Magali Fasseu

Aberrant Expression of OX1 Receptors for Orexins in Colon Cancers and Liver Metastases: an Openable Gate to Apoptosis

Resistance to apoptosis is a recurrent theme in colon cancer. We have shown previously that the 7-transmembrane spanning receptor OX1R for orexins promotes robust apoptosis in the human colon cancer cell line HT29 through an entirely novel mechanism involving phosphorylation of tyrosine-based motifs in OX1R. Here, we investigated the status of OX1R in a large series of human colorectal tumors and hepatic metastases. All primary colorectal tumors regardless of their localization and Dukes stages and all hepatic metastases tested expressed OX1R mRNA and/or protein. In sharp contrast, adjacent normal colonocytes or hepatocytes as well as control normal tissues were negative. Next, we showed that nine human colon cancer cell lines established from primary tumors or metastases expressed OX1R mRNA and underwent important apoptosis on orexin-A challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly used drug in colon cancer chemotherapy, 5-fluorouracil. When human colon cancer cells were xenografted in nude mice, orexin-A administered at day 0 strongly slowed the tumor growth and even reversed the development of established tumors when administered 7 days after cell inoculation. Orexin-A also acts by promoting tumor apoptosis in vivo because caspase-3 is activated in tumors on orexin treatment of nude mice. These findings support that OX1R is an Achilles heel of colon cancers, even after metastasis or chemoresistance. They suggest that OX1R agonists might be novel candidates for colon cancer therapy.

The development of early and mature B cells is impaired in mice deficient for the Ets‐1 transcription factor

The Ets‐1 transcription factor is essential for normal development of the natural killer and T cell lineages; however, its role in B cell development remains poorly understood. To address this issue, we used gene targeting to inactivate Ets‐1 in mice (Ets‐1–/–). We show here that the development of B cell precursors, particularly steps requiring pre‐B cell receptor function, is defective in Ets‐1–/– mice. Peripheral B cell subsets were analyzed in RAG2‐deficient mice reconstituted with Ets‐1–/– fetal liver cells. In such Ets‐1–/– chimeric mice, B cell precursors develop into IgM/IgD‐bearing cells, but B‐1a cells as well as transitional‐2 and marginal zone B cell subsets of the spleen are absent. In response to B cell receptor stimulation, Ets‐1–/– splenic B cells fail to express the CD69 and CD25 activation markers. Furthermore, despite activation of ERK and JNK signaling pathways, Ets‐1‐deficient B cells do not proliferate and die following BCR engagement. These findings demonstrate that the effect of Ets‐1 inactivation is not restricted to the terminal B cell differentiation stage, but also affects the development and function of earlier B cell subsets.

Transgenic Expression of the p16INK4a Cyclin-Dependent Kinase Inhibitor Leads to Enhanced Apoptosis and Differentiation Arrest of CD4−CD8− Immature Thymocytes

In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16INK4a is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16Ink4a controls thymocyte development by analyzing transgenic mice expressing the human p16INK4a into the T cell lineage. We show that forced expression of p16INK4a in thymocytes blocked T cell differentiation at the early CD4−CD8−CD3−CD25+ stage without significantly affecting the development of γδ T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2−/−). Upon anti-CD3ε treatment in vivo, p16INK4a-expressing RAG-2−/− thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16INK4a prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.

Advanced fibrosis: Correlation between pharmacokinetic parameters at dynamic gadoxetate-enhanced MR imaging and hepatocyte organic anion transporter expression in rat liver

