AMOTL1 Promotes Breast Cancer Progression and Is Antagonized by Merlin

AbstractThe Hippo signaling network is a key regulator of cell fate. In the recent years, it was shown that its implication in cancer goes well beyond the sole role of YAP transcriptional activity and its regulation by the canonical MST/LATS kinase cascade. Here we show that the motin family member AMOTL1 is an important effector of Hippo signaling in breast cancer. AMOTL1 connects Hippo signaling to tumor cell aggressiveness. We show that both canonical and noncanonical Hippo signaling modulates AMOTL1 levels. The tumor suppressor Merlin triggers AMOTL1 proteasomal degradation mediated by the NEDD family of ubiquitin ligases through direct interaction. In parallel, YAP stimulates AMOTL1 expression. The loss of Merlin expression and the induction of Yap activity that are frequently observed in breast cancers thus result in elevated AMOTL1 levels. AMOTL1 expression is sufficient to trigger tumor cell migration and stimulates proliferation by activating c-Src. In a large cohort of human breast tumors, we show that AMOTL1 protein levels are upregulated during cancer progression and that, importantly, the expression of AMOTL1 in lymph node metastasis appears predictive of the risk of relapse. Hence we uncover an important mechanism by which Hippo signaling promotes breast cancer progression by modulating the expression of AMOTL1

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Analysis of anti-human CD22 human mAbs affinity and specificity on human PBMCs. a) Affinity determination of anti-human CD22 mAbs using SPR by flowing various concentration of CD22 antibody over CD22 chip-bound. b) Flow cytometry analysis of CD22 mAbs on human PBMC. Human PBMC were labeled with anti human CD19 (APC) and purified CD22 mAbs (clone γ1λ1, γ3λ3-5, γ23κ5-2 or γ27λ26) coupled with Alexa Fluor 568 fluorochrome or with a commercial mouse mAb anti-human CD22 (Ms anti-human CD22). (PPT 666 kb

CpG-ODN and MPLA Prevent Mortality in a Murine Model of Post-Hemorrhage-Staphyloccocus aureus Pneumonia

Infections are the most frequent cause of complications in trauma patients. Post-traumatic immune suppression (IS) exposes patients to pneumonia (PN). The main pathogen involved in PN is Methicillin Susceptible Staphylococcus aureus (MSSA). Dendritic cells () may be centrally involved in the IS. We assessed the consequences of hemorrhage on pneumonia outcomes and investigated its consequences on DCs functions. A murine model of hemorrhagic shock with a subsequent MSSA pneumonia was used. Hemorrhage decreased the survival rate of infected mice, increased systemic dissemination of sepsis and worsened inflammatory lung lesions. The mRNA expression of Tumor Necrosis Factor-alpha (TNF-α), Interferon-beta (IFN-β) and Interleukin (IL)-12p40 were mitigated for hemorrhaged-mice. The effects of hemorrhage on subsequent PN were apparent on the pDCs phenotype (reduced MHC class II, CD80, and CD86 molecule membrane expression). In addition, hemorrhage dramatically decreased CD8+ cDCs- and CD8- cDCs-induced allogeneic T-cell proliferation during PN compared with mice that did not undergo hemorrhage. In conclusion, hemorrhage increased morbidity and mortality associated with PN; induced severe phenotypic disturbances of the pDCs subset and functional alterations of the cDCs subset. After hemorrhage, a preventive treatment with CpG-ODN or Monophosphoryl Lipid A increased transcriptional activity in DCs (TNF-α, IFN-β and IL-12p40) and decreased mortality of post-hemorrhage MSSA pneumonia

Forest management in Bolivia under Evo Morales: The challenges of post-neoliberalism

International audienceBased on a study of the forest sector in the northern Amazon region of Bolivia, this paper addresses the reasons why the Bolivian so-called "postneoliberal" project is an adjustment of neoliberalism rather than a departure from it. First, Bolivia has aimed not so much at breaking away radically from neoliberal frameworks as at reasserting the importance of the state and of the national scale in regulatory actions. Second, the neoliberal period has left behind the insuperable legacy of a neoliberal approach to nature: by allowing local stakeholders to access forest resources, it has reinforced a commercial relationship with the non-human world. Third, it is not easy to reverse the internationalization of forest governance. All in all, the Bolivian state's reterritorialization of the country's margins through the control of forest resources has perpetuated processes that were initiated during the hybrid neoliberal period (1993-2005), though the focus has shifted from commoditizing the forest for the benefit of local stakeholders to facilitating the redistribution of this resource on a national scale. Since 2006 Evo Morales's forest governance has tried to integrate this multiscale dimension to create a pragmatic management model that combines agrarian colonization, conservation and extractivism. This explains both the apparent contradictions of the new Bolivian forest policy and the difficulties in creating a radically new form of governance. The case of Bolivian forests shows the extent to which neoliberalism has shaped spaces and scales in a way that guarantees its own resilience

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

International audienceMonoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire

French club of Young Immunologists: One year and counting

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Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus

International audienceIn the thymus, a T-cell repertoire able to confer protection against infectious and noninfec-tious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the mat-uration efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2 + donors reacted to A2 + cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymo-cytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process

Contribution of the SYK Tyrosine kinase expression to human iNKT self-reactivity

International audienceInvariant Natural Killer T (iNKT) cells are particular T lymphocytes at the frontier between innate and adaptative immunities. They participate in the elimination of pathogens or tumor cells, but also in the development of allergic reactions and autoimmune diseases. From their first descriptions, the phenomenon of self-reactivity has been described. Indeed, they are able to recognize exogenous and endogenous lipids. However, the mechanisms underlying the self-reactivity are still largely unknown, particularly in humans. Using a CD1d tetramer-based sensitive immunomagnetic approach, we generated self-reactive iNKT cell lines from blood circulating iNKT cells of healthy donors. Analysis of their functional characteristics in vitro showed that these cells recognized endoge-nous lipids presented by CD1d molecules through their TCR that do not correspond to α-glycosylceramides. TCR sequencing and transcriptomic analysis of T cell clones revealed that a particular TCR signature and an expression of the SYK protein kinase were two mechanisms supporting human iNKT self-reactivity. The SYK expression, strong in the most self-reactive iNKT clones and variable in ex vivo isolated iNKT cells, seems to decrease the activation threshold of iNKT cells and increase their overall antigenic sensitivity. This study indicates that a modulation of the TCR intracellular signal contributes to iNKT self-reactivity

Ex vivo evolution of human antibodies by CRISPR-X: from a naive B cell repertoire to affinity matured antibodies

International audienceMethodology articl