CD3e expression in HTLV-1-infected individuals is associated with proviral load and Tax expression

Financial support: FUNDHERP, CTC, INCTC, FAPESP, CNPq and CAPES

New Mutations in Chronic Lymphocytic Leukemia Identified by Target Enrichment and Deep Sequencing

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy

Residual Expression of the Reprogramming Factors Prevents Differentiation of iPSC Generated from Human Fibroblasts and Cord Blood CD34+ Progenitors

Human induced pluripotent stem cells (hiPSC) have been generated from different tissues, with the age of the donor, tissue source and specific cell type influencing the reprogramming process. Reprogramming hematopoietic progenitors to hiPSC may provide a very useful cellular system for modelling blood diseases. We report the generation and complete characterization of hiPSCs from human neonatal fibroblasts and cord blood (CB)-derived CD34+ hematopoietic progenitors using a single polycistronic lentiviral vector containing an excisable cassette encoding the four reprogramming factors Oct4, Klf4, Sox2 and c-myc (OKSM). The ectopic expression of OKSM was fully silenced upon reprogramming in some hiPSC clones and was not reactivated upon differentiation, whereas other hiPSC clones failed to silence the transgene expression, independently of the cell type/tissue origin. When hiPSC were induced to differentiate towards hematopoietic and neural lineages those hiPSC which had silenced OKSM ectopic expression displayed good hematopoietic and early neuroectoderm differentiation potential. In contrast, those hiPSC which failed to switch off OKSM expression were unable to differentiate towards either lineage, suggesting that the residual expression of the reprogramming factors functions as a developmental brake impairing hiPSC differentiation. Successful adenovirus-based Cre-mediated excision of the provirus OKSM cassette in CB-derived CD34+ hiPSC with residual transgene expression resulted in transgene-free hiPSC clones with significantly improved differentiation capacity. Overall, our findings confirm that residual expression of reprogramming factors impairs hiPSC differentiation

Mesenchymal stromal cells up-regulate CD39 and increase adenosine production to suppress activated T-lymphocytes. Stem Cell Res 7: 66–74

Abstract Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A 2a (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases

Microarray profiles of ex vivo expanded hematopoietic stem cells show induction of genes involved in noncanonical Wnt signaling

Zanette, Dalila Lucíola. “Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta à informação no documento”.Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-04-13T17:59:31Z No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-04-13T18:12:41Z (GMT) No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5)Made available in DSpace on 2018-04-13T18:12:41Z (GMT). No. of bitstreams: 1 Zanette DL Microarray profiles of ex vivo expanded....pdf: 429718 bytes, checksum: 44af439761969d8585f1abad49511734 (MD5) Previous issue date: 2013FAPESP and CNPqUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilCentro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Clínica Médica Ribeirão Preto, SP, BrasilUniversidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Genética. Ribeirão Preto, SP, Brasil / Centro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, BrasilCentro de Terapia Celular, Centro Regional de Hemoterapia. Ribeirão Preto, SP, Brasil / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Clínica Médica Ribeirão Preto, SP, BrasilThe low number of hematopoietic stem cells (HSC) in umbilical cord blood (UCB) is directly related to increased risk of transplant failure. Effective ex vivo expansion of HSC has been tried for many years, with conflicting results because of the inability to reproduce in vitro HSC proliferation in the same way it occurs in vivo. We compared freshly isolated HSC with their expanded counterparts by microarray analysis and detected activation of the noncanonical Wnt (wingless-type MMTV integration site family) pathway. Study of early alterations during ex vivo UCB-HSC expansion could contribute to improvement of ex vivo expansion systems

Lymph node or perineural invasion is associated with low miR-15a, miR-34c and miR-199b levels in head and neck squamous cell carcinoma

Background: MicroRNAs (miRNAs or miRs) are post-transcriptional regulators of eukaryotic cells and knowledge of differences in miR levels may provide new approaches to diagnosis and therapy. Methods: The present study measured the levels of nine miRs in head and neck squamous cell carcinomas (HNSCC) and determined whether clinical pathological features are associated with differences in miR levels. SET (I2PP2A) and PTEN protein levels were also measured, since their levels can be regulated by miR-199b and miR-21, respectively. Nine miRs (miR-15a, miR-21, miR-29b, miR-34c, miR-100, miR-125b, miR-137, miR-133b and miR-199b) were measured by real time qRT-PCR in HNSCC samples from 32 patients and eight resection margins. SET (I2PP2A) and PTEN protein levels were estimated by immunohistochemistry in paired HNSCC tissues and their matched resection margins. Results: In HNSCC, the presence of lymph node invasion was associated with low miR-15a, miR-34c and miR-199b levels, whereas the presence of perineural invasion was associated with low miR-199b levels. In addition, miR-21 levels were high whereas miR-100 and miR-125b levels were low in HNSCC compared to the resection margins. When HNSCC line HN12, with or without knockdown of SET, were transfected with miR-34c inhibitor or miR-34c mimic, the miR-34c inhibitor increased cell invasion capacity while miR-34c mimic decreased the cell invasion. Conclusions: We showed that the levels of specific miRs in tumor tissue can provide insight into the maintenance and progression of HNSCC. General significance: MiRNAs are up- or down-regulated during cancer development and progression; they can be prognosis markers and therapeutic targets in HNSCC

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGAS gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). the RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. the differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, EPM, Discipline Hematol & Hemotherapy, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol, Diadema, BrazilUniv São Paulo, Fac Med Ribeirao Preto, São Paulo, BrazilLudwig Inst Canc Res, New York Branch, New York, NY USAUniversidade Federal de São Paulo, EPM, Discipline Hematol & Hemotherapy, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biol, Diadema, BrazilFAPESP: 04/13213-3FAPESP: 04/12855-1Web of Scienc

Number of expressed cancer/testis antigens identifies focal adhesion pathway genes as possible targets for multiple myeloma therapy

Considering that the importance of cancer/testis (CT) antigens in multiple myeloma (MM) biology is still under investigation, the present study aimed to: (1) identify genes differentially expressed in MM using microarray analysis of plasma cell samples, separated according to the number of expressed CTs; (2) examine possible pathways related to MM pathogenesis; (3) validate the expression of candidate genes by quantitative real-time PCR (RQ-PCR). Three samples predominantly positive (>6 expressed), including the U266 cell line, and three samples predominantly negative (0 or 1 expressed CT for the 13 analyzed CT antigens), were submitted for microarray analysis. Validation by RQ-PCR from 24 MM samples showed that the ITGAS gene was downregulated in predominantly positive (>6 expressed CTs, p = 0.0030) and in tumor versus normal plasma cells (p = 0.0182). The RhoD gene was overexpressed in tumor plasma cells when compared to normal plasma cells (p = 0.0339). Results of the microarray analysis corroborate the hypothesis that MM could be separated into predominantly positive and predominantly negative expression. The differential expression of ITGA5 and RhoD suggests disruption of the focal adhesion pathway in MM and offers a new target field to be explored in this disease.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo, Brazil)[04/13213-3]FAPESP[04/12855-1