Importance of spliceosomal RNP1 motif for intermolecular T-B cell spreading and tolerance restoration in lupus

We previously demonstrated the importance of the RNP1 motif-bearing region 131–151 of the U1-70K spliceosomal protein in the intramolecular T-B spreading that occurs in MRL/lpr lupus mice. Here, we analyze the involvement of RNP1 motif in the development and prevention of naturally-occurring intermolecular T-B cell diversification. We found that MRL/lpr peripheral blood lymphocytes proliferated in response to peptides containing or corresponding exactly to the RNP1 motif of spliceosomal U1-70K, U1-A and hnRNP-A2 proteins. We also demonstrated that rabbit antibodies to peptide 131–151 cross-reacted with U1-70K, U1-A and hnRNP-A2 RNP1-peptides. These antibodies recognized the U1-70K and U1-A proteins, and also U1-C and SmD1 proteins, which are devoid of RNP1 motif. Repeated administration of phosphorylated peptide P140 into MRL/lpr mice abolished T-cell response to several peptides from the U1-70K, U1-A and SmD1 proteins without affecting antibody and T-cell responses to foreign (viral) antigen in treated mice challenged with infectious virus. These results emphasized the importance of the dominant RNP1 region, which seems to be central in the activation cascade of B and T cells reacting with spliceosomal RNP1+ and RNP1- spliceosomal proteins. The tolerogenic peptide P140, which is recognized by lupus patients' CD4+ T cells and known to protect MRL/lpr mice, is able to thwart emergence of intermolecular T-cell spreading in treated animals

Formation des Personnels Animaliers

National audienceLa nouvelle directive donne des préconisations très précises en matière de formation et de compétences des personnels animaliers. Ces compétences peuvent provenir de la formation initiale, de formations spécifiques au métier d’animalier et de la formation continue des personnels et vont permettre d’exercer 4 fonctions différentes : concepteur (concevoir une procédure expérimentale), praticien (appliquer une procédure expérimentale), soigneur (soins aux animaux), « mise à mort des animaux ». Ces fonctions sont accessibles à des conditions définies notamment en ce qui concerne l’expérimentation animale (niveau A, B, C ou D). Une formation continue de 3 jours est obligatoire sur une période de six ans. L’adéquation entre les compétences et les missions des personnels doit être suivie via des carnets de compétences qui regroupent toutes les informations relatives à la formation des animaliers. Les anciens niveaux d’expérimentation animale ont une équivalence avec les nouveaux

Differential effect of FasL on GvHD disease according to its source

International audienceObjective: Hematopoietic stem cell transplantation is a potentially curative treatment for hematologic malignancies. An important limitation of its use is the frequent occurrence of graft versus host disease (GvHD). GvHD is associated with complex interactions between innate and adaptive immunity. The incidence of GvHD is 40 to 80% depending on donor-and transplant-characteristics. A number of pathways have been described on allogeneic T cell-mediated cytotoxicity, including the Fas/Fas ligand (FasL) pathway. However, the overall mechanistic findings on how FasL expression in donor cells affects target tissues remains poorly characterized. Methods: We used a well-defined CD4 and CD8-dependent sclerodermatous GvHD model. GvHD is induced by transplantation of low doses of T-and B-cell depleted BM cells and splenocytes from C57BL/6 mice into lethally irradiated BALB/c mice. Colon and skin samples were analyzed by flux cytometry and H&ES staining of fixed sections. Serum cytokines were measured by Multiplex ELISA. Results: Recipient from Fasl-KO BM mice exhibited exacerbate gut acute GvHD (diarrhea significantly higher compared to WT-BM recipient mice). Serum analysis at Day 10 post transplantation (PT) revealed a drastically reduced IL-18 level in these mice compared to WT-BM recipient mice. Cellular and histological analysis of colon biopsies showed significantly higher frequency of CD8 T cells and lymphocytic infiltrates in Fasl KO-BM recipient mice as compared to WT-BM. Recipients of FasL deficient T cells (transplanted with WT BM) displayed significantly reduced symptoms of acute (diarrhea) and chronic GvHD (skin lesion). Pathological skin analysis at Day 34 PT revealed that, unlike syngeneic control mice where cellular infiltrate was mostly of myeloid origin, the infiltrate in mice with GVHD included myeloid-cells and T-cells in similar proportion. In recipients of Fasl KO-splenocytes, proportions of Treg (FoxP3+CD25+) were significantly increased while those of IFN-γ + CD4 T-cells were significantly decreased as compared to WT-splenocytes recipients; suggesting a regulatory environment. Conclusion: These results indicate that FasL expression, either in BM or in in splenocytes, acts differentially in GvHD. FasL expression by donor myeloid cells confers protection from aGvHD via the production of IL-18. In contrast, FasL expression by donor T cells mediates cytotoxicity

