Evaluation of the integrity of human corneal epithelium maintained in organ-culture during eye banking using Corneamax®

Purpose: Mediums have been developed to conserve corneal endothelium in organ-culture during eye banking. CorneaMax® is used by 25% of Eye Bank in Europe. Only little is known about conservation of corneal epithelium with this medium during banking. Its preservation could be of interest in clinic to cure corneal disease with stem cells deficiency. Therefore, we wanted to examine the integrity of human corneal epithelium maintained in CorneaMax®. Methods: Human corneas, considered unsuitable for transplantation, were obtained from the Eye Bank in Lausanne. Average post-mortem time was 14 hours. Cornoscleral rings were maintained in organ-culture in Corneamax® at 32°C. Samples were formalin-fixed after period ranging from 0 (D0) to 35 days (D35, N=5 for each time points) and stained with H&E. Proliferation and apoptosis were evaluated by immunostaining with antibody against Ki67 and Caspase3 respectively. Results: Corneas, which were not in organ-cultured (D0), showed different morphology, including intact epithelium with 5 to 7 layers, but also completely denuded basement membrane. In two cases, at D0, the epithelium lost its adherence to the basal lamina of the cornea creating a large epithelial sheet. During the two first days, corneas and limbus area lost totally their epithelium, except for some remaining limbal basal cells. From day 2 to day 10, regeneration of the epithelium took place, starting from the limbal region in direction to the central cornea. From day 10 to day 35, corneal epithelium appeared as an atrophic epithelium, consisting of only two cell layers. Proliferation happened in the whole cornea during the 35 days of organ-culture, as shown by Ki67 positive cells. Apoptosis was rarely detected in the corneal epithelium. Conclusions: Corneas maintained in CorneaMax® showed a complete disappearance of the corneal epithelium during the two first days and a conservation of limbal basal cells in the limbal region. These remaining cells allowed a full regeneration of the tissue, leading to an atrophic epithelium, composed of only two cell layers. This atrophic epithelium could be seen in all the organ-cultured corneas during the 35 days of conservation. This study is a first step to develop medium in organ-culture in order to conserve corneal epithelial cells.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