PURPOSE To compare the value of enhancement and pharmacokinetic parameters measured at dynamic gadoxetate-enhanced magnetic resonance (MR) imaging in determining hepatic organic anion transporter expression in control rats and rats with advanced liver fibrosis. MATERIALS AND METHODS Institutional animal review board approval was received before the study began. Advanced liver fibrosis was created in rats by means of carbon tetrachloride injections over an 8-week period. In 17 rats with liver fibrosis and eight control rats, dynamic gadoxetate-enhanced MR images of the liver were obtained during 1 hour after injection of 0.025 mmol gadoxetate per kilogram of body weight. Enhancement parameters (maximum enhancement [Emax], time to peak [Tmax], and elimination half-life) were measured on enhancement-versus-time curves, and pharmacokinetic parameters (hepatic extraction fraction [HEF] and mean residence time [MRT]) were obtained by means of deconvolution analysis of the concentration-versus-time curves in the liver and the portal vein. The parameters were correlated at simple and multiple regression analysis with the expression of the hepatic anion uptake transporter organic anion-transporting polypeptide 1A1 (Oatp1a1), the hepatobiliary transporter multidrug resistance-associated protein 2 (Mrp2), and the backflux transporter Mrp4, as determined with reverse transcription polymerase chain reaction. RESULTS In rats with advanced liver fibrosis, the Emax, Tmax, HEF, and MRT decreased significantly relative to those in control rats, whereas the elimination half-life increased significantly. The enhancement and pharmacokinetic parameters correlated significantly with the expression of the transporters at simple regression analysis. At multiple regression analysis, HEF was the only parameter that was significantly associated with the expression of Oatp1a1 and Mrp2 (P < .001, r = 0.74 and P < .001, r = 0.70, respectively). CONCLUSION The pharmacokinetic parameter HEF at dynamic gadoxetate-enhanced MR imaging is independently correlated with hepatic organic anion transporter expression.

Sequential air–liquid exposure of human respiratory cells to chemical and biological pollutants

Although indoor air has wide ranging effects on human health, the effects of environmental, chemical, and biological pollutants on the respiratory system are not fully understood. In order to clarify the health effects of airborne pollutant exposure, it would appear that toxicological evidence is needed to complement epidemiological observations to support by providing biological plausibility. The aim of this study is to manage air-liquid successive exposures to different pollutants such as a chemical pollutant (formaldehyde--FA), and a biological contaminant (Aspergillus fumigatus--Asp) using our in vitro model. Human alveolar cells (A549) were exposed at the air-liquid interface in an exposure module, firstly to an environmental level of FA (50 μg/m³) (or air) for 30 min, and 14 h later to Asp (7×10⁸ spores/m³) (or air) for 30 min. After 10 h post-incubation, cellular viability was assessed. Inflammation biomarkers (IL-8, MCP-1) were assayed by ELISA and by RT-PCR. Whatever the conditions, no cytotoxic effect was observed. FA followed by air exposure did not induce modification of production and expression of cytokines, confirming results with a unique FA exposure. Air followed by Asp exposure tended to induce IL-8 expression whereas IL-8 production tended to increase after FA and Asp exposure compared to FA and air exposure. The reaction of cells to sequential exposure to FA and Asp was moderate. These results show the feasibility of our model for sequential exposures to different types of environmental pollutants, allowing using it for preliminary assessment of cellular activity modification induced by airborne contaminants.

Disruption of the lineage restriction of TCRβ gene rearrangements

Despite a common lymphoid recombinase, assembly of Ig genes is restricted to B cells, whereas TCR loci rearrange in T cells. Transcriptional promoters and enhancers are critical for the regulation of the recombination process. However, the specific function of such elements in conferring the lineage‐restriction of V(D)J recombination remains poorly understood. To gain further insights into the mechanism restricting TCRβ ‐chain rearrangements to T cells, we generated mice in which an 11 kb region — containing the β ‐chain constant region 2 and the TCRβ  enhancer (Eβ ) — was replaced with the B cell specific Ig heavy‐chain enhancer (Eμ ). Unlike the simple Eμ  to Eβ  replacement, this mutation allowed significant levels of Dβ  to Jβ  as well as Vβ  to DJβ  rearrangements in both T and B cells. Although the lineage restriction was disrupted, TCRβ  allelic exclusion was still efficient in mutated T cells. Together these results demonstrate that changes in the activity of regulatory elements located at the TCRβ  constant regions are sufficient to redirect the recombination pattern of TCRβ  variable gene segments.

Hepatic proliferation and angiogenesis markers are increased after portal deprivation in rats: a study of molecular, histological and radiological changes.

