SUMMARYMycobacterium tuberculosis produces latent infection or progressive disease. Indeed, latent infection is more common since it occurs in one-third of the world's population. We showed previously, using human material with latent tuberculosis, that mycobacterial DNA can be detected by in situ PCR in a variety of cell types in histologically-normal lung. We therefore sought to establish an experimental model in which this phenomenon could be studied in detail. We report here the establishment of such a model in C57Bl/6 ¥ DBA/2 F1 hybrid mice by the intratracheal injection of low numbers of virulent mycobacteria (4000). Latent infection was characterized by low and stable bacillary counts without death of animals. Histological and immunological study showed granulomas and small patches of alveolitis, with high expression of tumour necrosis factor alpha (TNFa), inducible nitiric oxide synthase (iNOS), interleukin 2 (IL-2) and interferon gamma (IFNg). In contrast, the intratracheal instillation of high numbers of bacteria (1 ¥ 10 6 ) produced progressive disease. These animals started to die after 2 months of infection, with very high bacillary loads, massive pneumonia, falling expression of TNF-a and iNOS, and a mixed Th1/Th2 cytokine pattern. In situ PCR to detect mycobacterial DNA revealed that the most common positive cells in latently-infected mice were alveolar and interstitial macrophages located in tuberculous lesions, but, as in latently-infected human lung, positive signals were also seen in bronchial epithelium, endothelial cells and fibroblasts from histologically-normal areas. Our results suggest that latent tuberculosis is induced and maintained by a type 1 cytokine pattern plus TNFa, and that mycobacteria persist intracellularly in lung tissue with and without histological evidence of a local immune response.

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SUMMARYMultinucleated giant cells (MGC) are a common feature of granulomas. The mechanism of their formation has been studied extensively, but their function has not been completely characterized. A new method for the in vivo production of MGC was developed involving subcutaneous injection of microscopic nitrocellulose particles with adsorbed mycobacterial antigens into the footpads of sensitized BALB/c mice (immune [I]-MGC), or by nitrocellulose administration to non-sensitized mice (foreign body [FB]-MGC). The development of granulomas with a highly enriched MGC population was observed 2 weeks after the nitrocellulose injection. MGC were larger with a greater number of nuclei in I-MGC than in FB-MGC. From days 7±28 after nitrocellulose administration, the production of interleukin-1a (IL-1a) and tumour necrosis factor-a (TNF-a) was demonstrated in both MGC types by in situ reverse transcription±polymerase chain reaction (RT±PCR) and immunohistochemistry. After 2 months, the MGC had ceased production of IL-1a and TNF-a, but the expression of transforming growth factor-b (TGF-b) was very high, occurring together with extensive ®brosis. These results suggest that MGC are an active source of in¯ammatory cytokines, which can contribute to the initiation, maintenance and down-regulation of granulomatous in¯ammation induced by immunological and inert substances.

SUMMARYProblems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current 6-month chemotherapy regimens used to treat tuberculosis, or of supplementing ineffective therapy. In this study we sought to de®ne the mechanism of action of two immunotherapies, both of which have previously been shown to prolong survival. Secondly, we wished to identify any clinically useful synergy between these therapies. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by type 1 cytokines plus tumour necrosis factor-a (TNF-a) and interleukin-1 (IL-1), followed by a phase of progressive disease. This progressive phase is accompanied by increasing expression of IL-4, and diminished expression of IL-1 and TNF-a. Animals in this late progressive phase of the disease (day 60) were treated with two injections (day 60 and day 90) of 0 . 1 or 1 . 0 mg of heat-killed Mycobacterium vaccae, or with 3b,17b-androstenediol (AED; 25 mg subcutaneously three times/week), or with both therapies. We show here using four techniques in parallel (morphometry, immunohistochemistry with automated cell counting, semiquantitative reverse transcription±polymerase chain reaction and enzyme-linked immunosorbent assays of cytokines in lung extracts) that treatment with M. vaccae causes a switch back towards a type 1 cytokine pro®le, restoration of expression of IL-1a and TNF-a, and a switch from pneumonia to granuloma. This is very similar to the changes previously seen after treatment with AED. However, there was no evidence for synergy between M. vaccae and AED.

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