Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis.

SummaryBacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-b-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a DvirB mutant. In HeLa cells, a DricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortus DricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.

These findings establish that activation of the proteolytic processing of shCD146, in particular by sCD146, constitutes a promising pathway to improve endothelial progenitors' regenerative properties for the treatment of cardiovascular diseases.

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.

Resumo -O objetivo deste trabalho foi avaliar a resposta da canola a fontes e doses de nitrogênio aplicadas na semeadura. O experimento foi conduzido em Latossolo Vermelho distroférrico típico, com textura muito argilosa. Utilizou-se delineamento experimental de blocos ao acaso, em arranjo fatorial 7x2, com sete doses de N em superfície na semeadura (0, 20, 40, 60, 80, 100 e 120 kg ha -1 ), duas fontes de N (sulfato de amônio e ureia) e quatro repetições. O experimento foi realizado com o híbrido Hyola 61, por dois anos, e foram avaliadas as seguintes variáveis: altura de planta, número de plantas por metro quadrado, massa de matéria seca da parte aérea, massa de síliquas por planta, massa de mil grãos, produtividade de grãos, e teores de proteína e de óleo nos grãos. As variáveis não foram influenciadas pelas fontes de N. A maior produtividade de grãos é alcançada com 88 kg ha -1 de N. Doses crescentes de N aumentam os teores de proteína e diminuem os de óleo nos grãos de canola.Termos para indexação: Brassica napus, adubação nitrogenada, sulfato de amônio, teor de óleo, teor de proteína, ureia. Grain yield and yield components in canola according to nitrogen sources and ratesAbstract -The objective of this work was to evaluate the response of canola to nitrogen sources and rates applied at sowing. The experiment was carried out on a dystroferric Red Ferralsol, with a very clayey texture. A randomized complete block design was used, in a 7x2 factorial arrangement, with seven N rates applied on soil surface at sowing (0, 20, 40, 60, 80, 100, and 120 kg ha -1 ), two N sources (ammonium sulfate and urea), and four replicates. The experiment was carried out with the Hyola 61 hybrid for two years, and the following variables were evaluated: plant height, number of plants per square meter, shoot dry matter mass, pod mass per plant, weight of a thousand grains, grain yield, and protein and oil contents in seeds. The variables were not affected by the N sources. The highest grain yield is obtained with 88 kg ha -1 N. Increasing N rates increase protein contents and reduce oil contents in canola seeds.Index terms: Brassica napus, nitrogen fertilization, ammonium sulfate, oil content, protein content, urea. IntroduçãoA canola (Brassica napus L. var. oleífera) é uma oleaginosa de inverno desenvolvida a partir do melhoramento genético da colza. Em razão dos preços vantajosos e de sua adaptabilidade às condições edafoclimáticas do Sul do Brasil, a canola vem se tornando, na região, juntamente com o milho safrinha (Zea mays L.) e o trigo (Triticum aestivum L.), uma excelente opção de cultivo de inverno, além de constituir uma sucessão adequada ao cultivo de soja (Glycine max L.).A canola é uma cultura altamente responsiva à aplicação de N (Rathke et al