Development of Humanized Mouse Model for Organ Transplantation

Purpose of Study: Solid organ transplantation has been a life-saving procedure for thousands of patients worldwide. Recent advances on improving donor-screening diagnostics have aimed at identification of the most compatible donor for the transplant recipient to maximize allograft survival. Current standards of donor selection relies on HLA typing and in vitro mixed lymphocyte reaction (MLR) which do not take into account the in vivo environment and recipient’s adaptive immune response. Humanized mouse models are an appealing alternative that permits personalized investigation of the immunocompatibility of potential donor tissues for the recipient human immune system without putting patients at risk. By utilizing genomics, molecular and cellular analyses of allogeneic immune response we analyze the efficiency of our novel humanized mouse model to assess the donor-recipient compatibility and determine that it to be significantly more sensitive than conventional screening methods. Methods Used: Human Leukocyte Antigen (HLA) typing and MLR for histocompatibility. Special strain of immunodeficient mice, NSG mice, subjected to irradiation (2Gy) and i.v injection of 8×10 peripheral blood mononuclear cells (PBMCs) from transplant recipients. For allogeneic immune response, humanized mice received 3×10 PBMCs from unrelated donors (UD) or related donors(RD). Whole genome transcriptome analysis and Real-Time PCR (RT-PCR) Transplant Rejection Array was used. Summary of Results: Humanized mice demonstrated that allogeneic UD challenge induced significant splenomegaly with infiltration of activated cytotoxic human CD8+ CD25+ T cells expressing Perforin, Granzyme B and Interferon gamma (IFN-γ). Amongst the RDs, RD1 showed minimal allogeneic response while RD2 promoted higher cytotoxic CD8+ T cells infiltration, indicating that RD1 has better immunocompatibility with the recipient than RD2. However, MLR and HLA typing had failed to differentiate the 2 RDs showing them to have equal immunocompatibility with the recipient. Conclusions: NSG-PBMC humanized mouse model was able to identify the related donor exhibiting minimal allogeneic response to the recipient. This model is significantly more immunologically sensitive than conventional MLR and HLA typing for selection of an immunocompatible donor for the transplant recipient

Achievement of generic and profesional competencies through virtual environments

This study is aimed to prove how Virtual Environments (VE) and Information and Communication Technologies (ICT) can be used as a tool to verify professional competencies. The incursion of virtual environments in education has shown that there is much potential in distance learning development. To find out how it influences the achievement of competencies, there was made an experimental study with a post-test design and control group. Students were divided into two groups; each of them was submitted to a different test. The results demonstrate that with the implementation of VE using ICT, the students who used the VE had a better performance than students who used the traditional evaluations. Confirmed with the 83% of the sample who achieved the highest levels (50% got strategical professional competencies, and 33% got autonomous professional competencies). Considering the study, the authors could notice that students do develop professional competencies along virtual environments, reflected not only in the level of competence achieved by the ones tested on the virtual environment but also in the average time they spend to do the test. Therefore, virtual environments have positives effects in the education field

miR-221 affects multiple cancer pathways by modulating the level of hundreds messenger RNAs.

microRNA miR-221 is frequently over-expressed in a variety of human neoplasms. Aim of this study was to identify new miR-221 gene targets to improve our understanding on the molecular tumor-promoting mechanisms affected by miR-221. Gene expression profiling of miR-221-transfected-SNU-398 cells was analyzed by the Sylamer algorithm to verify the enrichment of miR-221 targets among down-modulated genes. This analysis revealed that enforced expression of miR-221 in SNU-398 cells caused the down-regulation of 602 mRNAs carrying sequences homologous to miR-221 seed sequence within their 3'UTRs. Pathways analysis performed on these genes revealed their prominent involvement in cell proliferation and apoptosis. Activation of E2F, MYC, NFkB, and β-catenin pathways was experimentally proven. Some of the new miR-221 target genes, including RB1, WEE1 (cell cycle inhibitors), APAF1 (pro-apoptotic), ANXA1, CTCF (transcriptional repressor), were individually validated as miR-221 targets in SNU-398, HepG2, and HEK293 cell lines. By identifying a large set of miR-221 gene targets, this study improves our knowledge about miR-221 molecular mechanisms involved in tumorigenesis. The modulation of mRNA level of 602 genes confirms the ability of miR-221 to promote cancer by affecting multiple oncogenic pathways

High specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG positive Ewing sarcoma

