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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.

Purpose: To evaluate donor demographics, trends in donor tissue procurement and tissue storage over a long period., Methods: A retrospective, longitudinal, descriptive analysis was undertaken of data from the Hamburg Eye Bank Data Base (HEB-DB) that had been collected between 1981 and 2010. Data on 54 parameters of cornea donors [including clinical history, age, death cause, gender and death-to-explantation interval (DEI)] and of cultivated corneas (endothelial quality and development in culture, cultivation period, microbiological contamination) were retrieved. These data were analysed statistically, focusing on the historical development of the eye bank., Results: At the time of retrieval (June 2010), the HEB-DB contained data on 10 943 corneas (5503 donors). Most donors were men (65%) and had died from cardiopulmonary (n = 801)/cerebral (n = 261) failure or as the result of a polytraumatic accident/suicide (n = 602). Within these years, donor age, DEI and storage time increased. The percentage of stored corneas suitable for transplantation displayed a variable but increasing trend; in 2007, almost 75% of the stored corneas were transplanted. Between 1995 and June 2010, the median microbiological contamination rate was 5.3%. A change in the procurement procedure from enucleation to corneoscleral explantation in 2008 led to a briefly increased contamination rate., Conclusion:   Donor demographic data run parallel to the general demographic development. Our analysis indicates a dynamic development of the eye bank over the last 30 years and emphasizes the need for an active quality management in coping with the challenges of modern eye banking., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

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.

Purpose: Microbiological contamination is a common cause for elimination of organ-cultured donor corneas. The aims of the present study were to analyze contamination rates and identify risk factors for contamination., Methods: Retrospectively, the contamination rates of 4546 organ-cultured corneas and the causative species were studied. The impact of sex, age, death-to-explantation interval, explantation technique, cause of death, and mean monthly temperature on contamination rate was analyzed., Results: The median annual contamination rate was 5.3% (range: 3%-19%). Most contaminations were of fungal origin (61.9%), with Candida species (45%) being predominant. Bacterial contaminations (34.4%) were dominated by Staphylococcus species (12.8%). Sex, donor age, and mean monthly temperature had no statistically significant influence on the contamination rate. The median death-to-explantation interval of contaminated corneas (44 hours) was longer than that of sterile corneas (39 hours; P < 0.001; n = 4437). Cardiopulmonary failure was associated with the highest contamination rate (13.6%) of all death causes. The switch from whole globe to in situ excision was followed by a temporary increase in contamination rate (12.5%-19.4%)., Conclusions: Although the genesis of donor cornea contamination seems to be multifactorial, resident species from physiological skin flora are the main contaminants indicating that the donor corpses could be the main source of microbiological contamination. A change in the explantation technique was followed by an increase in the contamination rate.

Purpose: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients., Experimental Design: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples., Results: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of β1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [β(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival., Conclusion: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of β(1,6)-branched oligosaccharides for prostate cancer progression.

The purpose of this work was to investigate the role of the ubiquitin-proteasome network (UPN) in prostate cancer (PCA) and to elicit potential markers for this disease. The UPN represents a key factor in the maintenance of cellular homoeostasis as a result of its fundamental function in the regulation of intracellular protein degradation. Members of this network have a role in the biology of haematological and solid tumours. Tumour cells and normal epithelial cells from 22 prostatectomy specimens were isolated by laser microdissection. Prostate biopsy samples from healthy individuals served for technical calibration and as controls. Transcript levels of eight selected genes with E3 ubiquitin ligase activity (labelling target proteins for proteasome degradation) and two genes belonging to the proteasome-multienzyme complex itself were analysed by quantitative real-time RT-PCR. The proteasome genes PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L were significantly and coherently upregulated in PCA cells compared with the corresponding adjacent normal prostate tissue. Transcription of the E3 ubiquitin ligase SMURF2 was significantly higher in organ-confined tumours (pT2) compared with non-organ-confined cancers (pT3). The results indicate a role for PSMC4 and PSMB5 and the E3 ubiquitin ligase NEDD4L in prostate tumourigenesis, whereas SMURF2 downregulation could be associated with clinical progression. NEDD4L and SMURF2 both target transforming growth factor (TGF)-β for degradation. This reflects the pleiotropic role of the TGF-β signalling pathway acting as a tumour suppressor in normal and pre-cancerous cells, but having oncogenic properties in progressing cancer. Further studies have to elucidate whether these alterations could represent clinically relevant PCA-diagnostic and progression markers.

