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The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-α and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14(+) and poorly differentiated into CD1a(+) cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1β, interleukin-6, interleukin-10 and tumor necrosis factor-α upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host-pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence.

Aerobic (5-day-old cultures) and nonreplicating (dormant) Mycobacterium tuberculosis (5-, 12-, and 19-day-old cultures) bacteria were treated with rifampin (R), moxifloxacin (MX), metronidazole (MZ), amikacin (AK), or capreomycin (CP) for 7, 14, and 21 days. R-MX-MZ-AK and R-MX-MZ-CP killed both aerobic and dormant bacilli in 21 days, as shown by lack of regrowth in solid and liquid media. R-MX-MZ-AK and R-MX-MZ-CP also caused a strong decrease of nonreplicating bacilli in 7 days in a cell-based dormancy model.

Although multidrug-resistant (MDR) tuberculosis (TB) is a major public health problem in Eastern Europe, the factors contributing to emergence, spread and containment of MDR-TB are not well defined. Here, we analysed the characteristics of drug-resistant TB in a cross-sectional study in Abkhazia (Georgia) between 2003 and 2005, where standard short-course chemotherapy is supplemented with individualized drug-resistance therapy. Drug susceptibility testing (DST) and molecular typing were carried out for Mycobacterium tuberculosis complex strains from consecutive smear-positive TB patients. Out of 366 patients, 60.4% were resistant to any first-line drugs and 21% had MDR-TB. Overall, 25% of all strains belong to the Beijing genotype, which was found to be strongly associated with the risk of MDR-TB (OR 25.9, 95% CI 10.2-66.0) and transmission (OR 2.8, 95% CI 1.6-5.0). One dominant MDR Beijing clone represents 23% of all MDR-TB cases. The level of MDR-TB did not decline during the study period, coinciding with increasing levels of MDR Beijing strains among previously treated cases. Standard chemotherapy plus individualized drug-resistance therapy, guided by conventional DST, might be not sufficient to control MDR-TB in Eastern Europe in light of the spread of "highly transmissible" MDR Beijing strains circulating in the community.

Toll-like receptors (TLRs) sense conserved structures of pathogens and influence macrophage functions. Here we investigated the impact of TLR signaling on the modulation of macrophage defense mechanisms against infection of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis. We found that a synthetic derivative of the TLR2/6 agonist MALP-2 and the potent TLR4 agonist lipopolysaccharide inhibited the intracellular growth of MTB in murine macrophages. Likely the microbicidal effect was mediated by production of nitric oxide while it is still unclear the role played by release of TNF-α , IL-6, MIP-1β and IL-10. These results suggest that the activation of microbicidal defense via TLR ligands is an appealing target for the establishment on immune intervention against tuberculosis.

T helper type-1 response is essential to control Mycobacterium tuberculosis (MTB) infection but excessive antigen-mediated inflammation concurs to pathology. In mice challenged with MTB, the protection elicited by an Ag85B-encoding DNA vaccine, was lost when mice were boosted with Ag85B-protein in the absence of adjuvant. This effect was due to the expansion of a set of IFN-gamma secreting-CD4+ T cells highly responsive to Ag85B-protein but which lost the ability to interact with MTB-infected macrophages and control MTB growth. Ag85B-protein co-administration with the adjuvant LTK63 reduced the expansion of Ag85B-protein-responding CD4+ T cells and allowed the survival of those protective Ag85B-specific CD4+ T cells induced by the Ag85B-encoding DNA vaccine. Consequently, the protection against MTB-infection was restored. LTK63 caused also a marked augmentation of Ag85B-specific antibodies, in particular those belonging to the IgG2b isotype. The recovery of protection through a down-modulation of antigen-specific IFN-gamma response by an adjuvant is a novel finding which could be of relevance in tuberculosis vaccination.

