43 research outputs found
MĂ©canismes molĂ©culaires et impacts pathophysiologiques de la rĂ©gulation de l'expression de la cyclooxygĂ©nase-2 par les cytokines proinflammatoires interleukine-1[bĂȘta] et interleukine-17 dans les synoviocytes et les chondrocytes humains
ThÚse numérisée par la Direction des bibliothÚques de l'Université de Montréal
Design and implementation of the European-Mediterranean Postgraduate Programme on Organ Donation and Transplantation (EMPODaT) for Middle East/North Africa countries
This prospective study reports the design and results obtained after the EMPODaT project implementation. This project was funded by the Tempus programme of the European Commission with the objective to implement a common postgraduate programme on organ donation and transplantation (ODT) in six selected universities from Middle East/North Africa (MENA) countries (Egypt, Lebanon and Morocco). The consortium, coordinated by the University of Barcelona, included universities from Spain, Germany, Sweden and France. The first phase of the project was to perform an analysis of the current situation in the beneficiary countries, including existing training programmes on ODT, Internet connection, digital facilities and competences, training needs, and ODT activity and accreditation requirements. A total of 90 healthcare postgraduate students participated in the 1-year training programme (30 ECTS academic credits). The methodology was based on e-learning modules and face-to-face courses in English and French. Training activities were evaluated through pre- and post-tests, self-assessment activities and evaluation charts. Quality was assessed through questionnaires and semi-structured interviews. The project results on a reproducible and innovative international postgraduate programme, improvement of knowledge, satisfaction of the participants and confirms the need on professionalizing the activity as the cornerstone to ensure organ transplantation self-sufficiency in MENA countries
Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method
The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels) and reduced into smaller fat pieces of around 1â3Â mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in âProliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs) into osteoblastic lineage are passage dependentâ [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling tim
A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks
Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent âxyleneâ and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent âxyleneâ. Second minimize the time required to perform the extraction. Here we describe a method where:
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The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol
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The entire procedure takes about 15Â min
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Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot.
With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years
The immunomodulatory properties of human bone marrow-derived mesenchymal stromal cells are defined according to multiple immunobiological criteria
Objective: Human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are well known to modulate T cells. However, the molecular mechanisms that mark hBM-MSCs immunomodulation of T cells are not fully resolved. Materials and methods: hBM-MSCs harvested from sternum or iliac crest of five healthy donors and characterized in accordance with the International Society of Cellular Therapy (ISCT) guidelines are co-cultured with T cells. Additionally, modulatory effects of MSCs on T-cell viability, proliferation, cytokine profile, co-stimulatory pathway, activation and immunomodulation are also determined. Results: hBM-MSCs significantly reduced the expression of T-cell activation marker CD38 as well as co-stimulatory markers CD134 and CD154, whilst that of CD27 remained unchanged. BrdU, CFSE and Ki67 proliferation assays showed that hBM-MSCs reduced T-cell proliferation. Moreover, viability of T cells remained unchanged when co-cultured with hBM-MSCs. Finally, T cells when co-cultured with hBM-MSCs showed increased secretion of IL-10 and IL-11. Conclusion: Collectively, hBM-MSCs are able to modulate the main steps involved in T-cell response toward a tolerogenic state. Thus, establishing immunobiological criteria defining the immunosuppressive effect of hBM-MSCs is of importance to reach efficient immunotherapeutic intervention.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
Breast cancer cells and bone marrow mesenchymal stromal cells: a regulated modulation of the breast tumor in the context of immune response.