Background & Aims To determine the pathogenesis of liver nodules, and lesions similar to obliterative portal venopathy, observed after portosystemic shunts or portal vein thrombosis in humans. Methods We conducted an experimental study comparing portacaval shunt (PCS), total portal vein ligation (PVL), and sham (S) operated rats. Each group were either sacrificed at 6 weeks (early) or 6 months (late). Arterial liver perfusion was studied in vivo using CT, and histopathological changes were noted. Liver mRNA levels were quantified by RT-QPCR for markers of inflammation (Il10, Tnfa), proliferation (Il6st, Mki67, Hgf, Hnf4a), angiogenesis: (Vegfa, Vegfr 1, 2 and 3; Pgf), oxidative stress (Nos2, and 3, Hif1a), and fibrosis (Tgfb). PCS and PVL were compared to the S group. Results Periportal fibrosis and arterial proliferation was observed in late PCS and PVL groups. CT imaging demonstrated increased arterial liver perfusion in the PCS group. RT-QPCR showed increased inflammatory markers in PCS and PVL early groups. Tnfa and Il10 were increased in PCS and PVL late groups respectively. All proliferative markers increased in the PCS, and Hnf4a in the PVL early groups. Mki67 and Hnf4a were increased in the PCS late group. Nos3 was increased in the early and late PCS groups, and Hif1a was decreased in the PVL groups. Markers of angiogenesis were all increased in the early PCS group, and Vegfr3 and Pgf in the late PCS group. Only Vegfr3 was increased in the PVL groups. Tgf was increased in the PCS groups. Conclusions Portal deprivation in rats induces a sustained increase in intrahepatic markers of inflammation, angiogenesis, proliferation, and fibrosis.

609 LPS INDUCES AN UNEXPECTED ENDOPLASMIC RETICULUM UNFOLDED PROTEIN RESPONSE (UPRER) IN IMMUNE CELLS FROM PATIENTS WITH CIRRHOSIS

Methods: MRNAs obtained from PBMCs were stimulated by LPS or left un-stimulated for 4h. We first used the Affymetrix Human Exon Array to analyze regulation of 14,851 and 15,334 expressed genes out of the 32,778 on the array in four patients and four ‘healthy’ subjects respectively. Results were validated using RTqPCR to measure expression of 63 genes of interest in a prospective cohort of 57 patients and 9 ‘healthy’ subjects. Results: Gene profiling revealed a core of 741 genes that were similarly regulated by LPS in cirrhotic and ‘healthy’ PBMCs. Among them, 300 LPS-induced genes were enriched in GO categories ‘regulation of mononuclear cell proliferation’ and ‘cytokine activity’. 441 shared LPS-downregulated genes were involved in recognition of PAMPs, chemotaxis, cell migration and lysosome suggesting that LPS-induced immune paralysis is a universal response. Comparison of functions associated with the 1,356 genes that were specifically regulated by LPS in cirrhotic cells to functions of the 1,049 genes specifically regulated in ‘healthy’ cells allowed to define a cirrhosis specific phenotype. Unlike healthy cells, LPS-stimulated cirrhotic cells did not exhibit the interferon (type I and II)-mediated program (e.g. IRF1, IRF2, IRF7, IFNG, IFI6, STAT1, STAT2, IFITM2), which is critical for immune host defense and resolution of inflammation. Consistent with these results, CXCL10 and CXCL11, two interferoninduced chemokines were induced by LPS in ‘healthy’ but not in cirrhotic PBMCs. Moreover, LPS-stimulated cirrhotic cells had downregulation of endocytic trafficking genes, which may results in decreased TLR4 endocytosis and inhibition of TRIF-mediated interferon pathway. Conclusions: Both LPS-stimulated cirrhotic and ‘healthy’ PBMCs exhibit features of immune paralysis. Moreover, LPS stimulated cirrhotic PBMCs have a specific defect of induction of interferon target genes involved in host defense and resolution of inflammation. These findings may explain the severity of bacterial infections in cirrhosis.