Design of an "orthogonality breaking" polarimetric confocal microscope to study intracellular architecture dynamics

International audienc

Endotoxin regulates matrix genes increasing reactive oxygen species generation by intercellular communication between palmitate-treated hepatocyte and stellate cell

International audiencePrevious studies have shown that gut-derived bacterial endotoxins contribute in the progression of simple steatosis to steatohepatitis, although the mechanism(s) remains inaccurate to date. As hepatic stellate cells (HSC) play a pivotal role in the accumulation of excessive extracellular matrix (ECM), leading to collagen deposition, fibrosis, and perpetuation of inflammatory response, an in vitro model was developed to investigate the crosstalk between HSC and hepatocytes (human hepatoma cell) pretreated with palmitate. Bacterial lipopolysaccharide (LPS) stimulated HSC with phosphorylation of the p38 mitogen-activated protein kinase/NF-κB pathway, while several important pro-inflammatory cytokines were upregulated in the presence of hepatocyte-HSC. Concurrently, fibrosis-related genes were regulated by palmitate and the inflammatory effect of endotoxin where cells were more exposed or sensitive to reactive oxygen species (ROS). This interaction was accompanied by increased expression of the mitochondrial master regulator, proliferator-activated receptor gamma coactivator alpha, and a cytoprotective effect of the agent N-acetylcysteine suppressing ROS production, transforming growth factor-β1, and tissue inhibitor of metalloproteinase-1. In summary, our results demonstrate that pro-inflammatory mediators LPS-induced promote ECM rearrangement in hepatic cells transcriptionally committed to the regulation of genes encoding enzymes for fatty acid metabolism in light of differences that might require an alternative therapeutic approach targeting ROS regulation

Staphylococcus aureus induces DNA damage in host cell

International audienceStaphylococcus aureus causes serious medical problems in human and animals. Here we show thatS. aureus can compromise host genomic integrity as indicated by bacteria-induced histone H2AXphosphorylation, a marker of DNA double strand breaks (DSBs), in human cervix cancer HeLa andosteoblast-like MG-63 cells. This DNA damage is mediated by alpha phenol-soluble modulins (PSMα1–4),while a specific class of lipoproteins (Lpls), encoded on a pathogenicity island in S. aureus, dampens theH2AX phosphorylation thus counteracting the DNA damage. This DNA damage is mediated by reactiveoxygen species (ROS), which promotes oxidation of guanine forming 7,8-dihydro-8-oxoguanine (8-oxoG). DNA damage is followed by the induction of DNA repair that involves the ATM kinase-signalingpathway. An examination of S. aureus strains, isolated from the same patient during acute initial andrecurrent bone and joint infections (BJI), showed that recurrent strains produce lower amounts of Lpls,induce stronger DNA-damage and prompt the G2/M transition delay to a greater extent that suggest aninvolvement of these mechanisms in adaptive processes of bacteria during chronicization. Our findingsredefine our understanding of mechanisms of S. aureus-host interaction and suggest that the balancebetween the levels of PSMα and Lpls expression impacts the persistence of the infection

In vitro bone metastasis dwelling in a 3D bioengineered niche

International audienceBone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing

Smad1-Smad5 Ovarian Conditional Knockout Mice Develop a Disease Profile Similar to the Juvenile Form of Human Granulosa Cell Tumors

Granulosa cell tumors (GCTs) of the ovary are rare sex cord stromal tumors. Although generally indolent, GCTs recur, and if not diagnosed and treated in early stages, survival rates are significantly shortened. Very little is known regarding GCT etiology. Because of the low incidence of cases and lack of standard diagnostics, mouse models for granulosa cell tumors are a valuable tool for studying GCTs and provide models for developing diagnostic and treatment strategies. We recently developed a novel mouse model of metastatic granulosa cell tumors by genetic deletion of the bone morphogenetic protein signaling transcription factors (SMADs) in granulosa cells of the ovary. Histological and serum hormone analyses reveal that this mouse model most closely resembles the juvenile form of GCT. We further analyzed samples of human juvenile GCT (JGCT) for expression of anti-Müllerian hormone and activation of two major signaling pathways: TGFβ/SMAD2/3 and wingless-related mouse mammary tumor virus integration site (Wnt)/β-catenin. The TGFβ family is active in mouse Smad1-Smad5 double knockout tumors, and here we show that this pathway, but not the β-catenin pathway, is activated in samples of human JGCT. These data suggest that the SMAD family, possibly through disruption of SMAD1/5 or activation of SMAD2/3 may contribute to the pathogenesis of JGCT in humans