Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection.

Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.

Histone preopsonisation increases the respiratory burst response of phagocytes to Pneumocystis carinii.

Preincubation of Pneumocystis carinii with histone caused an increase in polymorphonuclear leucocyte and macrophage chemiluminescence when parasites were mixed with phagocytes compared with that obtained when unopsonised parasites were used. Toxic oxygen moieties released during the respiratory burst may cause tissue damage.

Plasmid analysis of Mycobacterium avium-intracellulare (MAI) isolated in the United Kingdom from patients with and without AIDS.

One hundred and forty-seven isolates (128 strains) of Mycobacterium avium-intracellulare (MAI) were screened by agarose gel electrophoresis for the presence of plasmids. Plasmids were characterised according to size and by Southern hybridisation analysis of intact and restriction endonuclease-digested DNA. Two cloned MAI plasmids, pLR7 and pLR20, were used as probes. There was no significant difference in the rate of plasmid carriage in MAI strains isolated from patients with the acquired immuno-deficiency syndrome (AIDS) and from non-AIDS patients in the UK, but a higher rate of plasmid carriage was observed in a panel of American strains from AIDS patients. Plasmids were grouped into two broad categories: small (mostly 14-30 kb) and large (greater than 150 kb). Southern blot analysis identified two distinct groups of small plasmids, the majority of which showed homology with pLR7. Plasmids from this group were significantly more common in strains of serotypes 4 and 8 which are particularly associated with AIDS.

Gastro-intestinal involvement in Mycobacterium avium-intracellulare infection of patients with HIV.

In a study of 866 faecal specimens from 437 persons, Mycobacterium avium-intracellulare (MAI) was isolated from 14.8% patients with AIDS and 1.3% patients with symptomatic HIV infection but not from any HIV seronegative or asymptomatic HIV seropositive persons. These data support the hypothesis that the gastro-intestinal tract is the portal of entry for MAI and confirm that MAI infection is a manifestation of late-stage HIV disease. Positive faecal cultures correlated well with disseminated disease. The use of faecal cultures for early diagnosis is therefore recommended.

Growth of Mycobacterium lepraemurinum in the mouse bone marrow: an ultrastructural study.

The ultrastruct of the mouse bone marrow during the first 8 weeks after intravenous inoculation of animals with 10(9) Mycobacterium lepraemurium is described. The bacteria were almost exclusively in macrophages, which became converted to epithelioid cells after 8 weeks, at which time they were very heavily infected. The nature of the exceptionally rapid increase in numbers of bacteria in the bone marrow compared with other tissues early in the infection is discussed. It is concluded that a short doubling time of bacteria situated in the marrow is a more probable explanation than recruitment from elsewhere in the animal.

Systemic Mycobacterium lepraemurium infection in mice: differences in doubling time in liver, spleen, and bone marrow, and a method for measuring the proportion of viable organisms in an inoculum.

Counts of acid-fast bacilli were made on homogenates of whole liver, whole spleen, and two femurs of CBA mice killed at various time intervals after intravenous infection with Mycobacterium lepraemurium. The growth curves so obtained showed that the bacillus multiplied faster in bone marrow than in liver or spleen. No evidence of redistribution during the early part of infection was obtained. The time of appearance of significant numbers of bacilli (10(7)) in the bone marrow was used to make estimates of viability of M. lepraemurium suspensions. Several applications of the techniques described are discussed.

Immunity to Plasmodium berghei in rats: passive serum transfer and role of the spleen.

Pools of rat antiserum to Plasmodium berghei had different levels of protective activity as assessed by a passive transfer test. Preincubation of parasite inocula with an effective pool before injection did not significantly enhance protective activity. Removal of the antiserum from preincubated parasite inocula abolished the bulk of protective activity. Similarly, antiserum effective in intact animals was largely ineffective in splenectomized recipients. These experiments suggest a minimal role for antibody acting directly on P. berghei-parasitized cells and reemphasize a significant role for the spleen in immunity to this plasmodium.

Mycobacterium bovis, BCG, modulation of murine antibody responses: influence of dose and degree of aggregation of live or dead organisms.

Mycobacteria have the ability to enhance or depress immune responses. This paper describes experiments designed to investigate the parameters determining the direction of modulation. It has been shown previously that 10(8) liver Mycobacterium bovis BCG depress the ability of mouse spleen cells to produce a primary antibody response in vitro to SRBC 2-3 weeks after i.v. injection, whereas the same number of dead organisms enhance this response. Using the same growth medium for the BCG (Glaxo glycerol-free medium), we now find that decreasing the BCG dose to mice from 10(8) to 10 (6) liver organisms results in enhanced responses and increasing the dose to more than 10(8) dead organisms results in depressed responses. It thus appears that bacterial load is the important factor determining whether depression or enhancement of the primary antibody response will occur, rather than the viability of the organisms per se. However, when the BCG was grown in Middlebrook 7H9 broth, doses as high as 4 X 10(9) dead BCG/mouse failed to depress although depressed responses were found if sufficient live organisms (7 X 10(8)) were injected. In view of the known growth characteristics of BCG in these 2 bacteriological media, it is suggested that the degree of aggregation of the injected suspension may also be of importance in determining whether or not depression will occur. A comparison of the effects of BCG injected untreated or after dispersion of bacterial aggregates supports this idea. Some degree of splenomegaly was always found in mice with depressed splenic responses but a large spleen did not necessarily yield cell suspensions with depressed responses.

