bone-marrow-derived-macrophage-production

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Aix-Marseille Université

To investigate the role of thymic output and viral fitness in immune reconstitution in HIV-1-infected children on antiretroviral therapy.

Immune reconstitution in HIV-1-infected children on antiretroviral therapy: role of thymic output and viral fitness.

Human immunodeficiency virus type 1 plasma viruses from 29 entry inhibitor-naive patients were characterized for their susceptibilities to T-20, AMD3100, and RANTES. A strikingly wide range of susceptibilities to T-20 was observed that was influenced by coreceptor usage but not by the susceptibilities of the viruses to inhibitors that target the chemokine receptors or by polymorphisms in the gp41 N helix.

Baseline susceptibility of primary human immunodeficiency virus type 1 to entry inhibitors.

To study the impact of antiretroviral therapy on the tropism of plasma HIV-1 virus populations during treatment response and virological escape.

Impact of antiretroviral treatment on the tropism of HIV-1 plasma virus populations.

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Functional proteomics mapping of a human signaling pathway.

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.

Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines.

Chronic myelomonocytic leukemia (CMML) is a hematological disease close to, but separate from both myeloproliferative disorders (MPD) and myelodysplastic syndromes and may show either myeloproliferative (MP-CMML) or myelodysplastic (MD-CMML) features. Not much is known about the molecular biology of this disease.

Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes.

The myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal haematological diseases characterized by ineffective haematopoiesis and predisposition to acute myeloid leukaemia (AML). The pathophysiology of MDSs remains unclear. A definition of the molecular biology of MDSs may lead to a better classification, new prognosis indicators and new treatments. We studied a series of 40 MDS/AML samples by high-density array-comparative genome hybridization (aCGH). The genome of MDSs displayed a few alterations that can point to candidate genes, which potentially regulate histone modifications and WNT pathways (e.g. ASXL1, ASXL2, UTX, CXXC4, CXXC5, TET2, TET3). To validate some of these candidates we studied the sequence of ASXL1. We found mutations in the ASXL1 gene in four out of 35 MDS patients (11%). To extend these results we searched for mutations of ASXL1 in a series of chronic myelomonocytic leukaemias, a disease classified as MDS/Myeloproliferative disorder, and found mutations in 17 out of 39 patients (43%). These results show that ASXL1 might play the role of a tumour suppressor in myeloid malignancies.

Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia.

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC(50) in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells.

Gene mutation is an important mechanism of myeloid leukemogenesis. However, the number and combination of gene mutated in myeloid malignancies is still a matter of investigation.

Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias.

Chronic myelomonocytic leukaemia (CMML) is a haematological disease currently classified in the category of myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) because of its dual clinical and biological presentation. The molecular biology of CMML is poorly characterized. We studied a series of 53 CMML samples including 31 cases of myeloproliferative form (MP-CMML) and 22 cases of myelodysplastic forms (MD-CMML) using array-comparative genomic hybridisation (aCGH) and sequencing of 13 candidate genes including ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2 and WT1. Mutations in ASXL1 and in the genes associated with proliferation (CBL, FLT3, PTPN11, NRAS) were mainly found in MP-CMML cases. Mutations of ASXL1 correlated with an evolution toward an acutely transformed state: all CMMLs that progressed to acute phase were mutated and none of the unmutated patients had evolved to acute leukaemia. The overall survival of ASXL1 mutated patients was lower than that of unmutated patients.

ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia.

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.

Effects of Coxiella burnetii on MAPKinases phosphorylation.

Variations in lipopolysaccharide (LPS), a bacterial outer membrane component, determine virulence of the obligate intracellular bacterium Coxiella burnetii, but the underlying mechanisms are unknown. We find that while avirulent C. burnetii LPS (avLPS) stimulates host p38α-MAPK signaling required for proper trafficking of bacteria containing compartments to lysosomes for destruction, pathogenic C. burnetii LPS (vLPS) does not. The defect in vLPS and pathogenic C. burnetii targeting to degradative compartments involves an antagonistic engagement of TLR4 by vLPS, lack of p38α-MAPK-driven phosphorylation, and block in recruitment of the homotypic fusion and protein-sorting complex component Vps41 to vLPS-containing vesicles. An upstream activator of p38α-MAPK or phosphomimetic mutant Vps41-S796E expression overrides the inhibition, allowing vLPS and pathogenic C. burnetii targeting to phagolysosomes. Thus, p38α-MAPK and its crosstalk with Vps41 play a central role in trafficking bacteria to phagolysosomes. Pathogenic C. burnetii has evolved LPS variations to evade this host response and thrive intracellularly.

Article	Year
Immune reconstitution in HIV-1-infected children on antiretroviral therapy: role of thymic output and viral fitness. AIDS (London, England)\| PubMed ID: 11919485	2002
Baseline susceptibility of primary human immunodeficiency virus type 1 to entry inhibitors. Journal of virology\| PubMed ID: 12502877	2003
Impact of antiretroviral treatment on the tropism of HIV-1 plasma virus populations. AIDS (London, England)\| PubMed ID: 12660527	2003
Functional proteomics mapping of a human signaling pathway. Genome research\| PubMed ID: 15231748	2004
Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines. Oligonucleotides\| PubMed ID: 17155913	2006
Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1 genes. BMC cancer\| PubMed ID: 18925961	2008
Mutations of polycomb-associated gene ASXL1 in myelodysplastic syndromes and chronic myelomonocytic leukaemia. British journal of haematology\| PubMed ID: 19388938	2009
Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells. Molecular cancer therapeutics\| PubMed ID: 19671755	2009
Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias. BMC cancer\| PubMed ID: 20678218	2010
ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia. British journal of haematology\| PubMed ID: 20880116	2010
Effects of Coxiella burnetii on MAPKinases phosphorylation. FEMS immunology and medical microbiology\| PubMed ID: 22092597	2012
Impaired stimulation of p38α-MAPK/Vps41-HOPS by LPS from pathogenic Coxiella burnetii prevents trafficking to microbicidal phagolysosomes. Cell host & microbe\| PubMed ID: 23245320	2012

Virginie Trouplin

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