Molecular mechanisms of follicular associated epithelium differentiation

Les muqueuses respiratoires, genitales et digestives sont continuellement exposées aux antigènes de l?alimentation, à la flore intestinale et aux pathogènes. Cela implique une activité immunologique intense et finement régulée dans ces tissus. On admet que la modulation de ces réponses immunitaires muqueuses s?effectue dans des organes sentinels spécifiques appelés o-MALT (organized mucosal associated lymphoid tissues). Ces processus de modulation et la biologie de ces sites immuno-inducteurs sont peu connus. Ceci est pourtant d?une grande relevance si l?on veut faire un design rationnel de drogues et de vaccins muqueux. Dans l?intestin grèle, ces organes sont composés de follicules multiples et sont appelés plaques de Peyer. Ils sont constitués de follicules enrichis en cellules B comprenant ou non un centre germinatif, de regions interfolliculaires comprenant des cellules T, et d?une région en d ome riche en cellules dendritiques, cellules B naives et cellules T CD4+, surmontée par un epithelium specialisé, le FAE (epithelium associé aux follicules). Le FAE contient des cellules M spécialisées dans le transport de macromolécules et micro-organismes de la lumière intestinale au tissu lymphoide sous-jacent. Ce transport des antigènes est une condition obligatoire pour induire une réponse immunitaire. Les cellules du FAE, outre les cellules M, expriment un programme de différenciation distinct de celui des cellules associées aux villosités. Ceci est characterisé par une baisse des fonctions digestives et de défenses, et l?expression constitutive des chimiokines: CCL20 et CCL25. Le but de l?étude présentée ici est de rechercher les facteurs cellulaires et/ou moléculaire responsables de cette différenciation. Certaines études ont démontré l?importance du contact entre le compartiment mésenchymateux et l?épithelium pour la morphogenèse de ce dernier. En particulier, les molécules de la matrice extracellulaire peuvent activer des gènes clefs qui, à leur tour, vont controler l?adhésion et la differenciation cellulaire. Dans l?intestin, les cellules mésenchymateuses différencient en myofibroblastes qui participent à l?élaboration de la matrice extracellulaire. Dans cette étude, nous avons décrit les différences d?expression de molécules de la matrices sous le FAE et les villosités. Nous avons également montré une absence de myofibroblastes sous le FAE. Suite à plusieurs évidences expérimentales, certains ont proposé une influence des composés présents dans la lumière sur la différenciation et/ou la maturation des plaques de Peyer. La chimiokine CCL20, capable de recruter des cellules initiatrices de la réponse immunitaire, constitue notre seul marqueur positif de FAE. Nous avons pu montrer que la flagelline, un composé du flagelle bactérien, était capable d?induire l?expression de CCL20 in vitro et in vivo. Cet effet n?est pas limité aux cellules du FAE mais est observé sur l?ensemble de l?épithelium intestinal. Molecular mechanisms of FAE differenciation. La signalement induit par la lymphotoxine ß est critique pour l?organogenèse des plaques de Peyer, car des souris déficientes pour cette molécules ou son récepteur n?ont ni plaque de Peyer, ni la plupart des ganglions lymphatiques. Nous avons obtenus plusieurs évidences que la lymphotoxine ß était impliquée dans la régulation du gène CCL20 in vitro et in vivo.<br/><br/>Mucosal surfaces of the respiratory, genital and digestive systems are exposed to food antigens, normal bacterial flora and oral pathogens. This justifies an intense and tuned immunological activity in mucosal tissues. The modulation of immune responses in the mucosa is thought to occur in specific sentinel sites, the organized mucosa associated lymphoid tissues (o-MALT). This immune modulation and the biology of these immune-inductive sites are poorly understood but highly important and relevant in the case of drugs and vaccines design. In the small intestine, these organs (gut associated lymphoid tissue : GALT) consists of single or multiple lymphoid follicles, the so-called Peyer?s patches (PP), with typical B cell-enriched follicles and germinal centers, inter-follicular T cell areas, and a dome region enriched in dendritic cells, naive B cells, and CD4+ T cells under a specialized follicle associated epithelium (FAE). To trigger protective immunity, antigens have to cross the mucosal epithelial barrier. This is achieved by the specialized epithelial M cells of the FAE that are able to take up and transport macromolecules and microorganisms from the environment into the underlying organized lymphoid tissue. The ontogeny of M cells remains controversial: some data are in favor of a distinct cell lineage, while others provide evidence for the conversion of differentiated enterocytes into M cells. In this study we mapped the proliferative, M cells and apoptotic compartments along the FAE. Enterocytes acquire transient M cell features as they leave the crypt and regain enterocyte properties as they move towards the apoptotic compartment at the apex of the FAE, favouring the hypothesis of a plastic phenotype. The follicle-associated epithelium (FAE) is found exclusively over lymphoid follicles in mucosal tissues, including Peyer?s patches. The enterocytes over Peyer?s patches express a distinct phenotype when compared to the villi enterocytes, characterized by the down regulation of digestive and defense functions and the constitutive expression of chemokines, i.e. CCL20 and CCL25. The purpose of this study was to investigate and identify the potential cells and/or molecules instructing FAE differentiation. Contact between the epithelial and the mesenchymal cell compartment is required for gut morphogenesis. Extracellular matrix molecules (ECM) can activate key regulatory genes which in turn control cell adhesion and differentiation. In the gut, mesenchymal cells differentiate into myofibroblats that participate to the elaboration of ECM. We have described a differential expression of extracellular matrix components under the FAE, correlating with the absence of subepithelial myofibroblats. Molecular mechanisms of FAE differenciation. Different studies proposed an influence of the luminal compartment in the differentiation and/or the maturation of PP. CCL20, a chemokine able to recruit cells that initiate adaptive immunity constitutes our first positive FAE molecular marker. We have shown that CCL20 gene expression is inducible in vitro and in vivo in intestinal epithelium by flagellin, a component of bacterial flagella. This effect was not restricted to the FAE. Lymphotoxin ß (LTß) signaling is critical for PPs organogenesis as LT deficient mice as well as LTß-receptor-/- mice lack PPs and most of the lymph nodes (LN). The continuous signaling via LTßR-expressing cells appears necessary for the maintenance throughout the life of PP architecture. We obtained in vitro and in vivo evidence that LTß signalling is involved in CCL20 gene expression.

Identifying the index lesion with template prostate mapping biopsies.

