Persistence of mycobacterium tuberculosis in people without clinical disease

Mycobacterium tuberculosis (MTB) can cause both active disease and latent tuberculosis (LTBI). Latent infection can reactivate and, is therefore, a potential reservoir of infection. Effective diagnosis and treatment of LTBI can only be achieved with a clear understanding of the pathogenesis of LTBI. This thesis attempts to develop an effective diagnostic method to detect LTBI and to clarify some aspects of its pathogenesis. Tuberculin skin tests performed on Sri Lankan school children revealed a high prevalence of LTBI (40.5%) in Jaffna compared to Hambanthota (7.3%). Geographical variations and dietary habits of the population in the two locations have influenced reactions to tuberculin significantly. Evaluation of antibody titre against 16kDa, 38kDa, 65kDa, 70kDa antigens using ELISA revealed a significantly increased IgG response in elderly people. Titres to 16kDa and 38kDa antigens were as high as in people with active disease. However, isoelectric focusing revealed that the antibody response in elderly people was mainly oligoclonal to 16kDa whereas the oligoclonal response in active disease was mainly to the 38kDa antigen. Polymerase chain reaction (PCR) detected MTB DNA in the sputum of 55% of elderly people, suggesting shedding of bacilli during LTBI. However, spoligotyping of the DNA ruled out transmission of infection within this community. Detection of MTB DNA using PCR and in situ PCR on autopsy material of people without clinical TB suggests that MTB can lurk in arrested granulomas and in normal tissues during LTBI. Latency was maintained at various extrapulmonary sites including the brain. MTB DNA was detected in the cytoplasm, and less frequently inside the nucleus of non-professional phagocytic cells and in macrophages. Acid-fast and immunohistochemical staining failed to detect bacilli in sections adjacent to those that were MTB DNA positive. Spoligotyping of MTB DNA in these tissues revealed a conserved pattern. In conclusion, this study has made some interesting findings, but raised many questions regarding the LTBI pathogenesis. Addressing these may help the development of effective diagnosis and treatment for LTBI

Mucosal immunity and novel tuberculosis vaccine strategies: route of immunisation-determined T-cell homing to restricted lung mucosal compartments

Despite the use of bacille Calmette–Guérin (BCG) for almost a century, pulmonary tuberculosis (TB) continues to be a serious global health concern. Therefore, there has been a pressing need for the development of new booster vaccines to enhance existing BCG-induced immunity. Protection following mucosal intranasal immunisation with AdHu5Ag85A is associated with the localisation of antigen-specific T-cells to the lung airway. However, parenteral intramuscular immunisation is unable to provide protection despite the apparent presence of antigen-specific T-cells in the lung interstitium. Recent advances in intravascular staining have allowed us to reassess the previously established T-cell distribution profile and its relationship with the observed differential protection. Respiratory mucosal immunisation empowers T-cells to home to both the lung interstitium and the airway lumen, whereas intramuscular immunisation-activated T-cells are largely trapped within the pulmonary vasculature, unable to populate the lung interstitium and airway. Given the mounting evidence supporting the safety and enhanced efficacy of respiratory mucosal immunisation over the traditional parenteral immunisation route, a greater effort should be made to clinically develop respiratory mucosal-deliverable TB vaccines

Within the Enemy’s Camp: contribution of the granuloma to the dissemination, persistence and transmission of Mycobacterium tuberculosis

Pulmonary tuberculosis, caused by Mycobacterium tuberculosis (M.tb) represents a leading global health concern, with 8.7 million newly emerging cases, and 1.4 million reported deaths annually. Despite an estimated one third of the world’s population being infected, relatively few infected individuals ever develop active clinical disease. The ability of the host to remain latently infected while preventing disease is thought to be due to the generation of a robust type 1 immune response in the lung, capable of controlling, but not clearing, M.tb. A key feature of the type 1 immune response to M.tb is the formation of immune cellular aggregates termed granuloma. The granuloma structure has long been considered a hallmark of host’s protective response toward M.tb. Historically, a correlative relationship between granuloma formation/maintenance and bacterial control has been seen in models where disrupted granuloma formation or structure was found to be fatal. Despite this established relationship much about the granuloma’s role in M.tb immunity remains unknown. Recent publications suggest that the granuloma actually aids the persistence of M.tb and that the development of a necrotic granuloma is essential to person-to-person transmission. Our group and others have recently demonstrated that enclosed within the granuloma is a population of immunologically altered antigen-presenting cells and T lymphocyte populations. Of note, the ability of these populations to produce type 1 cytokines such as interferon-gamma, and bactericidal products including nitric oxide, are significantly reduced, while remaining competent to produce high levels immunosuppressive interleukin-10. These observations indicate that although the chronic granuloma represents a highly unique environment, it is more similar to that of a tumor than an active site of bacterial control. In this review we will explore what is known about this unique environment and its contribution to the persistence of M.tb

Regulation of TB vaccine-induced airway luminal T cells by respiratory exposure to endotoxin.

Tuberculosis (TB) vaccine-induced airway luminal T cells (ALT) have recently been shown to be critical to host defense against pulmonary TB. However, the mechanisms that maintain memory ALT remain poorly understood. In particular, whether respiratory mucosal exposure to environmental agents such as endotoxin may regulate the size of vaccine-induced ALT population is still unclear. Using a murine model of respiratory genetic TB vaccination and respiratory LPS exposure, we have addressed this issue in the current study. We have found that single or repeated LPS exposure increases the number of antigen-specific ALT which are capable of robust secondary responses to pulmonary mycobacterial challenge. To investigate the potential mechanisms by which LPS exposure modulates the ALT population, we have examined the role of ALT proliferation and peripheral T cell recruitment. We have found that LPS exposure-increased ALT is not dependent on increased ALT proliferation as respiratory LPS exposure does not significantly increase the rate of proliferation of ALT. But rather, we find it to be dependent upon the recruitment of peripheral T cells into the airway lumen as blockade of peripheral T cell supplies markedly reduces the initially increased ALT. Thus, our data suggest that environmental exposure to airborne agents such as endotoxin has a profound modulatory effect on TB vaccine-elicited T cells within the respiratory tract. Our study provides a new, M.tb antigen-independent mechanism by which the respiratory mucosal anti-TB memory T cells may be maintained

Multifunctional profile of CD4⁺ T cells in the lung of infant and adult mice following BCG immunization.

<p>Infant and adult mice were immunized s.c. with BCG and sacrificed at 4 (A), 8 (B), or 16 (C) weeks following immunization. Cells from the lung were stimulated with <i>M</i>.<i>tb</i> CF + crude BCG for 24h or left unstimulated as a control. Cells were stained and analyzed by flow cytometry. Average proportions displayed in pie chart are of the CD4 T cells expressing specific cytokine combinations. Absolute numbers of CD4⁺ T cells in the tissues were calculated and displayed in bar graphs. Results are from one independent experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.</p

Ag-specific T cell responses in the lung after boost AdHu5Ag85A immunization in BCG primed mice.

<p>Infant and adult mice were BCG immunized, and the AdHu5Ag85A booster vaccine was administered i.n. at 16 weeks post-BCG (A). The mice were sacrificed 4 weeks (B, C, D) or 8 weeks (E, F, G) after boosting. Lung cells were stimulated either with <i>M</i>.<i>tb</i> CF + crude BCG for 24h (open bar), or Ag85A-specific CD4 or CD8 T cell peptide for 6h (black bar), or left unstimulated (B, C, E, F). Cells were stained and analyzed by flow cytometry. Absolute numbers of IFN-γ⁺CD4⁺ (B, E) and IFN-γ⁺CD8⁺ (C, F) T cells were calculated (unstimulated subtracted from stimulated). Ag85A CD8 peptide tetramer staining was performed on lung cells, and analyzed by flow cytometry (D, G). Absolute numbers of tet⁺CD8⁺ T cells were calculated. Results are from one experiment per timepoint, n = 4-5/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. All other comparisons (not indicated) were not significant.</p

Memory T cells in the lung and spleen of infant and adult mice following BCG immunization.

<p>Infant and adult mice were BCG immunized and sacrificed at 8 or 16 weeks. Cells from the lung (A) and spleen (B) were stained with extracellular antibodies for memory CD4 T cell markers and analyzed by flow cytometry. Absolute numbers of CD4⁺CD44⁺ cells that are T_eff/T_EM (CD127^-/+CD62L^-) or T_CM (CD127⁺CD62L⁺) in the tissues were calculated. Results are from one experiment per timepoint, n = 4/group/timepoint. Data are expressed as Mean ± SEM. *, p < 0.05.</p