Setting: Quality assurance for the World Health Organization (WHO)/International Union Against Tuberculosis and Lung Disease (The Union) global tuberculosis (TB) drug resistance surveillance programme., Objective: To monitor the quality of drug susceptibility testing (DST) in different countries., Methods: In 2002-2003 and 2005-2006, 20 Mycobacterium tuberculosis strains were sent by the WHO/Union Supranational Reference Laboratory of Rome to TB reference laboratories in Albania, Bahrain, Kosovo, Mozambique, Oman, Qatar and Turkey for external quality control (EQC)., Results: In 2002-2003, the specificity, sensitivity, efficiency, reproducibility and predictive values for resistance/susceptibility were >or=90% for streptomycin (SM), isoniazid (INH) and ethambutol (EMB). In 2005-2006, all statistical values were >or=96% for SM, INH, rifampicin and EMB., Conclusion: EQC improved the quality of M. tuberculosis DST in the participating countries.

A strain of Nocardia was isolated from cutaneous ulcers of a human immunodeficiency virus-infected patient in Italy. Comparative 16S rRNA gene sequence analysis revealed that the isolate represented a strain of Nocardia asiatica. Antimicrobial susceptibility testing was essential to guide the clinicians to successfully treat this infection.

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.

Long-term nonreplicating (dormant) Mycobacterium tuberculosis populations (26-day-old cells) were sterilized by metronidazole plus rifampin, but not by metronidazole or rifampin alone, after 7 and 11 days of exposure to the drugs. Lower or no drug activity was observed against 19- or 12-day-old dormant or 5-day-old actively replicating populations.

Moxifloxacin (MOX), an 8-methoxyquinolone compound, is now widely used for the treatment of bacterial infections and also accepted as 2nd-line drug for the treatment of multidrug-resistant (MDR) tuberculosis. To tentatively correlate the clinical outcome with in vitro results, we sought to set up susceptibility test conditions for Mycobacterium tuberculosis against MOX by using the reference agar proportion method, the BACTEC 460 radiometric system, and the recently validated nonradiometric BACTEC MGIT 960 system. Our aim was to determine the critical MOX test concentration to be used with the abovementioned methods for routine susceptibility testing. MICs were determined for 20 pan-susceptible strains, 10 MDR strains, and 10 fluoroquinolone-resistant strains with defined gyrA mutations. MOX MICs resulted in a bimodal pattern with values for gyrA mutants considerably higher than those for pan-susceptible and MDR strains. Our data showed that a concentration of 0.5 microg/mL allowed a clear-cut separation between susceptible and resistant strains when tested by all the studied methods. Confirmatory test with a subset of pan-susceptible and MDR isolates appeared to validate the selected critical concentration. The MOX-resistant strains were almost isolated from patients with prior fluoroquinolone exposure.

Increasing evidence is now available showing that CD1-restricted T cell responses against non-peptide mycobacterial antigens could play a role in the immune resistance against tuberculosis. BCG, widely used in anti-tubercular vaccination, shares various constituents with Mycobacterium tuberculosis, but does not provide full protection. In the present study we have investigated the pattern of group 1 CD1 molecule expression in adherent mononuclear cells (AMNC) of human peripheral blood, infected in vitro with BCG. Shortly after exposure to BCG, both BCG-positive and BCG-negative AMNC showed a moderate CD1 expression elicited by BCG-induced release of GM-CSF presumably acting through an autocrine and a paracrine mechanism. This was demonstrated using two-color flow cytometry with green fluorescent BCG and anti-CD1 PE-labeled antibodies. However, high CD1 expression induced by exogenously added GM-CSF in AMNC was reduced if target cells were cocultivated with BCG. Monoclonal antibodies against IL-10 partially restored CD1 expression, thus showing that IL-10, released from infected AMNC, is involved, at least in part, in CD1 negative modulation. Therefore, through a complex cytokine network, including not yet identified factor(s), BCG triggers but does not allow full expression of CD1 on AMNC. It cannot be excluded that this mechanism could play a role in the limited efficiency of BCG vaccination.