The Ity/Lsh/Bcg gene significantly affects mouse resistance to Mycobacterium lepraemurium.

Mouse resistance to infection with Mycobacterium lepraemurium was measured by counting the total number of intact acid-fast bacilli in the spleen 8 weeks after i.v. injection of a standard inoculation. The effect of Ityr on resistance to M. lepraemurium was confirmed and the results extended to two Ityr strains of mice, A and C57L, not previously tested. Resistance to M. lepraemurium was also examined in the F1, backcross and F2 generations of BALB/c X CBA crosses, and in the congenic strain B10.LLshr that is Ityr. In all experiments the results were consistent with the view that resistance to M. lepraemurium is significantly affected by a gene close to or identical to the Ity/Lsh/Bcg gene on mouse chromosome 1. Sex had a marked effect on resistance to M. lepraemurium, so that the males of some genetically resistant strains were almost as susceptible as some genetically susceptible females.

Rapid methods for counting mycobacteria--comparison of methods for extraction of mycobacterial adenosine triphosphate (ATP) determined by firefly luciferase assay.

A comparison of 5 different methods of extraction of adenosine 5'-triphosphate (ATP) from mycobacterial cells was carried out using Mycobacterium bovis, BCG as a model. ATP was measured using the luciferin-luciferase bioluminescence reaction. Boiling buffer extraction was the best method. The amount of ATP extracted correlated with the number of colony forming units over a wide range of count. Although great sensitivity in terms of number of bacilli detectable was not achieved the method was rapid and appears suitable for drug sensitivity testing of tubercle bacilli.

The effect of oral Mycobacterium vaccae on subsequent responses of mice to BCG sensitization.

It has been postulated that previous exposure to mycobacteria in the environment may be a contributing factor to the variable efficacy of BCG vaccination in protecting against human tuberculosis or leprosy, in different geographical regions. To test this hypothesis mice were given Mycobacterium vaccae in their drinking water for 3 weeks immediately, 27 days, or 54 days before they were injected subcutaneously with BCG. Spleen cells were examined, 50 days after injection of the mice, for ability to proliferate in vitro in response to killed mycobacteria added to the cultures. The results show that, depending on the timing of the exposure of the mice in vivo to M. vaccae before BCG injection and the dose of the mycobacterial challenge in vitro, oral administration of this species of environmental mycobacteria can either enhance, mask or interfere with the expression of sensitization by BCG. Thus, our data from mice support the hypothesis that the results of BCG vaccination for a particular human population are influenced by exposure to indigenous mycobacteria.

Immunologically mediated macrophage aggregation in monolayers of peritoneal cells from BCG-sensitized mice.

Aggregation of cultured macrophage monolayers derived from BCG-sensitized mice was produced if nonadherent cells and specific antigen (tuberculin) were present, particularly if the antigen was renewed in the form of tubercle bacilli. The evidence indicated that antigen-stimulated BCG-sensitized lymphocytes in these cultures produced a soluble factor, which in the presence of the renewed supply of antigen caused the aggregation. The phenomenon was irreversible and followed by death of the macrophages. The 'overlays' of aggregated monolayers would aggregate normal macrophages, provided that the recipient cultures contain-d their own (normal) lymphocytes as well as antigen; this suggested that cultures of BCG-sensitized peritoneal cells produced a factor able to effect aggregation via the activity of normal lymphocytes. Overlays from aggregated monolayers were able also to inhibit the migration of normal mouse macrophages; this and other evidence suggested a similar origin for the inhibition factor, but the latter's identity with the aggregation factor remains undecided. We conclude that the aggregation factor depends upon the presence of specific antigen both for its formation and its expression.

Phagosome/lysosome fusion: a possible prerequisite for the enhancement of antibody responses in vitro by BCG, Mycobacterium leprae and Corynebacterium parvum.

Primary in vitro antibody responses to SRBC were suppressed in cultures prepared from the spleens of CBA mice injected i.v. 20 days previously with 10(8) liver BCG. In contrast, cultures prepared from mice injected with dead BCG showed enhanced responses. In vitro spleen cell responses of the mice had returned to normal levels 4--6 weeks after their injection, but if dead BCG, M. leprae or C. parvum was added to the cultures, responses were enhanced. The enhancing effect of the added bacteria could be removed by adding also suramin, a drug known to inhibit in vitro fusion of lysosomes with phagosomes. It is suggested that the different in vivo effects of live and dead BCG may relate to differences in their handling by macrophages and more especially that the enhanced antibody forming cell response seen in the restimulated cultures of spleen cells from BCG primed mice, depends upon efficient intracellular fusion of lysosomes with the phagosomes containing the added dead bacteria.

Suppressed or enhanced antibody responses in vitro after BCG treatment of mice: importance of BCG viability.

Mycobacterium bovis, BCG, is known to be capable of either enhancing or suppressing various immune responses. Using a standard technique and number of organisms, some of the parameters predetermining whether enhancement or supression will occur have been investigated. Dead BCG given intravenously into mice caused an enhancement of the antibody response in vitro to sheep erythrocytes. In contrast, the same number of viable organisms caused suppression if given intravenously but enhancement if given subcutaneously. The inclusion of 25% or more killed organisms in an intravenous inoculum of fully viable organisms changed suppression to enhancement. Treatment of BCG infected mice with streptomycin lessened the suppression but did not change it to enhancement. The possible causes of suppression are discussed.