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International audience; Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as pathogenesis of diabetes or endothelial dysfunction leading to cardiovascular events. Therefore, the identification of new anti-AGE molecules or extracts aims at preventing such pathologies. Many Clusiaceae and Calophyllaceae species are used in traditional medicines to treat arterial hypertension as well as diabetes. Focusing on these plant families, an anti-AGE plant screening allowed us to select Mammea neurophylla for further phytochemical and biological studies. Indeed, both DCM and MeOH stem bark extracts demonstrated in vitro their ability to prevent inflammation in endothelial cells and to reduce vasoconstriction. A bioguided fractionation of these extracts allowed us to point out 4-phenyl- and 4-(1-acetoxypropyl)coumarins and procyanidins as potent inhibitors of AGE formation, potentially preventing endothelial dysfunction. The fractionation steps also led to the isolation of two new compounds, namely neurophyllols A and B from the DCM bark extract together with thirteen known mammea A and E coumarins (mammea A/AA, mammea A/AB, mammea A/BA, mammea A/BB, mammea A/AA cycloD, mammea A/AB cycloD, disparinol B, mammea A/AB cycloE, ochrocarpin A, mammea A/AA cycloF, mammea A/AB cycloF, mammea E/BA, mammea E/BB) as well as δ-tocotrienol, xanthones (1-hydroxy-7-methoxyxanthone, 2-hydroxyxanthone) and triterpenes (friedelin and betulinic acid). During this study, R,S-asperphenamate, previously described from fungal origin was also purified.

Polyclonal anti-lymphoyctes antibodies were first successfully used in the 1970 in organ transplantation, but ten years later, monoclonal antibodies (mAb) emerged as a new class of immunosuppressive agents in transplantation with the potential to target highly specifically immune cells responsible for acute rejection. Some have proved their efficacy, such as mAb recognizing CD3- and CD25-positive T cells and have been extensively studied in clinical trials. Others such as mAb against CD52 and CD20, are still under investigation; finally, the next challenge is, based on our improved understanding of the mechanisms of immune recognition and allograft rejection, to use these mAb either alone or in combination with standard immunosuppressive regimens to manipulate the allogenic response to reach antigen-specific tolerance desired in solid-organ transplantation.

Introduction Like other tissues in the body, the human dental pulp is equipped with a network of immune cells that can be mobilized against pathogens when they invade the tooth. Very little data, mostly obtained with classic histologic methods, have reported their quantities and relative percentages. The objective of this study was to characterize and precisely quantify immunocompetent cells in healthy human dental pulp by using fluorescence-activated cell sorting, together with identifying specific cell subsets in the leukocyte (CD45 + ) cells. Methods Healthy human third molars were collected from 42 young patients. Dental pulps were separated from the hard tissues and prepared for flow cytometry or immunostaining analyses. Results CD45 + cells represented 0.94% ± 0.65% of cells obtained from the enzymatic digestion of whole dental pulps ( n = 34). CD16 + CD14 + granulocytes/neutrophils (50.01% ± 9.08%, n = 7) were found to represent the major subpopulation in CD45 + cells followed by CD3 + T lymphocytes (32.58% ± 11%, n = 17), CD14 + monocytes (8.93% ± 5.8%, n = 7), and HLA-DR high Lin1 - dendritic cells (4.51% ± 1.12%, n = 7). Minor subpopulations included CD3 − CD56 + natural killer cells (2.63% ± 1.15%, n = 7) and CD19 + B lymphocytes (1.65% ± 0.89%, n = 17). We further identified cells harboring a phenotype compatible with Foxp3/CD25-expressing regulatory T lymphocytes (CD45 + CD3 + CD4 + CD127 low ). Fluorescence-activated cell sorting analysis and confocal microscopy also revealed expression of HO-1 in HLA-DR + cells. Conclusions For the first time, this study identifies and precisely quantifies the relative proportion of immunocompetent cells potentially involved in tissue homeostasis of healthy human dental pulp.

Editorial; International audience

International audience; Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Background We recently reported that in a rat major histocompatibility complex (MHC) mismatched heart allograft model, treatment with CD40Ig, a chimeric molecule that blocks CD40L, leads to indefinite allograft survival mediated by CD8CD45RC Tregs [1]. Although essential, the exact role of TCR/MHC/peptide interaction in Treg activity is still unknown. We therefore characterize the allogeneic peptide(s) recognized and the TCR usage of the CD8 CD45RC Tregs.

Material and methods A lewis 1W rat heart is grafted in a MHC-mismatched Lewis 1A rat and infected with 2.10pi of adenovirus recombinant for CD40Ig molecule (AdCD40Ig) the day of the graft. Tregs, effector CD4CD25 T lymphocytes (TL), and plasmacytoide dendritic cells (pDC) from spleen are sorted by FACS Aria for in vitro tests. For in vivo studies, 4,5.10vg of FGL2-recombinant adenovirus associated virus (AAVFGL2) are intramuscularly or intravenously injected in recipients 30 days before the graft. Splenocytes are transferred to sublethaly irradiated rats by i.v injection the day before the graft.