The role of direct cell-cell interactions mediating selective bone metastasis by breast cancer cells (BCCs) niche is still mostly unknown.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
G-quadruplex forming sequences in the genes coding for cytochrome P450 enzymes and their potential roles in drug metabolism
International audienceThe majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in cytochrome genes regulation, and emphasize the importance of G-quadruplexes in drug metabolism
An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis
Deparaffinization of formalin-fixed paraffin
embedded (FFPE) tissues with xylene currently remains
a major challenge to the biomedical community. We
developed an efficient xylene-free protocol to isolate
proteins from archived FFPE human tissue sections. A
total of 79 different types of FFPE tissue sections of 8
”m thickness were obtained from various archived FFPE
specimens. Deparaffinization was conducted by gently
washing each section with around 1 ml of hot distilled
water (â80°C). The deparaffinized tissues were
homogenized in lysis buffer, and the isolated proteins
were quantified and efficiently resolved using western
blot analysis for the presence of Protein kinase B
(PKB/AKT) and ÎČ-actin. Moreover, a significant amount
of proteins was successfully isolated with an average of
2.31 ”g/”l. The migration pattern of AKT and ÎČ-actin
obtained from the specimens was similar to the positive
control obtained from protein lysates prepared from in
vitro cultured MDA231 cancer cell lines. AKT was
successfully identified in all specimens, and ÎČ-actin
protein was resolved with an efficiency higher than 80%.
The entire extraction procedure requires only 20
minutes. This newly developed technique is an efficient,
safe, cost-effective, and rapid method to isolate proteins
from FFPE tissue sections adequate for molecular
analysis
Immunological modulation following bone marrow-derived mesenchymal stromal cells and Th17 lymphocyte co-cultures
Objective and design: The objective of the study is to uncover the influence of human bone marrow-derived mesenchymal stem cells (BM-MSCs) on the generation of Th17 lymphocytes in co-cultures of both BM-MSCs and T cells. Materials and methods: BM-MSCs, characterized according to the international society for cellular therapy (ISCT) criteria, were co-cultured with T cells isolated from peripheral blood. The expression levels of IL-17 receptor, RORÎłt and IL-23 receptor were evaluated using flow cytometry. The levels of cytokines involved in Th17 immunomodulation were measured using multiplex assay. Treatment: Inflammatory primed and non-primed BM-MSCs were co-cultured with either activated or non-activated T cells either at (1/80) and (1/5) ratio respectively. Results: MSC/T-cell ratio and inflammation significantly influenced the effect of BM-MSCs on the generation of Th17 lymphocytes. Cocultures of either primed or non-primed BM-MSCs with activated T cells significantly induced IL-17A-expressing lymphocytes. Interestingly, the expression of the transcription factor RORÎłt was significantly increased when compared to levels in activated T cells. Finally, both cell ratio and priming of BM-MSCs with cytokines substantially influenced the cytokine profile of BM-MSCs and T cells. Conclusion: Our findings suggest that BM-MSCs significantly modulate the Th17 lymphocyte pathway in a complex manner.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
Th17 immune response to adipose tissueâderived mesenchymal stromal cells
Adipose tissueâderived mesenchymal stromal cells (ASCs) hold the promise of achieving successful immunotherapeutic results due to their ability to regulate different Tâcell fate. ASCs also show significant adaptability to environmental stresses by modulating their immunologic profile. Cellâbased therapy for inflammatory disease requires a detailed understanding of the molecular relation between ASCs and Th17 lymphocytes taking into account the influence of inflammation and cell ratio on such interaction. Accordingly, a doseâdependent increase in Th17 generation was only observed in high MSC:Tâcell ratio with no significant impact of inflammatory priming. ILâ23 receptor (ILâ23R) expression by T cells was not modulated by ASCs when compared to levels in activated T cells, while RORâÎłt expression was significantly increased reaching a maximum in high (1:5) unprimed ASC:Tâcell ratio. Finally, multiplex immunoassay showed substantial changes in the secretory profile of 15 cytokines involved in the Th17 immune response (ILâ1ÎČ, ILâ4, ILâ6, ILâ10, ILâ17A, ILâ17F, ILâ22, ILâ21, ILâ23, ILâ25, ILâ31, ILâ33, IFNâÎł, sCD40, and TNFâα), which was modulated by both cell ratio and inflammatory priming. These findings suggest that Th17 lymphocyte pathway is significantly modulated by ASCs that may lead to immunological changes. Therefore, future ASCâbased immunotherapy should take into account the complex and detailed molecular interactions that depend on several factors including inflammatory priming and cell ratio