Identification of a muropeptide precursor transporter from gut microbiota and its role in preventing intestinal inflammation.

The gut microbiota is a considerable source of biologically active compounds that can promote intestinal homeostasis and improve immune responses. Here, we used large expression libraries of cloned metagenomic DNA to identify compounds able to sustain an anti-inflammatory reaction on host cells. Starting with a screen for NF-κB activation, we have identified overlapping clones harbouring a heterodimeric ATP-binding cassette (ABC)-transporter from a Firmicutes. Extensive purification of the clone's supernatant demonstrates that the ABC-transporter allows for the efficient extracellular accumulation of three muropeptide precursor, with anti-inflammatory properties. They induce IL-10 secretion from human monocyte-derived dendritic cells and proved effective in reducing AIEC LF82 epithelial damage and IL-8 secretion in human intestinal resections. In addition, treatment with supernatants containing the muropeptide precursor reduces body weight loss and improves histological parameters in Dextran Sulfate Sodium (DSS)-treated mice. Until now, the source of peptidoglycan fragments was shown to come from the natural turnover of the peptidoglycan layer by endogenous peptidoglycan hydrolases. This is a report showing an ABC-transporter as a natural source of secreted muropeptide precursor and as an indirect player in epithelial barrier strengthening. The mechanism described here might represent an important component of the host immune homeostasis.

Adlercreutzia equolifaciens Is an Anti-Inflammatory Commensal Bacterium with Decreased Abundance in Gut Microbiota of Patients with Metabolic Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) affects about 20-40% of the adult population in high-income countries and is now a leading indication for liver transplantation and can lead to hepatocellular carcinoma. The link between gut microbiota dysbiosis and NAFLD is now clearly established. Through analyses of the gut microbiota with shotgun metagenomics, we observe that compared to healthy controls, Adlercreutzia equolifaciens is depleted in patients with liver diseases such as NAFLD. Its abundance also decreases as the disease progresses and eventually disappears in the last stages indicating a strong association with disease severity. Moreover, we show that A. equolifaciens possesses anti-inflammatory properties, both in vitro and in vivo in a humanized mouse model of NAFLD. Therefore, our results demonstrate a link between NAFLD and the severity of liver disease and the presence of A. equolifaciens and its anti-inflammatory actions. Counterbalancing dysbiosis with this bacterium may be a promising live biotherapeutic strategy for liver diseases.

Fecal Microbiota Transplant from Human to Mice Gives Insights into the Role of the Gut Microbiota in Non-Alcoholic Fatty Liver Disease (NAFLD).

Non-alcoholic fatty liver diseases (NAFLD) are associated with changes in the composition and metabolic activities of the gut microbiota. However, the causal role played by the gut microbiota in individual susceptibility to NAFLD and particularly at its early stage is still unclear. In this context, we transplanted the microbiota from a patient with fatty liver (NAFL) and from a healthy individual to two groups of mice. We first showed that the microbiota composition in recipient mice resembled the microbiota composition of their respective human donor. Following administration of a high-fructose, high-fat diet, mice that received the human NAFL microbiota (NAFLR) gained more weight and had a higher liver triglycerides level and higher plasma LDL cholesterol than mice that received the human healthy microbiota (HR). Metabolomic analyses revealed that it was associated with lower and higher plasma levels of glycine and 3-Indolepropionic acid in NAFLR mice, respectively. Moreover, several bacterial genera and OTUs were identified as differently represented in the NAFLR and HR microbiota and therefore potentially responsible for the different phenotypes observed. Altogether, our results confirm that the gut bacteria play a role in obesity and steatosis development and that targeting the gut microbiota may be a preventive or therapeutic strategy in NAFLD management.

Gene expression profiling in clinically localized prostate cancer: a four-gene expression model predicts clinical behavior.

PURPOSE: New diagnostic and prognostic molecular markers are required for prostate cancer, one of the most common male malignancies in Western countries. Gene expression profiling may help to identify genes involved in prostate carcinogenesis, yield clinical biomarkers, and improve tumor classification. EXPERIMENTAL DESIGN: To identify fundamental differences between normal and neoplastic prostate tissue, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of 291 selected genes in samples of normal prostate and of well-documented primary, clinically localized prostate tumors. RESULTS: Forty-six genes showed significantly different expression in tumors relative to normal prostate. The dysregulated genes belong notably to the extracellular membrane and extracellular membrane remodeling categories and are involved in angiogenesis. Furthermore, we obtained a four-gene (XLKD1/LYVE1, CGA, F2R/PAR1, and BCL-G) model that discriminated between the seven patients with and the seven patients without relapse, independently of stage and grade. CONCLUSIONS: Some dysregulated genes are good candidates for use as molecular markers and/or therapeutic targets. Furthermore, differential gene expression profiling of clinically localized prostate tumors from relapsing and nonrelapsing patients identified a set of four genes with a pattern of expression that defines a molecular signature that could predict the clinical behavior of this disease.

