Interactions between dendritic cells and Mycobacterium tuberculosis, the aetiological agent of tuberculosis in humans, are thought to be central to anti-mycobacterial immunity. We have previously shown that M. tuberculosis binds to human monocyte-derived dendritic cells mostly through the C-type lectin DC-SIGN (dendritic-cell-specific intercellular molecule-3-grabbing non-integrin)/CD209, and we have suggested that DC-SIGN may discriminate between mycobacterial species through recognition of the mannose-capping residues on the lipoglycan lipoarabinomannan of the bacterial envelope. Here, using a variety of fast- and slow-growing Mycobacterium species, we provide further evidence that mycobacteria recognition by DC-SIGN may be restricted to species of the M. tuberculosis complex. Fine analyses of the lipoarabinomannan molecules purified from these species show that the structure and amount of these molecules alone cannot account for such a preferential recognition. We propose that M. tuberculosis recognition by DC-SIGN relies on both a potential difference of accessibility of lipoarabinomannan in its envelope and, more probably, on the binding of additional ligands, possibly including lipomannan, mannose-capped arabinomannan, as well as the mannosylated 19 kDa and 45 kDa [Apa (alanine/proline-rich antigen)] glycoproteins. Altogether, our results reveal that the molecular basis of M. tuberculosis binding to DC-SIGN is more complicated than previously thought and provides further insight into the mechanisms of M. tuberculosis recognition by the immune system.

By labeling surface carbohydrates we found that a pool of lipoglycans, cell wall associated, is exposed at the cell surface of mycobacteria and thus, most probably, inserted in the outer leaflet of the outer membrane. In contrast, plasma membrane anchored lipoglycans are not accessible to surface labeling. This result supports the role of lipoglycans as key immunomodulatory molecules but raises the question of their transport from the plasma membrane, where they are synthesized, to the outermost layers of the envelope, where they can act as modulins. The data are discussed in term of consequences for cell envelope organization. KeywordsMycobacterium; lipoglycan; localization; cell envelope; transporter The lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), are major immunomodulatory molecules of the mycobacterial cell envelope 1-4 . Mannose-capped LAM (ManLAM) is a potential ligand for the entry of Mycobacterium tuberculosis into phagocytic cells via the host C-lectins 5 . In addition, purified ManLAM reproduces several properties of M. tuberculosis that may contribute to the inhibition of the host defense response and define ManLAM as a major virulence factor of the pathogen 5 . In contrast, phosphoinositol-capped LAM (PILAM) and LM stimulate innate immunity via signaling through Toll-like receptor 2 (TLR2) 5 . Lipoglycans are delivered from infected macrophages, via exosomes or apoptotic vesicles, to non-infected bystander dendritic cells 6-8 and thus can modulate the functions of the latter, via binding C-lectins 9 or TLR-2, even though they are not necessarily receptor ligands on the whole bacterium 10 . Nevertheless, their role as mycobacterial cell surface adhesins or as soluble molecules released by phagocytic cells implies that they are exposed to Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public AccessAuthor Manuscript Tuberculosis (Edinb) the cell surface or, at least, located in the outermost part of the cell envelope. However, their precise localization remains unclear. They are not covalently attached to the cell envelope, but they have never been found in culture supernatants or in the surface-exposed material obtained by gentle mechanical treatment of cells with glass beads and/or detergent treatment 11,12 , suggesting that they are instead imbedded in the cell wall.In the present study, the exposition of lipoglycans to the cell surface of mycobacteria was investigated by cell labeling with biotin. The validity of this approach relies on the assumption that labeling is indeed restricted to surface components. BCG cells were grown as ...

We recently designed the CIME cocktail consisting of 10 drugs to assess the activity of the major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A), a phase II enzyme (UGT1A1/6/9), two drug transporters (P-gp and OATP1B1) and a component of the renal function ( Videau et al. 2010 ). The present work aimed at studying the usefulness of the CIME cocktail in the rat.The CIME cocktail was given per os to three male and three female rats, or incubated with rat liver microsomes. Parent substrates and metabolites were quantified by LC-MS/MS in plasma, urine and hepatic microsomal media, and phenotyping index were subsequently calculated.The CIME cocktail could therefore be used in the rat to phenotype rapidly and simultaneously CYP3A1/2 with omeprazole/omeprazole-sulfone, midazolam/1'-hydroxymidazolam or 4-hydroxymidazolam and/or dextromethorphan/3-methoxymorphinan, CYP2C6/11 with tolbutamide/4-hydroxytolbutamide, CYP2D1/2 with omeprazole/5-hydroxyomeprazole or dextromethorphan/dextrorphan, and UGT1A6/7 with acetaminophen/acetaminophen-glucuronide. Our results confirmed also several known gender differences and brought new information on the urinary excretion of rosuvastatin. However, the major rat CYPs, CYP2C11 and CYP2C12, are not specifically assessed. An optimized version of the CIME cocktail should therefore be designed and would be of major importance to more largely phenotype DMPK enzymes in rats to study DMPK variability factors such as disease, age, or to exposure to inductors or inhibitors.