Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0G0.6 days) than those isolated from CB (2.6G1.3 days) and AF (2.3G1.0 days) (P!0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 ؋ 10 3 M. tuberculosis CFU per ml of sputum. (5,(9)(10)(11)29). The use of these drugs as second-line antituberculosis agents is recommended for treating multidrug-resistant (MDR) tuberculosis (TB) (3,8). As with other antimicrobial agents, the use of FQ can generate resistant mutants (5, 6, 11-13). The principal target of the FQ in M. tuberculosis is DNA gyrase, a type II topoisomerase composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively (2,11,33). Mutations in the so-called quinolone resistance-determining region (QRDR) of gyrA are the primary mechanisms of FQ resistance in M. tuberculosis. Amino acids at positions 88, 90, 91, and 94 of gyrA are those most frequently substituted in the FQ-resistant M. tuberculosis clinical isolates (1,11,12,19,22,26,27,(31)(32)(33); mutations of gyrB have rarely been reported (15). Fluoroquinolones (FQ) are antimicrobial agents with good in vitro and in vivo activities against Mycobacterium tuberculosisDespite the increasing use of these drugs in TB therapy, standard 3-week culture-based FQ susceptibility testing in solid media is not always performed (11). PCR-based techniques provide new possibilities for the rapid diagnosis of FQ resistance; however, it can be difficult to put them into practice in the mycobacteriology laboratory (7,24,33,34). To this end, we developed a reverse hybridization-based line probe assay for rapid detection of gyrA mutations in M. tuberculosis. The test was ...

Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4+ T cells producing IFN-gamma and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4+ T cell proliferation and IFN-gamma production in response to Ag85B protein, despite restraint of Th1 response by CD8+ T cell-dependent mechanisms and activation of CD4+ T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4+ T cells lost the ability to produce IFN-gamma and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4+ T cells determined dilution or loss of the protective Ag85B-specific CD4+ induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective anti-tuberculosis immune response, and actually concur to disease progression.

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The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC-specific C-type lectin intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC-SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)-10 synthesis. Here, we confirm that DC-SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 and show that it is not expressed in DC derived from monocytes cultured in GM-CSF and interferon-alpha (IFN-alpha). We also demonstrate that DC-SIGN(-) DC cultured in GM-CSF and IFN-alpha are able to phagocytose BCG and to undergo a maturation program as well as DC-SIGN(+) DC cultured in IL-4 and GM-CSF. We also show that BCG causes the impairment of IL-12 and the induction of IL-10 secretion by DC, irrespective of DC-SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of naïve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC-SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria-induced immunosuppression cannot be attributed to the engagement of a single receptor.