PURPOSE: The natural history of prostate cancer might be driven by the index lesion. We determined the percent of men in whom the index lesion could be defined using transperineal template prostate mapping biopsies. MATERIALS AND METHODS: Included in study were consecutive men undergoing transperineal template prostate mapping biopsies with biopsies grouped into 20 zones. Men with clinically significant disease in only 1 prostate area were considered to have an identifiable index lesion. We evaluated the impact of using 2 definitions of clinically significant disease (Gleason grade pattern 4 and/or lesion volume 0.5 cc or greater) and 2 clustering rules (stringent and tolerant) to define the index lesion. RESULTS: Included in study were 391 men with a median age of 62 years (IQR 58-67) and a median prostate specific antigen of 6.9 ng/ml (IQR 4.8-10.0). Of the men 269 (69%) were previously diagnosed with prostate cancer. By deploying a median of 1.2 cores per ml (IQR 0.9-1.7) cancer was diagnosed in 82.9% of the men (324 of 391) with a median of 6 positive cores (IQR 2-9), a median maximum cancer core length of 5 mm (IQR 3-8) and a total cancer core length per zone of 7 mm (IQR 3-13). Insignificant disease was found in 26.3% to 42.9% of cases. When a stringent spatial relationship was used to define individual lesions, 44.4% to 54.6% of patients had 1 index lesion and 12.7% to 19.1% had more than 1 area with clinically significant disease. These proportions changed to 46.6% to 59.2% and 10.5% to 14.5%, respectively, when less stringent spatial clustering was applied. CONCLUSIONS: Transperineal template prostate mapping biopsies enable the index lesion to be localized in most men with clinically significant disease. This information may be important to select appropriate candidates for targeted therapy and to plan a tailored treatment strategy in men undergoing radical therapy.

Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen–positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

Abstract Background: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions: miR-143 is as a new target for prostate cancer treatment.

Comparison of quality of life after stereotactic body radiotherapy and surgery for early-stage prostate cancer

Background: As the long-term efficacy of stereotactic body radiation therapy (SBRT) becomes established and other prostate cancer treatment approaches are refined and improved, examination of quality of life (QOL) following prostate cancer treatment is critical in driving both patient and clinical treatment decisions. We present the first study to compare QOL after SBRT and radical prostatectomy, with QOL assessed at approximately the same times pre- and post-treatment and using the same validated QOL instrument. Methods: Patients with clinically localized prostate cancer were treated with either radical prostatectomy (n = 123 Spanish patients) or SBRT (n = 216 American patients). QOL was assessed using the Expanded Prostate Cancer Index Composite (EPIC) grouped into urinary, sexual, and bowel domains. For comparison purposes, SBRT EPIC data at baseline, 3 weeks, 5, 11, 24, and 36 months were compared to surgery data at baseline, 1, 6, 12, 24,and 36 months. Differences in patient characteristics between the two groups were assessed using Chi-squared tests for categorical variables and t-tests for continuous variables. Generalized estimating equation (GEE) models were constructed for each EPIC scale to account for correlation among repeated measures and used to assess the effect of treatment on QOL. Results: The largest differences in QOL occurred in the first 1-6 months after treatment, with larger declines following surgery in urinary and sexual QOL as compared to SBRT, and a larger decline in bowel QOL following SBRT as compared to surgery. Long-term urinary and sexual QOL declines remained clinically significantly lower for surgery patients but not for SBRT patients. Conclusions: Overall, these results may have implications for patient and physician clinical decision making which are often influenced by QOL. These differences in sexual, urinary and bowel QOL should be closely considered in selecting the right treatment, especially in evaluating the value of non-invasive treatments, such as SBRT.

Is high dose rate brachytherapy reliable and effective treatment for prostate cancer patients? A review of the literature.

The intrinsic physical and radiobiological characteristics of High Dose Rate Brachytherapy (HDR-BT) are well suited to the treatment of prostate cancer. HDR-BT was initially used as a boost to external beam brachytherapy, but has subsequently been employed as the sole treatment, which is termed HDR monotherapy. This review summarizes the clinical outcomes and toxicity results of the principal studies and discusses the radiobiological basis supporting its use.

Detection of Clinically Significant Prostate Cancer Using Magnetic Resonance Imaging-Ultrasound Fusion Targeted Biopsy: A Systematic Review.