A plasmid DNA containing two different expression cassettes was prepared to independently drive antigen 85B (85B) of Mycobacterium tuberculosis and HIV-Tat in C57BL/6 mice. In vivo expression of the plasmid was demonstrated by efficient transcription of 85B and Tat mRNAs in mouse fibroblasts. DNA-85B or DNA-(85B-Tat) were immunogenic and protected mice to the same extent against M. tuberculosis infection, with a decrease in the numbers of CFU lung-1 in comparison with nonimmunized animals down to levels (0.64 log10 CFU) not significantly different from protection conferred by bacillus Calmette-Guérin vaccine (0.97 log10 CFU decrease). Multipromoter plasmids, which permit the reduction of the total amount of DNA injected, can be useful for DNA vaccination against tuberculosis.

The Mycobacterium tuberculosis genome encodes 13 sigma factors. We have previously shown that mutations in some of these transcriptional activators render M. tuberculosis sensitive to various environmental stresses and can attenuate the virulence phenotype. In this work, we focused on extracytoplasmic factor sigmaE and studied the effects induced by the deletion of its structural gene (sigE) in the infection of human monocyte-derived dendritic cells (MDDC). We found that the wild-type M. tuberculosis strain (H37Rv), the sigE mutant (ST28), and the complemented strain (ST29) were able to infect dendritic cells (DC) to similar extents, although at 4 days postinfection a reduced ability to grow inside MDDC was observed for the sigE mutant ST28. After mycobacterium capture, the majority of MDDC underwent full maturation and expressed both inflammatory cytokines, such as tumor necrosis factor alpha, and the regulatory cytokines interleukin-12 (IL-12), IL-18, and beta interferon (IFN-beta). Conversely, a higher level of production of IL-10 was observed in ST28-infected MDDC compared to H37Rv- or ST29-infected cell results. However, in spite of the presence of IL-10, supernatants from ST28-infected DC induced IFN-gamma production by T cells similarly to those from H37Rv-infected DC culture. On the other hand, IL-10 impaired CXCL10 production in sigE mutant-infected DC and, indeed, its neutralization restored CXCL10 secretion. In line with these results, supernatants from ST28-infected cells showed a decreased capability to recruit CXCR3+ CD4+ T cells compared to those obtained from H37Rv-infected DC culture. Thus, our findings suggest that the sigE mutant-induced secretion of IL-10 inhibits CXCL10 expression and, in turn, the recruitment of activated-effector cells involved in the formation of granulomas.

Setting: After the collapse of the Soviet Union, countries in the region faced a dramatic increase in tuberculosis cases and the emergence of drug resistance., Objective: To discuss the relevance of the DOTS strategy in settings with a high prevalence of drug resistance., Design: Retrospective analysis of one-year treatment outcomes of short-course chemotherapy (SCC) and results of drug susceptibility testing (DST) surveys of six programmes located in the former Soviet Union: Kemerovo prison, Russia; Abkhasia, Georgia; Nagorno-Karabagh, Azerbaijan; Karakalpakstan, Uzbekistan; Dashoguz Velayat, Turkmenistan; and South Kazakhstan Oblast, Kazakhstan. Results are reported for new and previously treated smear-positive patients., Results: Treatment outcomes of 3090 patients and DST results of 1383 patients were collected. Treatment success rates ranged between 87% and 61%, in Nagorno-Karabagh and Kemerovo, respectively, and failure rates between 7% and 23%. Any drug resistance ranged between 66% and 31% in the same programmes. MDR rates ranged between 28% in Karakalpakstan and Kemerovo prison and 4% in Nagorno-Karabagh., Conclusion: These results show the limits of SCC in settings with a high prevalence of drug resistance. They demonstrate that adapting treatment according to resistance patterns, access to reliable culture, DST and good quality second-line drugs are necessary.

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 x 10(3) M. tuberculosis CFU per ml of sputum.

The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.

Recovery of Mycobacterium tuberculosis from sputa treated with cetyl-pyridinium chloride (CPC) and stored for 20 +/- 9 days was significantly higher than that from sputa that were untreated and processed by the N-acetyl-L-cisteine-NaOH method. Addition of CPC is useful for isolation of M. tuberculosis from sputa subjected to long-term storage received from remote areas of the world.

