Maturation markers on DCs exposed to LPS and/or ManLAM and PIM from H37Rv.

<p>Immature DCs were stimulated with mycobacterial glycolipids (ManLAM and PIM at the concentration of 10 µg/ml and 5 µg/ml, respectively) or/and LPS (100 ng/ml) as indicated in the diagrams. After 48 h cells were harvested, stained for maturation markers CD80, CD86, and MHC class II and analyzed by flow cytometry. For each treatment mean fluorescence intensities (MFI) were related to the levels obtained for stimulation with LPS and expressed as %. The median percentage change in MFI is shown as a line. The box defines the 75th and 25th percentiles and the whiskers define the maximum and minimum values of 7–11 donors/group. The horizontal, dashed lines represent surface marker expression obtained for LPS treated DC. Groups significantly different from LPS-treated control are labeled with asterisks. Vertical bars designate significant differences between treatment groups. Wilcoxon matched pair test was used to assess statistical significance (*P<0.05, **P<0.01).</p

Inhibition of ManLAM-induced production of pro-inflammatory cytokines in DCs by ManLAM-specific MAb.

<p>ManLAM from <i>Mtb</i> H37Rv and LPS were pre-incubated for 1 h with ManLAM-specific MAb (KITB24) or isotype-matched control MAb. Next, MAb pre-incubated glycolipids (ManLAM and LPS at the concentration of 10 µg/ml and 100 ng/ml, respectively) were added to immature DCs and 12 h production of TNF (A) and IL-12p40 (B) was estimated in supernatants by ELISA. Experiment was carried out in triplicates, values are mean ± SD. Two-tailed, unpaired <i>t</i>-test was used to assess the statistical significance (***P<0.001).</p

Cytokine production by DCs exposed to LPS and/or ManLAM and PIM from <i>Mtb</i> H37Rv.

<p>After 12 h exposure to ManLAM and/or PIM (at the concentration of 10 µg/ml and 5 µg/ml, respectively), TNF, IL-12p40, IL-6 and IL-10 in DC culture supernatants were assayed by ELISA. The level of cytokines released upon exposure to LPS (100 ng/ml) is shown as 100% of activation (dashed line). Background levels (not-treated cells) are 0%. The median percentage change in cytokine production is shown as a line. The box defines the 75th and 25th percentiles and the whiskers define the maximum and minimum values of 3–9 donors/group. Groups significantly different from LPS-treated control are labeled with asterisks. Vertical bars designate significant differences between treatment groups. Wilcoxon matched pair test was used to assess statistical significance (*P<0.05, **P<0.01).</p

MLVA Based Classification of <em>Mycobacterium tuberculosis</em> Complex Lineages for a Robust Phylogeographic Snapshot of Its Worldwide Molecular Diversity

<div><p>Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including <em>Mycobacterium tuberculosis</em> complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, <em>M. bovis</em>/MIT49, <em>M.canettii</em>/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS<em>6110</em> insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS<em>6110.</em> These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.</p> </div

Two MST phylogenetic trees done with 95 Mozambican strains based on 12-loci MIRU-VNTRs (A), and 24-loci MIRU-VNTRs (B).

<p>Two MST phylogenetic trees done with 95 Mozambican strains based on 12-loci MIRU-VNTRs (A), and 24-loci MIRU-VNTRs (B).</p

Phylogenetic tree constructed with MrBayes3 software (

<p><a href="http://mrbayes.csit.fsu.edu/" target="_blank">http://mrbayes.csit.fsu.edu/</a><b>).</b> The tree is done with the 37 MIRU-VNTR prototypes of <i>M. tuberculosis</i> sensu stricto.</p

Some cases of discrepancies in classification between the newly defined lineages based on 12-loci MIRU-VNTRs and the Brudey's classification scheme in SpolDB4 (see footnote for extended explanation).

a<p>As an extended explanation, one may refer to the example of case F, where profile 1 corresponding to MIT310 (LAM7-TUR lineage on the basis of spoligotyping), and profile 2 corresponding to MIT430 (T3-ETH lineage on the basis of spoligotyping), both correspond to the Euro American-40 sublineage. Indeed, profile 1 with deletion of the block 20–24 and profile 2 with deletion of the block 10–19 could indicate a possible common ancestor with all spacers in positions 10 to 24 being present. As indicated by our IS<i>6110</i>AD-typing data (see text), this hypothetical ancestor would be harboring a copy of IS<i>6110</i> between the spacers 19 and 20. Depending on the adjacent deletion located on the left or the right side of this IS<i>6110</i> would result in the 2 different spoligotype patterns observed here, i.e., profile 1 or 2. Hence, albeit phylogenetically very close, these 2 isolates would be classified as LAM7-TUR and T3-ETH, in the Brudey's classification scheme in SpolDB4.</p>b<p>MIT; MIRU International Type according to the SITVITWEB database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041991#pone.0041991-Demay1" target="_blank">[5]</a>.</p

Additional file 2: of Predominance of Uganda genotype of Mycobacterium tuberculosis isolated from Ugandan patients with tuberculous lymphadenitis

Table S2. Correlation of the major spoligotypes with HIV status

MST tree done with 12 MIRU-VNTR loci of 176 strains from MIRU-VNTR<i>plus</i>

<p><b>database</b> (<a href="http://www.miru-vntrplus.org/MIRU/index.faces" target="_blank">http://www.miru-vntrplus.org/MIRU/index.faces</a>).</p

Characterization of Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping-0

<p><b>Copyright information:</b></p><p>Taken from "Characterization of Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping"</p><p>http://www.biomedcentral.com/1471-2180/7/76</p><p>BMC Microbiology 2007;7():76-76.</p><p>Published online 9 Aug 2007</p><p>PMCID:PMC1988810.</p><p></p>ics software using the unweighted pair group method. The 178 CAS1 strains studied showed an overall homology of >70%. No MIRU clusters were observed between any of the 189 'unique' strains studied