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Objective: To study the effects and mechanisms of recombinant Mycobacterium smegmatis (rMS) carrying pZM03 (a co-expression plasmid encoding human granulysin [GLS] and murine interleukin 12 [IL-12]) on murine M. tuberculosis infection., Design: BALB/c mice infected with M. tuberculosis were treated with normal saline, M. smegmatis, pZM03 or rMS. The number of viable bacteria in the lungs and spleens were counted to observe the therapeutic effects. The levels of IL-12 and interferon-gamma (IFN-gamma) in serum, and IFN-gamma and tumour necrosis factor-alpha (TNF-alpha) released from spleen lymphocytes were detected to observe the T-helper 1 (Th1) response. Secretory IgA (SIgA) in bronchoalveolar lavage fluid was measured to observe the mucosal immunity. The lungs and spleens were prepared for pathological analysis., Results: The rMS group showed a significantly reduced number of colony-forming units compared to the other groups. The expression of GLS in the tissue, and increased levels of IL-12, IFN-gamma, TNF-alpha and SIgA, were found in the rMS group. The pathological changes in the lungs of the rMS group were localised, while those in the control group were extensive., Conclusion: rMS had immunotherapeutic effects associated with a switch to the Th1 response and the antibacterial activity of GLS.

Splenic tuberculosis is an unusual clinical phenomenon, especially in immunocompetent hosts. It often demonstrates diagnostic complexity, which makes the identification of the agent difficult. We encountered the case of a middle-aged immunocompetent male who claimed to be suffering from pain in the left hypochondriac region without any indication of cough, hemoptysis, weight loss or fever. When physically examined, he had splenomegaly without any other clinical findings. This was further confirmed by imaging. A splenectomy was performed, and samples were taken for histopathological examination and microbiological analysis. Gross examination of the specimen showed multiple nodules coalescing to form a large yellowish-white mass of solid consistency. Histopathological examination showed large areas of caseation surrounded by multiple granulomas of epitheloid cells and Langhan's type giant cells throughout the splenic pulp. PCR verified the diagnosis of Mycobacterium tuberculosis infection. No primary focus of infection was detected in the lungs or any other organs.

A 40-year old Thai male presented with epigastric discomfort and weight loss without fever for 2 months. On examination, there was no hepatosplenomegaly or lymphadenopathy. His ultrasonogram showed multiple lesions in the spleen with enlarged abdominal lymph nodes. Pathology revealed caseating granulomas of the spleen and lymph nodes. No AFB were seen but PCR was positive for M. tuberculosis complex. The fever resolved within 3 days of surgery and the patient was treated with antituberculous drugs.

Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.

A DNA vaccine encoding Ag85A from Mycobacterium tuberculosis was administered to guinea pigs by epidermal gene gun bombardment and its protective efficacy was determined. Vaccination with Ag85A DNA twice significantly reduced the severity of pulmonary pathology and number of pulmonary colony-forming units (CFU) (p<0.01). When immunogenic synthetic Ag85A peptide was used as a booster, lung pathology was improved significantly and pulmonary CFU were reduced dramatically. Neither Ag85A DNA nor BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. When the vaccinated guinea pigs were followed up for 7 months, the pulmonary lesions became fibrotic in guinea pigs vaccinated with Ag85A DNA plus Ag85A peptide, or BCG Tokyo, and no tubercle bacilli were detected. The protective efficacy of the tuberculosis Ag85A DNA vaccine was improved significantly by peptide boosting. It is concluded that dosage and peptide boosting are important in the induction of higher protective efficacy by a tuberculosis DNA vaccine.

To gain a better understanding of the pathological role of natural killer (NK) T cells in murine tuberculosis, NKT knockout (KO) mice (J(alpha)281(-/-)mice) were utilized. Eight-week-old NKT KO mice of BALB/c origin and wild-type (WT) mice were infected with Mycobacterium tuberculosis Kurono strain by the airborne route using an airborne infection apparatus, and their capacity to control mycobacterial growth, granuloma formation, and cytokine production were examined. The NKT KO mice developed granulomatous lesions in the lungs; there was no statistically significant difference in pulmonary granuloma size between NKT KO and WT mice (p<0.01). The average CFU values increased 3 weeks post-infection, but decreased 9 and 11 weeks post-infection, in the lungs of NKT KO mice. When stimulated with Kurono strain in vitro, splenic cells from NKT KO mice produced less IFN-gamma than did those from WT mice. Expression of mRNA for IL-2, IL-4, IL-6, IL-10 and IL-12 p40 was slightly lower in NKT KO mice compared with WT mice. Our data indicate that NKT cells play a detrimental role in late-phase mycobacterial infection, although Th1 cells are essential in early-phase mycobacterial infection.