CONTEXT: The current standard for diagnosing prostate cancer in men at risk relies on a transrectal ultrasound-guided biopsy test that is blind to the location of the cancer. To increase the accuracy of this diagnostic pathway, a software-based magnetic resonance imaging-ultrasound (MRI-US) fusion targeted biopsy approach has been proposed. OBJECTIVE: Our main objective was to compare the detection rate of clinically significant prostate cancer with software-based MRI-US fusion targeted biopsy against standard biopsy. The two strategies were also compared in terms of detection of all cancers, sampling utility and efficiency, and rate of serious adverse events. The outcomes of different targeted approaches were also compared. EVIDENCE ACQUISITION: We performed a systematic review of PubMed/Medline, Embase (via Ovid), and Cochrane Review databases in December 2013 following the Preferred Reported Items for Systematic reviews and Meta-analysis statement. The risk of bias was evaluated using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. EVIDENCE SYNTHESIS: Fourteen papers reporting the outcomes of 15 studies (n=2293; range: 13-582) were included. We found that MRI-US fusion targeted biopsies detect more clinically significant cancers (median: 33.3% vs 23.6%; range: 13.2-50% vs 4.8-52%) using fewer cores (median: 9.2 vs 37.1) compared with standard biopsy techniques, respectively. Some studies showed a lower detection rate of all cancer (median: 50.5% vs 43.4%; range: 23.7-82.1% vs 14.3-59%). MRI-US fusion targeted biopsy was able to detect some clinically significant cancers that would have been missed by using only standard biopsy (median: 9.1%; range: 5-16.2%). It was not possible to determine which of the two biopsy approaches led most to serious adverse events because standard and targeted biopsies were performed in the same session. Software-based MRI-US fusion targeted biopsy detected more clinically significant disease than visual targeted biopsy in the only study reporting on this outcome (20.3% vs 15.1%). CONCLUSIONS: Software-based MRI-US fusion targeted biopsy seems to detect more clinically significant cancers deploying fewer cores than standard biopsy. Because there was significant study heterogeneity in patient inclusion, definition of significant cancer, and the protocol used to conduct the standard biopsy, these findings need to be confirmed by further large multicentre validating studies. PATIENT SUMMARY: We compared the ability of standard biopsy to diagnose prostate cancer against a novel approach using software to overlay the images from magnetic resonance imaging and ultrasound to guide biopsies towards the suspicious areas of the prostate. We found consistent findings showing the superiority of this novel targeted approach, although further high-quality evidence is needed to change current practice.

Localized prostate cancer in Norway, the United States, and Spain: between-country differences of variables before treatment among patients eligible for curative treatment

Between-country differences in medical and sociodemographic variables, and patient-related outcomes (PROs) before treatment might explain published variations of side effects after radical prostatecomy (RP) or radiotherapy (RAD) for prostate cancer (PCa). This hypothesis was tested among 1908 patients from the United States, Spain, and Norway. Significant between-country differences were observed for most factors investigated before treatment. The observations should be considered in comparison of the frequency and severity of internationally published studies. Background: In men with PCa, large variations of PROs after RP or high-dose RAD might be related to betweencountry differences of medical and sociodemographic variables, and differences in PROs before treatment in the sexual and urinary domains. Patients and Methods: In 1908 patients with localized PCa from Norway, the United States, or Spain, the relation between medical (prostate-specific antigen, Gleason score, cT-category) and sociodemographic variables (age, education, marital status) before treatment was investigated. Using the Expanded Prostate Cancer Index Composite questionnaire, PROs before treatment within the sexual and urinary domains were also considered. Results: Compared with the European patients, American patients were younger, fewer had comorbid conditions, and more had a high education level. Fifty-three percent of the US men eligible for RP had low-risk tumors compared with 42% and 31% among the Norwegian and the Spanish patients, respectively. Among the Spanish RAD patients, 54% had had low-risk tumors compared with 34% of the American and 21% of the Norwegian men planned for RAD, respectively. Compared with the European patients, significantly fewer US patients reported moderate or severe sexual dysfunction and related problems. In most subgroups, the number of patients with sexual or urinary dysfunction exceeded that of patients with bother related to the reported dysfunction. Conclusion: Statistically significant between-country differences were observed in medical and sociodemographic variables, and in PROs before treatment within the sexual and urinary domains. Large differences between reported dysfunction and related problems within the sexual and urinary domains indicate that dysfunction and bother should be reported separately in addition to calculation of summary scores. The documented differences, not at least regarding PROs, might in part explain the large variation of side effects after treatment evident in the medical literature