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There is high medical need for safe long-term immunosuppression monotherapy in kidney transplantation. Selective targeting of post-transplant alloantigen-(re)activated effector-T cells by anti-TNF antibodies after global T cell depletion may allow safe drug minimization, however, it is unsolved what might be the best maintenance monotherapy. In this open, prospective observational single-centre trial, 20 primary deceased donor kidney transplant recipients received 2x20 mg Alemtuzumab (d0/d1) followed by 5 mg/kg Infliximab (d2). For 14 days all patients received only tacrolimus, then they were allocated to either receive tacrolimus (TAC, n = 13) or sirolimus (SIR, n = 7) monotherapy, respectively. Protocol biopsies and extensive immune monitoring were performed and patients were followed-up for 60 months. TAC-monotherapy resulted in excellent graft survival (5yr 92%, 95%CI: 56.6-98.9) and function, normal histology, and no proteinuria. Immune monitoring revealed low intragraft inflammation (urinary IP-10) and hints for the development of operational tolerance signature in the TAC- but not SIR-group. Remarkably, the TAC-monotherapy was successful in all five presensitized (ELISPOT+) patients. However, recruitment into SIR-arm was stopped (after n = 7) because of high incidence of proteinuria and acute/chronic rejection in biopsies. No opportunistic infections occurred during follow-up. In conclusion, our novel fast-track TAC-monotherapy protocol is likely to be safe and preliminary results indicated an excellent 5-year outcome, however, a full-scale study will be needed to confirm our findings. EudraCT Number: 2006-003110-18.

CD133+ cells are hemangioblasts that have capacity to generate into both hematopoietic and endothelial cells (ECs). Hypoxia/normoxia has shown to be the regulator of the balance between stemness and differentiation. In this study we performed Agilent's whole human genome oligo microarray analysis and examined the differentiation potential of the bone-marrow-derived CD133+ cells after hypoxic/normoxic preconditioning of CD133+ cells. Results showed that there was no significant increase in erythroid colony forming unit (CFU-E) and CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte formation with cells treated under hypoxia/normoxia. However, a significant increment of EC forming unit at 24 h (143.2 ± 8.0%) compared to 0 h (100 ±11.4%) was observed in CFU-EC analysis. Reverse transcription–polymerase chain reaction and immunostaining analysis showed that the differentiated cells diminished hematopoietic stem cell surface markers and acquired the gene markers and functional phenotype of ECs. The transcriptome profile revealed a cluster of 232 downregulated and 498 upregulated genes in cells treated for 24 h under hypoxia. The upregulated genes include angiogenic genes, angiogenic growth factor genes, angiogenic cytokine and chemokine genes, as well as angiogenic-positive regulatory genes, including FGFBP1 , PDGFB , CCL15 , CXCL12 , CXCL6 , IL-6 , PTN , EREG , ERBB2 , EDG5 , FGF3 , FHF2 , GDF15 , JUN , L1CAM , NRG1 , NGFR , and PDGFB . On the other hand, angiogenesis inhibitors and related genes, including IL12A , MLLT7 , STAB1 , and TIMP2 , are downregulated. Taken together, hypoxic/normoxic preconditioning may lead to the differentiation of CD133+ cells toward endothelial lineage, which may improve the current clinical trial studies.

12 microRNA expression profiling platforms are compared for their reproducibility, sensitivity, accuracy and specificity, and the strengths and weaknesses of each platform are discussed. MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription–quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

Background Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. Methods Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). Results Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. Conclusions The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Introduction Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. Methods We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. Results High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP- N -acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. Conclusions In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

About 10% of hip endoprostheses will loosen after 10 years. Prosthesis loosening is caused by two different pathomechanisms: aseptic loosening (AL) and septic loosening (SL). This study evaluated differences in gene expression in AL and SL. Eight hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from two patients with SL and three patients with AL. Tissue parts directly adjacent to the site of RNA isolation were analyzed immuno/histopathologically in order to overcome the problem of tissue heterogeneity. Thirty-three genes were found constantly differentially expressed, among which were cd11b, cd18, cd68, osteopontin and ferritin heavy-chain upregulated in AL and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin2 and nidogen upregulated in SL. The most striking finding was the strong upregulation (from 20-fold to 323-fold) of megakaryocyte stimulating factor (msf) in all aseptic cases and one of the two septic cases, which was confirmed by real-time reverse transcription-polymerase chain reaction. In this study, msf is linked to prosthesis loosening for the first time. The upregulation in AL suggests an important pathogenetic role: the msf splice product lubricin is responsible for the lubrication of healthy joints, but its excellent lubrication ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52–54°C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Nat. Methods 11, 809–815 (2014); published online 29 June 2014; corrected after print 30 July 2014 In the version of this article initially published, the author Linda Wong was omitted from the author list. The error has been corrected in the HTML and PDF versions of the article.