The only available vaccine against tuberculosis (TB) is Bacillus Calmette-Guérin (BCG) whose efficacy in preventing pulmonary tuberculosis is however controversial. Here, we show that BCG infection of monocytes causes their differentiation into mature dendritic cells (DCs) lacking CD1 molecules expression, coupled with suboptimal up-regulation of HLA class II, CD80 and CD40 molecules and a marked unresponsiveness to lipopolysaccharide stimulation. In addition, alloreactive naïve T lymphocytes primed by these subverted DCs did not undergo defined functional polarization, as witnessed by their inability to produce IFN-gamma. Since efficient antigen presentation and IFN-gamma production by mycobacterial-specific T lymphocytes are required for protection against Mycobacterium tuberculosis, our data might provide additional explanation for the low efficacy of BCG vaccination.

Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-alpha) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMphi) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-alpha, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMphi, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.

The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.

Programmed cell death (PCD) is an active way for plant cells marching to death, which plays an important role in plant development and stress responses. Cytological, biochemical, molecular and physiological methods for measuring plant PCD were reviewed. Application of flow cytometer to plant PCD research was also covered.

The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the Valpha14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of Valpha14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1(+) Valpha14 NKT population preferentially producing IFN-gamma predominated at an early stage (day 8), which was substituted by an NK1.1(-) population preferentially producing IL-4 at later stages (day 30). Despite the fact that Valpha14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in Valpha14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that Valpha14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-alpha in liver and lungs of Valpha14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-alpha mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for Valpha14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-gamma-inducing cytokines, IFN-alpha beta and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-alpha beta. Indeed, IFN-alpha beta neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4(+), and CD8(+) cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-alpha beta-induced expression of CXCL10, neutralization of IFN-alpha beta significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-alpha beta may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

The activity of moxifloxacin was enhanced by the addition of ethionamide but not by that of cycloserine, thiacetazone, capreomycin, para-aminosalicylic acid, or linezolid in BALB/c mice infected with a strain of Mycobacterium tuberculosis resistant to isoniazid, rifampin, and six other drugs. These observations are important for the therapy of multidrug-resistant tuberculosis.

The potential pathogenic role of Mycobacterium tuberculosis H37Rv fadD33, a gene encoding an acyl-CoA synthase that is underexpressed in the attenuated strain H37Ra, was investigated. In a first approach, fadD33 was cloned and expressed in strain H37Ra to restore gene expression and fadD33-complemented bacteria were used to investigate whether fadD33 might confer any growth advantage to M. tuberculosis H37Ra in an infection model of BALB/c mice. No differences were found in the growth rates of M. tuberculosis H37Rv, H37Ra and fadD33-complemented H37Ra in the lungs and spleen. In contrast, in the liver, where the attenuated strain H37Ra showed impaired growth compared to the virulent strain H37Rv, complementation of the attenuated strain H37Ra with fadD33 restored bacterial replication. In a further approach, the fadD33 gene of strain H37Rv was disrupted by allelic exchange mutagenesis and the virulence of the mutant strain was tested by mouse infection. It was found that disruption of fadD33 decreased M. tuberculosis H37Rv growth in the liver, but not in the lungs or spleen, and complementation of the fadD33-disrupted mutant with fadD33 restored bacterial replication in the liver, but did not affect replication in the lungs and spleen. These findings suggest that fadD33 plays a role in M. tuberculosis virulence by supporting bacterial growth in the liver.

The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography. The GroES preparation showed high purity by electrophoresis and immunoblotting. Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system. BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M. avium. Neither GroES nor CpG alone provided any protection against subsequent challenge with M. avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M. avium after intranasal challenge with a low dose of the organism. This indicates that intranasal administration of GroES and CpG oligodeoxynucleotides increases the resistance of BALB/c mice to M. avium infection.

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt-MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS-matured DC. In addition, Mt-MoDC produced TNF-alpha and IL-10, but were unable to secrete IL-12. The generation of Mt-MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat-killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt-MoDC revealed an impaired antigen-presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt-MoDC were unable to secrete cytokines, in particular IL-4 and IFN-gamma, suggesting that they could be ineffective in helping the macrophage-mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.