinomas. To obtain the necessary RNA quantities for whole genome cDNA microarrays, the isolated total RNAs were amplified by T7-based RNA-polymerase in vitro transcription. The microarray analyses (Human Unigene Set RZPD3.1) resulted in 216 differentially expressed genes (191 down-regulated, 25 up-regulated). Among these were several known prostate cancer relevant genes, such as AMACR, TARP, LIM, GPR160 (all up-regulated), CAV1, NTN1, MT1X; CLU, TRIM29, SPARCL1 and HSPB8 (all down-regulated).

Limited specificity of promoter constructs for gene therapy in osteosarcoma.

Osteosarcoma (OS), a malignant bone neoplasia in childhood, has poor prognosis if metastases appear in the lung. A novel therapeutic approach could consist in a gene therapeutic treatment of OS metastases. However, if promiscuous viral vectors are applied for the delivery of potentially toxic transgenes, their misdelivery into normal tissues could cause severe complications. This problem could be circumvented by application of OS-specific promoters for transgene expression control. We analysed the function of promoters described to be tumour-, osteosarcoma- or osteoblast-specific. Expression rates driven by osteoblast- specific fragments from the collagen1A1-promoter, the human Osteocalcin-promoter, the bone-sialoprotein promoter and the beta-catenin promoter depending on vitamin supplementation were analysed in five OS cell lines, in normal lung fibroblasts and in a non-osteoblastic prostate cancer cell line (LNCaP) by dual luciferase assays. In addition, an unspecific but doxycyclin-repressible promoter construct (pAd.3r-luc) was examined. We found that all constructs were active in OS cell lines to varying extents. The complete human Osteocalcin promoter and the bone-sialoprotein promoter were partially induced by vitamin D3 or C respectively while the pAd.3r-luc activity could be shut down by doxycyclin. In contrast, the human Osteocalcin-promoter was not activated by vitamin D3 in LNCaP cells; its action remained relatively low. Interestingly, excepting the beta-catenin promoter, we measured strong activities of all promoters in lung fibroblast cells. Our study demonstrates that promoter activity should be evaluated not only for the target cells of the gene therapeutic approaches, but also for neighbouring normal tissues. Unspecific but repressible promoters could represent an alternative.

ESR1 amplification in breast cancer by optimized RNase FISH: frequent but low-level and heterogeneous.

Prevalence of ESR1 amplification in breast cancer is highly disputed and discrepancies have been related to different technical protocols and different scoring approaches. In addition, pre-mRNA artifacts have been proposed to influence outcome of ESR1 FISH analysis. We analyzed ESR1 gene copy number status combining an improved RNase FISH protocol with multiplex ligation-dependent probe amplification (MLPA) after laser microdissection. FISH showed a high prevalence of ESR1 gains and amplifications despite RNase treatment but MLPA did not confirm ESR1 copy number increases detected by FISH in more than half of cases. We suggest that the combination of the ESR1-specific intra-tumor heterogeneity and low-level copy number increase accounts for these discrepancies.