International audience; Background and objectives. In contrast to the improvement in our understanding of the pathogenesis and presentation of autosomal recessive polycystic kidney disease (ARPKD), data regarding the issue of kidney and liver transplantation in patients with ARPKD remain particularly scarce. Here, we report the results and outcome of renal and/or liver transplantation in a series of patients with ARPKD. Methods. Fourteen ARPKD patients (age: 3-25 years) who underwent renal transplantation with or without liver transplantation were retrospectively identified in five French nephrology departments. The patients' medical charts were reviewed and relevant data were collected. Results. The clinical and radiological presentation of the 14 patients was highly variable illustrating the heterogeneity of ARPKD. Six patients underwent kidney and/or liver transplantation in adulthood. First renal graft survival was 92, 78 and 14% at 1, 5 and 10 years after renal transplantation, respectively. Mortality rate was relatively high (3/14; 21%) in these young patients and was directly related to infectious complications (recurrent angiocholitis) of severe Caroli's disease (dilatation of intra- and/or extra-hepatic bile ducts), a typical feature of ARPKD. Conclusions. Our data suggest that ARPKD patients evaluated for renal transplantation should be carefully screened for severe Caroli's disease. Even though the limited number of patients included in our study precludes any definite recommendation, pre-emptive liver transplantation may be a therapeutic option in ARPKD patients with severe Caroli's disease evaluated for renal transplantation.

Communication orale O-330; International audience

Parkinson’s disease (PD) is a neurodegenerative condition that affects 1% of the population over 65 years of age. The two pathological hallmarks of PD are a loss of dopaminergic neurons in the substantia nigra (SN) and the presence of cytoplasmic eosinophilic inclusions termed Lewy bodies (LBs), whose main component is phosphorylated α-synuclein.1 This degeneration of SN neurons leads to a dopamine deficiency responsible for the major motor symptoms. Nevertheless, it has become increasingly evident that PD is a multicentric neurodegenerative process that also affects neuronal structures outside the SN.2 In this context, various reports performed on surgical or autopsy specimens have shown that the enteric nervous system (ENS) is affected during PD.3 4 However, it is still a matter of debate whether these alterations occur early in the course of the disease. This is mainly due to a lack of accessibility to the ENS in the living patients. Therefore, demonstrating (1) the ability to study the ENS using routine colonic biopsies and (2) the presence of lesions characteristics …

Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.

International audience; We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors. This has been demonstrated by specific binding assays and chemical cross-linking experiments using 125I-labeled growth factors. It appears, therefore, that the human genome contains at least two distinct genes, for TK14 and FLG, that code for related fibroblast growth factor receptors.

MHC class I-specific inhibitory receptors are expressed by a subset of memory-phenotype CD8+ T cells. Similar to NK cells, MHC class I-specific inhibitory receptors might subserve on T cells an important negative control that participates to the prevention of autologous damage. We analyzed here human CD8+ T cells that express the Ig-like MHC class I-specific inhibitory receptors: killer cell Ig-like receptor (KIR) and CD85j. The cell surface expression of Ig-like inhibitory MHC class I receptors was found to correlate with an advanced stage of CD8+ T cell maturation as evidenced by the reduced proliferative potential of KIR+ and CD85j+ T cells associated with their high intracytoplasmic perforin content. This concomitant regulation might represent a safety mechanism to control potentially harmful cytolytic CD8+ T cells, by raising their activation threshold. Yet, KIR+ and CD85j+ T cells present distinct features. KIR+CD8+ T cells are poor IFN-γ producers upon TCR engagement. In addition, KIR are barely detectable at the surface of virus-specific T cells during the course of CMV or HIV-1 infection. By contrast, CD85j+CD8+ T cells produce IFN-γ upon TCR triggering, and represent a large fraction of virus-specific T cells. Thus, the cell surface expression of Ig-like inhibitory MHC class I receptors is associated with T cell engagement into various stages of the cytolytic differentiation pathway, and the cell surface expression of CD85j or KIR witnesses to the history of qualitatively and/or quantitatively distinct T cell activation events.

Background Indoleamine 2, 3-dioxygenase (IDO) converts tryptophan in various catabolites and has been shown to induce immune tolerance in different immune-mediated diseases, including organ transplantation. One of these tolerogenic metabolites is anthranilic acid. Tranilast is a clinically approved, structural and functional analogue of anthranilic acid that has been recently shown to be effective in murine models of multiple sclerosis and rheumatoid arthritis. We examined the effect of tranilast in a rat cardiac allograft model. Materials and methods Lewis 1W rat hearts were grafted in MHC-mismatched Lewis 1A rats. The receiver is orally treated with 650mg/kg of tranilast daily for 30 days. Total splenocytes and purified spleen cell subtypes sorted by FACS Aria were transferred to sublethaly irradiated rats by i.v injection the day before transplantation. Results

Methods All patients suffered from MS and had not received immune drugs since at least 6 months. The frequency and the phenotype of B cell subsets have been analysed using different specific markers: CD19CD27 memory B cells, CD19CD38CD24 mature naive B cells and CD19 CD24CD38 transitional B cells. Their capacity to secrete IL-10 was analysed in vitro 48 hours after stimulation by CD40 ligand and CpG ODN.

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25-52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ(+) T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8(+) T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8(+) T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals.