Detection and treatment of subclinical tuberculosis

SummaryReduction of active disease by preventive therapy has the potential to make an important contribution towards the goal of tuberculosis (TB) elimination. This report summarises discussions amongst a Working Group convened to consider areas of research that will be important in optimising the design and delivery of preventative therapies. The Working Group met in Cape Town on 26th February 2012, following presentation of results from the GC11 Grand Challenges in Global Health project to discover drugs for latent TB

Cytosolic Phospholipase A(2) Enzymes Are Not Required by Mouse Bone Marrow-Derived Macrophages for the Control of Mycobacterium tuberculosis In Vitro

During the course of infection Mycobacterium tuberculosis predominantly resides within macrophages, where it encounters and is often able to resist the antibacterial mechanisms of the host. In this study, we assessed the role of macrophage phospholipases A(2) (PLA(2)s) in defense against M. tuberculosis. Mouse bone marrow-derived macrophages (BMDMs) expressed cPLA(2)-IVA, cPLA(2)-IVB, iPLA(2)-VI, sPLA(2)-IIE, and sPLA(2)-XIIA. The expression of cPLA(2)-IVA was increased in response to M. tuberculosis, gamma interferon, or their combination, and cPLA(2)-IVA mediated the release of arachidonic acid, which was stimulated by M. tuberculosis in activated, but not unactivated, macrophages. We confirmed that arachidonic acid is highly mycobactericidal in a concentration- and pH-dependent manner in vitro. However, when M. tuberculosis-infected macrophages were treated with PLA(2) inhibitors, intracellular survival of M. tuberculosis was not affected, even in inducible nitric oxide synthase-deficient macrophages, in which a major bactericidal mechanism is removed. Moreover, intracellular survival of M. tuberculosis was similar in cPLA(2)-IVA-deficient and wild-type macrophages. Our results demonstrate that the cytosolic PLA(2)s are not required by murine BMDMs to kill M. tuberculosis

Development of OVαβ TCR expressing thymocytes.

<p>The strong skewing of thymocytes expressing the high affinity MBPαβ TCR towards the CD4 lineage is not seen in OVαβTCR expressing mice. Expression of only the MBPβ chain does not markedly alter T cell development. (<b>A</b>) Single cell suspensions of thymocytes (top) and lymph node cells (bottom) from OVαβ, MBPαβ, MBPβ transgenic and transgene negative mice were stained with antibodies against CD4, CD8 and TCR Cβ. Only TCR Cβ⁺ cells are shown in the lymph node cell plots. The number indicates the percent of cells in the quadrant. Only subtle variations in the percent of cells within each quadrant were noted during repeat experiments. (<b>B</b>) CD4 SP thymocytes from OVαβ TCR transgenic mice express a high percentage of endogenous TCRα chains, whereas MBPαβ TCR transgenic mice predominantly express the transgene encoded Vα2 chain. Single cell suspensions of thymocytes from the indicated mouse strain were stained with antibodies against CD4, CD8 and individually with each of the four available TCR Vα specific antibodies. The percent of cells within the CD4 single positive population expressing each of the Vαs is shown. More than 1×10⁶ events were collected per sample. One of two experiments is shown. Greater than 98% of T cells in the OVαβ, MBPαβ and MBPβ mice expressed the transgene encoded Vβ8.2 TCR. (<b>C</b>) Increased levels of proliferation in response to the Ac1-11 peptide indicate a substantial number of antigen specific T cells develop in the OVαβ mice. CD4 T cells were enriched by negative selection. Double cultures were set up with 2.5×10⁴ CD4⁺ T cells and 1×10⁵ T cell depleted, irradiated B10.PL splenocytes plus the indicated amount of the MBP Ac1-11 peptide. Assays were pulsed with 3[H]thymidine after 48 hours and harvested after an additional 24 hours. Each condition was set up in triplicate and data was averaged. One example of three experiments is shown.</p

Thymocytes expressing the OVαβ TCR are poorly positively selected even in the absence of endogenous TCRs.

<p>(<b>A</b>) As compared to thymocytes expressing the high affinity MPBαβ TCR, the OVαβ TCR is very inefficiently positively selected, which is reflected in a much lower percentage of CD4SP thymocytes. (<b>B</b>) OVαβ TCR transgenic double positive (DP) thymocytes have a minimal upregulaton of CD69, which is an indicator of productive TCR-MHC interactions. (<b>C</b>) FACS analysis of lymph node cells shows that, despite poor positive selection, CD4 T cells accumulate in the OVαβ TCR transgenic mouse.</p

The relative affinity of the OVαβ and MBPαβ TCRs.

<p>Antibody blocking studies show that the OVαβ TCR is of much lower affinity for the I-A^u:Ac1-11 peptide ligand as compared to the MBPαβ TCR. (<b>A</b>) Purified CD4⁺ T cells from MBPαβ TCR RAG1^−/− and OVαβ TCR RAG1^−/− mice were incubated with the indicated amount of antibody against Vβ8 (F23.1) and then challenged with titrated amounts of the Ac1-11 peptide presenting by B10.PL APCs. (<b>B</b>) Purified T cells were activated with 3uM Ac1-11, presented by B10.PL APCS, in the presence of titrated amounts of blocking antibody. Assays were pulsed after 48 hours and harvested at 72 hours. Each condition was done in triplicate and the data was averaged. One representative experiment of three is shown. (<b>C</b>) For clarity, the data from (B) are presented as the percent of the maximal response of the T cells without the blocking antibody.</p

Development of spontaneous EAE in TCR transgenic mice.

<p>(<b>A</b>) RAG1 deficient mice were monitored for twelve months for overt signs of EAE (hind leg paraparesis). The day of the onset of EAE in individual mice is indicated on the chart. (<b>B</b>) More than 60% of MBPαβ mice and nearly all OVαβ TCR mice remained healthy throughout the course of the study, whereas 100% of the TCR transgenic RAG1 deficient became sick. Onset of disease was at an average of eleven weeks of age in all three susceptible mice.</p

Analysis of lymphocytes from MBPβ transgenic mice cultured with the AC1-11 peptide.

<p>Total lymphocytes from MBPβ-only transgenic mice were bulk cultured with the MBP-derived AC1-11 peptide. Several additional rounds of activation were done by culturing with T cell depleted B10.PL splenocytes loaded with the AC1-11 peptide. FACS was carried out seven days after the final activation. (<b>A</b>) Nearly all remaining cells were CD4⁺ and expressed the transgene-encoded Vβ8 TCRβ chain. Across several experiments, approximately 30% of the T cells were not Vα2 positive. (<b>B</b>) The Vα2⁺ and Vα2⁻ T cells were sorted and then stimulated by the addition of irradiated, T cell depleted B10.PL splenocytes and titrated amounts of the AC1-11 peptide. Cells were pulsed with 3[H]thymidine after 48 hours and harvested after a total of 72 hours. Approximately 5×10⁴ T cells and 1×10⁵ APCs were used per well. Assays were carried out in triplicate and averaged. Results shown are representative of three independent experiments. (<b>C</b>) Predicted amino acid sequence of the CDR3α region of the Vα6 (TRAV6-7/DV9*02) – Jα13 (TRAJ13*01) TCR cloned from the Vα2⁻ population. Gene nomeclature based on the IMTG Marie-Paule convention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017702#pone.0017702-Lefranc1" target="_blank">[46]</a>.</p

Acid-Susceptible Mutants of Mycobacterium tuberculosis Share Hypersusceptibility to Cell Wall and Oxidative Stress and to the Host Environment▿

Mycobacterium tuberculosis can persist in macrophage phagosomes that acidify to a pH of ∼4.5 after activation of the macrophage with gamma interferon. How the bacterium resists the low pH of the acidified phagosome is incompletely understood. A screen of 10,100 M. tuberculosis transposon mutants for mutants hypersensitive to pH 4.5 led to the discovery of 21 genes whose disruption attenuated survival of M. tuberculosis at a low pH (41). Here, we show that acid-sensitive M. tuberculosis mutants with transposon insertions in Rv2136c, Rv2224c, ponA2, and lysX were hypersensitive to antibiotics, sodium dodecyl sulfate, heat shock, and reactive oxygen and nitrogen intermediates, indicating that acid resistance can be associated with protection against other forms of stress. The Rv2136c mutant was impaired in intrabacterial pH homeostasis and unable to maintain a neutral intrabacterial pH in activated macrophages. The Rv2136c, Rv2224c, and ponA2 mutants were attenuated in mice, with the Rv2136c mutant displaying the most severe level of attenuation. Pathways utilized by M. tuberculosis for acid resistance and intrabacterial pH maintenance are potential targets for chemotherapy