Anti-ManLAM antibodies circulating in Multi Drug Resistant (MDR) and Drug-sensitive tuberculosis (TB) patients exert differential effect on fagocytosis and costimulatory potential of Dendritic Cells.

SCHIERLOH P; COLMAN E; LANDONI V; LABORDE E; BASILE J; BALBOA L; SABIO Y GARCÍA C; ROMERO M; GEFFNER L; BARRERA L; CASTAGNINO J; SASIAIN MC

Congreso; First French - Argentine Immunology Congress; 2010

Sociedad Argentina de Inmunologia y Sociedad Francesa de Inmunologia

Mannosilated lipoarabinomannan (ManLAM) is an abundant, complex and bioactive lipoglycan present in cell wall of pathogenic slow-growing mycobacteria (1,2). This molecule exerts its immunomodulatory effect through recognition by innate immune receptors expressed on host cells (3,4). Anti-ManLAM antibodies (αManLAM Abs) have been detected in serum from tuberculosis patients (TB) but its role in physiopathology remains poorly studied (5-8). In order to address the role of αManLAM Abs on dendritic cell (DC) function and factors/mechanisms involved, αManLAM Abs were characterized in serum samples from 20 MDR and 20 Drug-sensitive (DS) TB patients and 17 healthy donors (N), thereafter, well characterized serum subgroups were employed to perform bioassays on monocytes derived DC from normal blood donors. Immunochemical characterization of circulating ManLAM Abs For αManLAM Abs quantification in sera, we developed an in-house indirect ELISA using purified ManLAM (Colorado State Univ.) as capture antigen (5,6). An optimal 1/1000 serum dilution were established in preliminary experiments. Polivalent goat antihuman IgG/A/M Ab conjugated with HRP (Sigma) was used as 2º reagent. We confirm the presence of circulating ManLAM Abs in 85% of TB sera (34/40). No significant differences were observed among mean titres of MDR (N=20) and DS-TB (N=20). In order to analyze chemical nature (lipidic vs glycosidic) of αManLAM epitopes, high titer sera (MDR N=6; DS N=6) were diluted 1/1000 and pre-incubated with excess of total lipidic extract of Mtb (Tlip, Colorado State Univ.) or yeast derived mannan (Sigma) before αManLAM ELISA assay (8). Competition ELISA showed that both kinds of ManLAM epitopes were recognized by most sera without clear association to any TB group. Next, to analyze cell wall epitope-display, we developed a whole Bacteria ELISA method by using intact M.tuberculosis (Mtb) 6006 local strain (ANLIS-Malbran) as antigen. We establish an optimal 1/500 serum dilution and optimal sensitivity/specificity range for this method. All αManLAM high titer sera were above cut-off value (100% positivity) and whole bacteria binding correlates well with αManLAM titre (p