Purpose: Deletions of 8p and gains of 8q belong to the most frequent cytogenetic alterations in prostate cancer. The target genes of these alterations and their biological significance are unknown., Experimental Design: To determine the relationship between chromosome 8 changes, and prostate cancer phenotype and prognosis, a set of 1.954 fully annotated prostate cancers were analyzed in a tissue microarray format by fluorescence in situ hybridization., Results: Both 8p deletions and 8q gains increased in number during different stages of prostate cancer progression. 8p deletions/8q gains were found in 26.1%/4.8% of 1,239 pT(2) cancers, 38.5%/9.8% of 379 pT(3a) cancers, 43.5%/8.9% of 237 pT(3b) cancers, 40.7%/14.8% of 27 pT(4) cancers, 39.1%/34.8% of 23 nodal metastases, 51.9%/33.3% of 27 bone metastases, and 45.5%/59.9% of 22 hormone refractory cancers (P < 0.0001 each). Both 8p deletions and 8q gains were also significantly associated with high Gleason grade and with each other (P < 0.0001 each). In primary tumors, 8p deletions were seen in only 27.3% of 1,882 cancers without 8q gain but in 57.4% of 122 tumors with 8q gain (P < 0.0001). Among cancers treated with radical prostatectomy, 8p deletions (P = 0.003) and 8q gains (P = 0.02) were associated with biochemical tumor recurrence. However, multivariate analysis (including prostate-specific antigen, pT/pN stage, Gleason score, and surgical margin status) did not reveal any statistically independent effect of 8p or 8q alterations on biochemical tumor recurrence., Conclusions: 8p deletions and 8q gains are relatively rare in early stage prostate cancer but often develop during tumor progression. The prognostic effect does not seem to be strong enough to warrant clinical application.

The transcription factor ERG is highly upregulated in the majority of prostate cancers due to chromosomal fusion of the androgen responsive promoter of TMPRSS2 to the ERG reading frame. Our aim was to identify this gene fusion in urine samples from prostate cancer patients prior to radical treatment and to compare fusion status with clinicopathological variables. Urine fractions from 55 patients (with and without prior prostatic massage) were analyzed for the presence of TMPRSS2:ERG isoforms using real-time qPCR. Sixty-nine percent of urine samples following prostatic massage were positive for TMPRSS2:ERG isoforms a or b, five out of which were positive for both, vs 24% of samples obtained without prior massage. Isoform a seems to be most prevalent and some patients may be positive for more than one fusion variant, reflecting the multifocality of prostate cancer. Prostatic massage prior to sampling, analysis of pelleted urine material and detection of cDNA provided the highest sensitivity. Positive statistical correlations were identified between TMPRSS2:ERG fusion and high s-PSA, pathological stage and Gleason score. Our findings contribute to the increasing elucidation of the role of TMPRSS2:ERG in the development of prostate cancer.

Background: Insufficient sensitivity and specificity of prostate biopsies for cancer detection., Objectives: Based on evidence from our microarray analyses, we hypothesized that considerable molecular changes precede morphologically detectable malignant transformation of prostate epithelial tissues. The identification of such changes could lead to novel strategies in the clinical management of prostate cancer., Design, Setting, and Participants: Histologically normal, fresh prostate tissue from prostate cancer patients, healthy donors, and cancer suspect patients with continuous negative biopsies were analyzed., Measurements: To identify molecular changes between 29 tumor-free prostate tissues from healthy donors and 27 patients with proven prostate cancer, we performed a global microarray screening. Based on this screening as well as literature data, we selected a subset of 29 genes for validation by arrayed real-time reverse transcription-polymerase chain reaction (RT-PCR) using histologically tumor-free biopsy samples from 114 patients representing three prostate cancer risk groups., Results and Limitations: We identified five genes (FOS, EGR1, MYC, TFRC, and FOLH1), which displayed significant differential expression between morphologically normal prostate tissues from men of each of the three risk groups. These results were independent from age, prostate-specific antigen (PSA), frequency and timing of previous prostate biopsies, tissue composition, tumor stage, and tumor grade. In univariate logistic regression analyses, the transcript levels of these genes were found to be highly indicative for the presence or absence of cancer in the entire prostate. The study was designed as a proof of principle. The clinical relevance of our results has to be evaluated in a larger clinical setting., Conclusions: Our results suggest a measurable molecular cancer phenotype in histologically normal prostate tissue indicating the presence of prostate cancer elsewhere in the organ.

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.

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.

Objectives: Gene expression analyses have become an important approach to understand the biology of cancer. However, transcript level patterns and RNA quality could rapidly change in response to ischemic and mechanical stress. Studies have shown that this occurs both perioperatively and after surgical removal of organs., Methods: To better understand the relative importance of perioperative and postoperative gene expression changes, we performed quantitative reverse transcription-polymerase chain reactions on the transcripts of 91 cancer-related genes from normal and cancerous prostate tissues from 10 patients at eight different time points during surgical manipulation and after removal of the prostate., Results: The mRNA levels of 8 (EGR1, p21, KRT17, PIM1, S100P, TNFRSF, WFDC2, and TRIM29) of 91 genes changed significantly with time of surgery in normal and tumor tissue. Remarkably, all eight genes were up-regulated, a reaction that was most prominent during the early intraoperative period. Additional changes occurred but were much less prominent during the first postoperative hour., Conclusions: Our results substantially challenge the utility of immediate postoperative tissue sampling. At least for prostate cancer, the data suggest that preoperative tissue collection by core biopsies is optimal for studying molecular changes in normal and neoplastic prostate tissues.

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).