New spiro-piperidines as 5-HT2B receptor antagonists.

A functional screening highlighted a series of spiro-piperidines as 5-HT2B receptor antagonists. Preliminary structure-activity relationship has been explored driving to potent antagonists (IC50 = 1 nM) and indicating directions for further explorations.

Differential expression of 37 selected genes in hormone-refractory prostate cancer using quantitative taqman real-time RT-PCR.

Progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. The development of more effective treatments depends on our understanding of the molecular events associated with the hormone-refractory stage. We quantified, among 90 screened genes, the expression of 37 target genes, using real-time quantitative RT-PCR. Gene expression was studied in 13 samples of HPRC compared to 33 clinically localised cancers and normal prostate tissue. We identify 19 genes with significant differential expression in HRPC compared to localised prostate cancer. Genes with decreased expression included receptors for growth factors, MMR genes and the serine protease hepsin. Analysis of increased gene expression confirmed the importance of AR upregulation and highlighted genes not previously linked to HRPC, including enzymes involved in steroid synthesis and the antiapoptotic factor survivin. Progression of prostate cancer to the hormone-refractory state is associated with differential gene expression, which may prove useful for both understanding disease progression and the development of new therapeutic approaches.

Extensive analysis of the 13q14 region in human prostate tumors: DNA analysis and quantitative expression of genes lying in the interval of deletion.

BACKGROUND: Loss of heterozygosity (LOH) on chromosome arm 13q14 is one of the most consistent genetic alterations in sporadic prostate cancer. This alteration may be involved in prostate oncogenesis through inactivation of one or more tumor suppressor genes (TSGs). Candidate gene expression is an approach to focus the search for TSGs in this region. METHODS: We tested 41 human sporadic prostate tumors for 13q14 LOH by using seven polymorphic markers overlapping the critical region and used a real-time quantitative RT-PCR assay to study the same tumors for expression of the 31 genes located in this genomic region (localized by the Human Genome Project Working Draft). RESULTS: Allelic loss on at least one locus was found in 18 (41%) of the 41 tumor DNAs. Only four genes (ITM2B, CHC1L, KIAA0970, and LOC51131), located in the region most frequently deleted in prostate carcinoma, showed a significant difference in expression between normal and neoplastic prostate tissues. CONCLUSIONS: Given their location in the LOH hotspot, as indicated by our genomic analysis, ITM2B, CHC1L, KIAA0970, and LOC51131 are candidate tumor suppressor genes in this region. ITM2B that showed a significant association (P < 0.005) between expression and LOH at the corresponding locus could, furthermore, be the main target of the observed LOH at 13q in prostate tumors.

Extensive analysis of the 7q31 region in human prostate tumors supports TES as the best candidate tumor suppressor gene.

Loss of heterozygosity (LOH) on chromosome arm 7q31 is found in many prostate tumors. Such alterations are generally associated with inactivation of tumor suppressor genes. It has been shown previously that the main region of LOH at 7q31 spans the interval between the D7S486 and D7S2460 microsatellite loci, which contains several candidate tumor suppressor genes (TSG) such as TES, CAV2, CAV1, MET, CAPZA2, ST7 and WNT2. We tested 41 human sporadic prostate tumors for 7q31 LOH by using 5 polymorphic markers overlapping the critical region and used a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay to study the expression of the 7 candidate TSGs located in this genomic region. We found that CAV1, CAV2, MET and TES mRNA expression was lower in prostate tumors than in normal prostate tissues. Our immunohistochemical results and previously published data on the compartmental expression of these messenger RNAs in stromal and epithelial cells suggest that TES is the best candidate tumor suppressor gene at 7q31.

Quantification of expression of netrins, slits and their receptors in human prostate tumors.

Recently, DCC (Deleted in Colorectal Cancer) protein has been forwarded as a receptor for netrin. The Netrin/DCC complex is critical for axon guidance and cell migration. In the developing nervous system, netrin protein secreted by midline cells attracts commissural axons by activating the DCC receptor on growth cones. This attraction can be switched to repulsion or silenced completely, depending on the DCC binding partner. The potential suppressor function of DCC in prostate tumorigenesis, through a still unknown mechanism, prompted us to quantify the expression of several genes involved in this axon guidance pathway. The relative expression levels of DCC, NEO1, NTN1, NTN2L, NTN4, UNC5C, Slit1, Slit2, Slit3, Robo1 and Robo2 were simultaneous quantified in 48 tumors and 7 normal prostate tissues by using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). A reduction in DCC, NEO1, NTN1 and NTN4 expression was observed in prostate tumors, while many of the same prostate tumors over-expressed either Slit genes or their receptors, Robo.