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.Barbara und Hubertus Trettner foundationDeutsche Forschungsgemeinschaft DFG 391665916Deutsche Stiftung fur junge Erwachsene mit KrebsDr. Leopold und Carmen Ellinger foundationDr. Rolf M. Schwiete foundationDr. Rudolf und Brigitte Zenner StiftungFriedrich-Baur foundationGerman Cancer Aid DKH-70112257German Cancer Aid DKH-108128German Cancer Aid DKH-70112018German Cancer Aid DKH-70113421

Ante-mortem and Post-mortem Inspection and Relationship between Findings in a North Albanian Pig Slaughterhouse

Simple Summary In European Union abattoirs, the safety of meat is dependent on the favorable opinion from an official veterinarian, in accordance with the current legislation. From this perspective, the feedback generated from the ante-mortem visit and the post-mortem inspection can be investigated to control the health and welfare conditions of the animals in the pre-slaughter phases. From this perspective, we evaluated the ante-mortem and post-mortem inspection outcomes of slaughtered pigs in northern Albania and correlated the results in order to gain insight into the conditions and injuries of pigs slaughtered outside the European context and to extend knowledge on the possible relationship between ante-mortem and post-mortem relief. Dyspnea and tail, skin, and ear lesions were the most frequently observed conditions before slaughter, while pleuritis, pneumonia, liver alterations, white spots on the liver, and pericarditis were the most frequent lesions after slaughter. A significant increase in the total number of post-mortem findings was also observed as the number of ante-mortem findings increased. Overall, the prevalence of the findings observed in this study falls within the broad range of the data in the literature, but additional information should be collected during meat inspection so as to better understand the relationship between ante- and post-mortem outcomes. In June 2014, Albania was granted EU candidate status, thus starting a process of compliance with the membership criteria. In this context, a modern meat inspection approach in line with the European legislation was applied to a pig slaughterhouse in northern Albania in order to investigate the ante-mortem (AM) and post-mortem (PM) conditions and the relationship between these findings. For this purpose, 3930 pigs divided into 35 batches were evaluated over a 3-month period. The most frequent AM conditions recorded were tail lesions and dyspnea (9.1%), followed by skin (8.9%) and ear lesions (8.5%), while in the PM inspections, pleuritis was the most frequently observed condition (10.2%), followed by pneumonia (8.5%), liver alterations (5.7%), milk spot liver (3.8%), and pericarditis (3.3%). With the exception of liver alterations, the other PM lesions mentioned were positively associated with lesions on the ears (OR = 1.036; p < 0.001) and skin (OR = 1.026; p = 0.011) and dyspnea (OR = 1.021; p = 0.005), confirming the link between these variables and the health and welfare conditions of pigs on farms. Overall, the evidence that emerged from this Albanian slaughterhouse can be considered in line with other European contexts, especially in light of the considerable variability in the data present in the literature

Integrative clinical transcriptome analysis reveals TMPRSS2‐ERG dependency of prognostic biomarkers in prostate adenocarcinoma

In prostate adenocarcinoma (PCa), distinction between indolent and aggressive disease is challenging. Around 50% of PCa are characterized by TMPRSS2‐ERG (T2E)‐fusion oncoproteins defining two molecular subtypes (T2E‐positive/negative). However, current prognostic tests do not differ between both molecular subtypes, which might affect outcome prediction. To investigate gene‐signatures associated with metastasis in T2E‐positive and T2E‐negative PCa independently, we integrated tumor transcriptomes and clinicopathological data of two cohorts (total n = 783), and analyzed metastasis‐associated gene‐signatures regarding the T2E‐status. Here, we show that the prognostic value of biomarkers in PCa critically depends on the T2E‐status. Using gene‐set enrichment analyses, we uncovered that metastatic T2E‐positive and T2E‐negative PCa are characterized by distinct gene‐signatures. In addition, by testing genes shared by several functional gene‐signatures for their association with event‐free survival in a validation cohort (n = 272), we identified five genes (ASPN, BGN, COL1A1, RRM2 and TYMS)—three of which are included in commercially available prognostic tests—whose high expression was significantly associated with worse outcome exclusively in T2E‐negative PCa. Among these genes, RRM2 and TYMS were validated by immunohistochemistry in another validation cohort (n = 135), and several of them proved to add prognostic information to current clinicopathological predictors, such as Gleason score, exclusively for T2E‐negative patients. No prognostic biomarkers were identified exclusively for T2E‐positive tumors. Collectively, our study discovers that the T2E‐status, which is per se not a strong prognostic biomarker, crucially determines the prognostic value of other biomarkers. Our data suggest that the molecular subtype needs to be considered when applying prognostic biomarkers for outcome prediction in PCa