Background: One of the most widely studied gene therapeutic strategies for cancer is the introduction of tumour-suppressor genes-generally p53-into the target cells. As the genes of p53 and/or retinoblastoma (Rb) are mutated in the major part of osteosarcomas (OS), we aimed to study the effect of p53 and Rb transgenes on a panel of five different osteosarcoma cell lines., Methods: OS cell lines were transduced by adenoviral vectors delivering the transcription units of the wildtype p53 and the Rb gene. Effects of the transgenes alone and at additional cytostatic stress were studied by proliferation, alive/dead and cell cycle assays., Results: The individual cells lines displayed divergent reactions to p53- or Rb-transgene delivery reaching from cell death (SaOs-2, U2OS at p53 transduction) over stopped or lowered cell division (MG-63, K-HOS, SJSA-1 at p53 and Rb transduction) to nearly unhindered cell growth (U2OS at Rb transduction). In those OS cell lines reacting with lowered cell division to p53 or Rb delivery, cytostatics only moderately intensified the transgene effects. Surprisingly, these reactions were apparently not dependent on the functional status of the cellular p53 and/or Rb genes or on differences in the infectability of the cell lines by the adenoviral vectors. Most interestingly, the respective effects of the p53 or Rb transgenes were not multiplied by simultaneous transduction of both tumour-suppressor genes., Conclusions: The application of wildtype tumour-suppressor gene therapy on genetically variable osteosarcomas may be efficient only in yet not identified genetic subgroups of this tumour entity. Hyperactive tumour-suppressor transgenes could be an alternative., (Copyright (c) 2004 John Wiley & Sons, Ltd.)

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.

In a patient with partial androgen insensitivity syndrome (AIS), we identified a single inherited presumably silent nucleotide variation (AGC -> AGT) in exon 8 (codon 888) of the AR gene. However, in the patient's genital skin fibroblasts, a considerably shortened transcript of 5.5 kb (normal: 10.5 kb) was detected, which misses a part of exon 8 and a prominent portion of the 3'-untranslated region. The translation product includes eight missense amino acids from codon 886 onward followed by a premature stop codon. As shown by in vitro expression analysis, the mutant protein lacks any residual function. However, reverse transcribed PCRs and sequence data indicate the existence of two additional splicing variants of 6.4 kb and 7.8-kb length both in patient and normal control genital skin fibroblasts. These splicing variants comprise the complete coding region but a shortened 3'-untranslated region. Thus, a distinct alternative pre-messenger RNA-processing event leading to two additional transcripts occurs generally in genital skin fibroblasts. In addition, this process partially prevents aberrant splicing in the patient and produces a small fraction of normal, functionally intact AR-protein that could explain the partial masculinization in this patient. This first report of an exonic splicing mutation in the AR-gene indicates a physiologic relevance of the regular AR-messenger RNA variants with shortened 3'-untranslated regions and their functional translation products in human genital development.

Impaired virilisation during embryonic development and pubertal arrest in patients with androgen insensitivity syndromes (AIS) is usually caused by mutations in the androgen receptor (AR)- or the 5alpha-reductase II (5RII) gene. However, identical mutations may lead to strikingly different phenotypes. To investigate whether this may be caused by individually altered transcription rates in fibroblasts from the genital region (GF) from affected patients, we applied competitive reverse transcribed PCRs (competitive RT-PCR) targeting AR- and 5RII-transcripts. We could demonstrate that AR- and 5RII-mRNA concentrations in cells from patients with partial and complete AIS and missense mutations in the AR- or 5RII-gene are normal or only moderately lowered compared to equally aged normal controls. However, in a patient bearing a premature stop-codon in the AR-gene a considerably lowered AR-transcript level was detected. We conclude, that in patients with incomplete virilisation disorders due to missense mutations, transcription regulation of AR and 5RII generally follows normal patterns. Accordingly, the premature stop-codon found in one patient's AR-gene may rather cause reduced transcript stability than an impairment of transcription activity. Therefore, altered AR- and 5RII-transcription rates in fibroblasts from the GF do not seem to be the cause for the variable genotype-phenotype correlation in androgen insensitivity syndrome.

Various mutations within the human androgen receptor gene have been documented to cause defective sexual differentiation in karyotypic male individuals. In this study, we report a previously undescribed point mutation at the donor splice-site of the second intron of the androgen receptor gene in a patient with a completely female phenotype. The sequence alteration was detected by single-strand-conformation-analysis-PCR and genomic sequencing. Applying competitive reverse transcribed PCR, cDNA sequencing and Western blotting, we could demonstrate considerable aberrations of structure and concentration of the transcript and its translation product in the patient's fibroblasts from the genital region. (1) In the transcript, exon 1 and 3 are directly linked to each other, the complete second exon is skipped. The mRNA predictively suffers a codon frame-shift in exon 3 associated with a premature termination between codons 598 and 599, leading to a truncated androgen receptor protein lacking any in vivo function. (2) Steady-state concentration levels of transcript and protein are abnormally low. Our observations highlight the influence of exon-flanking intron sequences on proper expression and function of gene products.