Molecular profiling of benign prostatic hyperplasia using a large scale real-time reverse transcriptase-polymerase chain reaction approach.

PURPOSE: Benign prostatic hyperplasia (BPH) is characterized by a hyperplastic growth of epithelial and stromal cells in the prostate. Despite the high prevalence of the disease little is known regarding the molecular etiology of BPH. Therefore, a comparison of gene expression patterns between normal prostate, BPH and prostate cancer could provide insights into the pathogenic mechanisms of the disease and identify candidate genes that could be targeted for therapeutic use. MATERIALS AND METHODS: Prostate tissue specimen were obtained from 30 patients undergoing adenomectomy for BPH. Adenoma weight was less than 60 gm in 15 patients and more than 60 gm in the remainder. Normal prostate tissue was obtained from 15 patients undergoing radical prostatectomy for cancer from areas selected for absent tumor and BPH. Two pools of organ confined prostate cancer were also analyzed. We quantified in the 5 pools of tissues the expression of 327 genes using real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: A total of 23 genes showed increased expression in BPH with a fold change of at least 2.5 between normal prostate and the 2 BPH groups, of which most were normal or down-regulated in prostate cancer. Seven genes showed decreased expression in BPH with a fold change of at least 3.5 between normal prostate and BPH. Most of them were also normal or down-regulated in prostate cancer. CONCLUSIONS: We identified a set of genes up-regulated in BPH compared to normal prostate tissue and often prostate cancer, including genes previously implicated in BPH and others not previously linked to this disease to our knowledge. Further investigations are now warranted to determine the clinical relevance and therapeutic potential of these genes.

IL-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques.

The main failure of antiretroviral therapy is the lack of restoration of HIV-specific CD4(+) T cells. IL-7, which has been shown to be a crucial cytokine for thymopoiesis, has been envisaged as an additive therapeutic strategy. However, in vitro studies suggest that IL-7 might sustain HIV replication in thymocytes and T lymphocytes. Therefore, in the present study, we evaluated the effect of IL-7 on both T cell renewal and viral load in SIVmac-infected young macaques in the absence of antiretroviral therapy. This evaluation was conducted during the asymptomatic phase in view of a potential treatment of HIV patients. We show that IL-7 induces both a central renewal and a peripheral expansion of T lymphocytes associated with cell activation. No alarming modulation of the other hemopoietic cells was observed. No increase in the viral load was shown in blood or lymph nodes. These data strengthen the rationale for the use of IL-7 as an efficient immunotherapy in AIDS.

Positive regulation of CXCR4 expression and signaling by interleukin-7 in CD4+ mature thymocytes correlates with their capacity to favor human immunodeficiency X4 virus replication.

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) variants in infected individuals is associated with poor prognosis. One of the possible causes of this emergence might be the selection of X4 variants in some specific tissue compartment. We demonstrate that the thymic microenvironment favors the replication of X4 variants by positively modulating the expression and signaling of CXCR4 in mature CD4(+) CD8(-) CD3(+) thymocytes. Here, we show that the interaction of thymic epithelial cells (TEC) with these thymocytes in culture induces an upregulation of CXCR4 expression. The cytokine secreted by TEC, interleukin-7 (IL-7), increases cell surface expression of CXCR4 and efficiently overcomes the downregulation induced by SDF-1 alpha, also produced by TEC. IL-7 also potentiates CXCR4 signaling, leading to actin polymerization, a process necessary for virus entry. In contrast, in intermediate CD4(+) CD8(-) CD3(-) thymocytes, the other subpopulation known to allow virus replication, TEC or IL-7 has little or no effect on CXCR4 expression and signaling. CCR5 is expressed at similarly low levels in the two thymocyte subpopulations, and neither its expression nor its signaling was modified by the cytokines tested. This positive regulation of CXCR4 by IL-7 in mature CD4(+) thymocytes correlates with their high capacity to favor X4 virus replication compared with intermediate thymocytes or peripheral blood mononuclear cells. Indeed, we observed an enrichment of X4 viruses after replication in thymocytes initially infected with a mixture of X4 (NL4-3) and R5 (NLAD8) HIV strains and after the emergence of X4 variants from an R5 primary isolate during culture in mature thymocytes.