Differential effect of FasL on GvHD disease according to its source

International audienceObjective: Hematopoietic stem cell transplantation is a potentially curative treatment for hematologic malignancies. An important limitation of its use is the frequent occurrence of graft versus host disease (GvHD). GvHD is associated with complex interactions between innate and adaptive immunity. The incidence of GvHD is 40 to 80% depending on donor-and transplant-characteristics. A number of pathways have been described on allogeneic T cell-mediated cytotoxicity, including the Fas/Fas ligand (FasL) pathway. However, the overall mechanistic findings on how FasL expression in donor cells affects target tissues remains poorly characterized. Methods: We used a well-defined CD4 and CD8-dependent sclerodermatous GvHD model. GvHD is induced by transplantation of low doses of T-and B-cell depleted BM cells and splenocytes from C57BL/6 mice into lethally irradiated BALB/c mice. Colon and skin samples were analyzed by flux cytometry and H&ES staining of fixed sections. Serum cytokines were measured by Multiplex ELISA. Results: Recipient from Fasl-KO BM mice exhibited exacerbate gut acute GvHD (diarrhea significantly higher compared to WT-BM recipient mice). Serum analysis at Day 10 post transplantation (PT) revealed a drastically reduced IL-18 level in these mice compared to WT-BM recipient mice. Cellular and histological analysis of colon biopsies showed significantly higher frequency of CD8 T cells and lymphocytic infiltrates in Fasl KO-BM recipient mice as compared to WT-BM. Recipients of FasL deficient T cells (transplanted with WT BM) displayed significantly reduced symptoms of acute (diarrhea) and chronic GvHD (skin lesion). Pathological skin analysis at Day 34 PT revealed that, unlike syngeneic control mice where cellular infiltrate was mostly of myeloid origin, the infiltrate in mice with GVHD included myeloid-cells and T-cells in similar proportion. In recipients of Fasl KO-splenocytes, proportions of Treg (FoxP3+CD25+) were significantly increased while those of IFN-γ + CD4 T-cells were significantly decreased as compared to WT-splenocytes recipients; suggesting a regulatory environment. Conclusion: These results indicate that FasL expression, either in BM or in in splenocytes, acts differentially in GvHD. FasL expression by donor myeloid cells confers protection from aGvHD via the production of IL-18. In contrast, FasL expression by donor T cells mediates cytotoxicity

Reactivity of rabbit antibodies directed to peptide 131–151 of the U1-70K protein with RNP1-peptides and spliceosomal proteins

<p><b>Copyright information:</b></p><p>Taken from "Importance of spliceosomal RNP1 motif for intermolecular T-B cell spreading and tolerance restoration in lupus"</p><p>http://arthritis-research.com/content/9/5/R111</p><p>Arthritis Research & Therapy 2007;9(5):R111-R111.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2212579.</p><p></p> The antiserum was diluted 1/20,000 and incubated first for 1 h at 37°C and then overnight at 4°C with increasing amounts of different RNP1-peptides used as competitors in the fluid-phase. The homologous peptide 131–151 was used as control. The mixtures were then added to microtiter plates pre-coated with 2 μM of immunizing peptide 131–151. The results are expressed as the percentage of inhibition of the ELISA reaction measured without competitor peptide. U1-70K, U1-A, U1-C, and SmD1 proteins were subjected to electrophoresis, transferred to nitrocellulose, and colored with Ponceau red (lanes 1–4). The blotted strips were then incubated with the serum (diluted 1/500) from rabbits that received either peptide 131–151 (lanes 5–8) or CFA alone (lanes 9–12). IgG antibodies only were tested. ECL reagents were used to reveal positive reactions

Brief peptide P140 therapy does not affect immune responses to viral challenge in MRL/lpr mice

<p><b>Copyright information:</b></p><p>Taken from "Importance of spliceosomal RNP1 motif for intermolecular T-B cell spreading and tolerance restoration in lupus"</p><p>http://arthritis-research.com/content/9/5/R111</p><p>Arthritis Research & Therapy 2007;9(5):R111-R111.</p><p>Published online 26 Oct 2007</p><p>PMCID:PMC2212579.</p><p></p> P140-treated (closed symbols) and untreated MRL/lpr mice (open symbols) (10 mice/group) were challenged intra-nasally with infectious influenza virus. Thirteen days after viral challenge, the proliferative response of PBLs to increasing concentrations of HA peptide 307–319 was measured . Results are expressed as SI. Bars show the mean ± SEM. The average tritiated thymidine incorporation in the absence of peptide and in the presence of Con-A was 50 and 4,000 cpm, respectively. Weight loss pattern after intra-nasal viral challenge. The weight of MRL/lpr mice treated or untreated with P140 peptide was compared

Novel insights into residual hematopoiesis from stem cell populations in pediatric B-acute lymphoblastic leukemia

International audienceNo abstract availabl

Extracellular vesicles produced by NFAT3-expressing cells hinder tumor growth and metastatic dissemination

International audienceMetastases are the main cause of cancer-induced deaths worldwide. To block tissue invasion, development of extracellular vesicles (EVs) as therapeutic carriers, appears as an exciting challenge. To this aim, we took advantage of the anti-invasive function of NFAT3 transcription factor we identified previously in breast cancer and addressed the opportunity to transfer this inhibitory function by EVs. We show here that EVs produced by poorly invasive NFAT3-expressing breast cancer cell lines are competent to block in vitro invasion of aggressive cancer cells from different origins and, in cooperation with macrophages, inhibit cell proliferation and induce apoptosis. Moreover, this inhibitory effect can be improved by overexpression of NFAT3 in the EVs-producing cells. These results were extended in a mouse breast cancer model, with clear impact of inhibitory EVs on tumor growth and metastases spreading. This work identifies EVs produced by NFAT3-expressing breast cancer cells as an anti-tumoral tool to tackle cancer development and metastases dissemination

Lentiviral transduction of CD34(+) cells induces genome-wide epigenetic modifications.

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34(+) cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34(+) cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34(+) cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols