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.

Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP).

In order to explore the effect on bone marrow macrophages of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP), mice were injected intravenously with a preparation of such liposomes at a dose known to deplete spleen and liver macrophages. Two days later, the macrophages in the marrow of the femoral bones were quantified by flow cytometry using a macrophage-specific monoclonal antibody (F4/80), and their ultrastructure and phagocytic activity towards zymosan particles was assessed. To determine the effect on erythropoiesis of liposome-encapsulated Cl2MDP-induced changes in bone marrow macrophages, red blood cell parameters and the formation of erythroid burst-forming unit (BFU-E)-derived colonies in vitro were evaluated. In mice injected with liposome-encapsulated Cl2MDP, there was a 54% and 67% decrease in the total number of bone marrow macrophages as compared to uninjected controls and mice treated with empty liposomes, respectively. Moreover, residual macrophages showed an abnormal ultrastructure, with reduced numbers of crystalloid inclusions and increased numbers of large myelin figures. However, the phagocytic activity of these cells was unimpaired or slightly enhanced. In mice injected with liposome-encapsulated Cl2MDP there was an approximately 60% decrease in the percentage and total number of circulating reticulocytes and a 54% reduction in the BFU-E number, demonstrating deregulation of erythropoiesis under conditions of macrophage loss and impairment. The results suggest that mice treated with liposome-encapsulated Cl2MDP are a model for studying the role of macrophages in erythropoiesis.

Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease.

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.

Bacille Calmette-Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-gamma).

Immunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type-1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN-gamma (BCG-IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time- and dose-dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG-IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG-plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG-IFN compared with infection with BCG-plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN-gamma by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Assessment of immunity to mycobacterial infection with luciferase reporter constructs.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

Role of macrophages in acetaminophen (paracetamol)-induced hepatotoxicity.

Research into the pathogenesis of acetaminophen (paracetamol)-induced hepatotoxicity has concentrated on the generation of toxic metabolites by the hepatocytes. It has, however, recently been shown that human macrophages cultured with acetaminophen secrete increased quantities of tumour necrosis factor (TNF). This study examines whether macrophages have a direct role in acetaminophen toxicity, using a mouse model in which it is possible to eliminate more that 99 per cent of hepatic macrophages by previously injecting liposomes containing dichloromethylene disphosphonate (DMDP). Acetaminophen-induced liver damage was assessed biochemically and histologically. It was shown that the liver damage occurring 0.5, 1, and 2 h after an intraperitoneal injection of acetaminophen was significantly less in mice previously injected with liposomes containing DMDP than in previously untreated mice, or mice previously injected with empty liposomes. By 4 h there was no difference between the groups. We conclude that macrophages play an early and probably a direct role in mediating the liver damage due to acetaminophen. This is consistent with the role that macrophages have been shown to play in the pathogenesis of alcohol-induced liver damage.

Genetic and serovar typing of clinical isolates of the Mycobacterium avium-intracellulare complex.

SETTING: One hundred and thirty-four Mycobacterium avium-intracellulare complex (MAC) isolates were obtained from 121 patients in the UK. OBJECTIVE: To compare serotyping and genetic analysis for species identification of MAC isolates from patients with and without the acquired immunodeficiency syndrome (AIDS). DESIGN: Clinical MAC isolates were cultured and analyzed by serotyping, the commercially available Accuprobe kit, hybridization with genes coding for the 19 kDa and 38 kDa antigens of M. tuberculosis and fingerprinting with the pMB22 probe derived from M. paratuberculosis. RESULTS: Species classification on the basis of genetic analysis was similar to serovar typing, with only exceptional discrepancies. Serovar prevalence was different in the two groups of patients, and different from those reported in other countries. MAC isolates from AIDS patients were exclusively M. avium, whereas patients without AIDS had MAC infections with M. avium and M. intracellulare in about equal proportion. M. intracellulare clinical isolates were genetically more heterogeneous than M. avium. Only M. intracellulare hybridized with the 38 kDa gene probe. CONCLUSIONS: Serovars are strongly linked with species in clinical MAC isolates, confirming results previously obtained with reference strains. M. intracellulare can be easily identified by the presence of a 38 kDa gene.

Defective antigen processing associated with familial disseminated mycobacteriosis.

To gain insights into a possible immune defect predisposing to disseminated mycobacteria infection, we studied three of six surviving children with disseminated Mycobacterium avium complex infection, who had no recognized form of immunodeficiency. We used mycobacteria isolated from the patients and diphtheria, tetanus and pertussis vaccine (DTP) to study antigen-specific T lymphocyte responses. We observed that interferon-gamma (IFN-gamma) production by T cells in response to antigens (both mycobacteria and DTP) in these patients with disseminated infection was greatly impaired. This defect did not seem to be the result of T cell unresponsiveness, as phytohaemagglutinin (PHA) stimulation was able to induce high levels of IFN-gamma comparable to those seen in control patients with localized infection. Further experiments showed that peripheral blood mononuclear cells (PBMC) from patients with disseminated infection were able to present influenza haemagglutinin (HA) peptides to specific T cell clones. However, this ability was lost when the whole HA protein was used as source of antigen. Taken together, these observations support the notion that the primary immune defect in these patients with disseminated mycobacterial infection rests in the antigen-processing functions of their antigen-presenting cells (APC). These findings may provide clues to the wider problem of susceptibility to mycobacteria and other intracellular pathogens and have implications in designing therapy for these patients.

Ribosomal internal transcribed spacer sequences are identical among Mycobacterium avium-intracellulare complex isolates from AIDS patients, but vary among isolates from elderly pulmonary disease patients.

Sequencing 280 bp of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes in a collection of 46 clinical isolates of the Mycobacterium avium-intracellulare complex (MAI complex) identified nine different sequences, grouping these isolates in nine 'ITS sequevars'. This analysis extends the subdivision within the MAI complex to 18 ITS sequevars and also improves discrimination from other mycobacterial species. Evaluation of the sequevar grouping among different clinical sources revealed strong association of the M. avium sequevar Mav-B with AIDS and with lymphadenitis in children (18 out of 20 and 3 out of 3 respectively). Isolates from elderly patients with pulmonary disease and not suspected of being HIV infected belonged predominantly to M. intracellulare ITS sequevars and sequevars not assigned to either M. avium or M. intracellulare. On the other hand, animal isolates were of both the Mav-A and Mav-B sequevars. We conclude that ITS sequevar typing is an accurate way of identifying distinct MAI complex strains. The observed differences between clinical sources suggest that ITS sequevars reflect possibly important, biologically and clinically relevant polymorphisms between MAI complex organisms.

The effect of Kupffer cell elimination on ethanol-induced liver damage in mice.

C57BL/10 mice develop inflammatory and necrotic changes in the liver, as well as raised serum ALT activities, after 9 days of exposure to ethanol vapour. If mice were injected twice with liposomes containing dichloromethylene diphosphonate (DMDP), with an interval of 5 days between the injections, there was complete elimination of Kupffer cells (hepatic macrophages) for a 9-day period starting 1 day after the first injection. The inflammatory and necrotic changes were significantly reduced in mice injected with liposomes containing DMDP as compared to uninjected mice or mice injected with empty liposomes; serum ALT activities were also significantly reduced. No significant difference was seen in serum tumour necrosis factor-alpha levels between the different groups. Kupffer cells therefore play a significant role in the development of the liver damage resulting from exposure to ethanol. Acetaldehyde production by Kupffer cells is one way in which these cells can damage hepatocytes and further work needs to be done to investigate this and other mechanisms.

Familial disseminated atypical mycobacterial infection in childhood: a human mycobacterial susceptibility gene?

Inherited defects in specific components of the immune system have provided many clues to the immunological mechanisms underlying resistance to microbial infection. We report a familial immune defect predisposing to disseminated atypical mycobacterial infection in childhood. 6 children with disseminated atypical mycobacterial infection and no recognised form of immunodeficency were identified. Four, including two brothers, come from a village in Malta, and two are brothers of Greek Cypriot origin. They presented with fever, weight loss, lymphadenopathy, and hepatosplenomegaly. They had anaemia and an acute phase response. A range of different mycobacteria (Mycobacterium fortuitum, M chelonei, and four strains of M avium intracellulare complex) were isolated. Treatment with multiple antibiotics failed to eradicate the infection, although treatment with gamma interferon was associated with improvement. Three have died and the surviving children have chronic infection. Tumour necrosis factor-alpha production in response to endotoxin and gamma-interferon was found to be defective in affected patients and their parents. T-cell proliferative responses to mycobacterial and recall antigens were reduced in parents of affected children and gamma-interferon production was diminished in the affected patients and their parents. Clinical and immunological features suggest that these patients are phenotypically similar to Lsh/Ity/Bcg susceptible mice. Understanding of this defect may provide insights into the mechanisms responsible for susceptibility to mycobacteria.

Macrophages are a major source of acetaldehyde in circulating acetaldehyde-albumin complexes formed after exposure of mice to ethanol.

C57BL mice were depleted of macrophages by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (DCMDP), and control mice were uninjected or injected with empty liposomes. One day after injection, a proportion of the DCMDP-treated and control mice was continuously exposed to ethanol vapor for 4 days. Albumin fractions were separated from the sera of both ethanol-unexposed and ethanol-exposed animals and tested for cytotoxicity against a monolayer of A9 cells using two indicators of cytotoxicity: detachment of adherent cells and a decrease in the ability of cells to reduce tetrazolium. The results show that, in mice exposed to ethanol, macrophages are a major source of the acetaldehyde in circulating cytotoxic acetaldehyde-albumin complexes and presumably also of free acetaldehyde.

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

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.

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.

A modified broth dilution assay for antibiotic sensitivity testing of Mycobacterium avium-intracellulare using paraffin slide cultures.

Mycobacterium avium-intracellulare (MAI) can utilize paraffin wax as the sole carbon source in basal media. Paraffin slide culture (Para SL/C) has been employed for isolation and speciation of MAI derived from clinical sources. We have evaluated an adaptation of this method for antimicrobial sensitivity testing. Sixteen clinical isolates of MAI were tested against ciprofloxacin amikacin, and azithromycin by Para SL/C and compared with sensitivities obtained with a conventional broth microtiter procedure. The system can be performed rapidly over a median time interval of 6-8 days. The MIC was defined as the lowest concentration of antimicrobial agent necessary to inhibit growth on paraffin wax coated slides. With Para SL/C, the MIC values were determined at the time when the corresponding control tubes showed confluent growth. The procedure was reproducible with all of the agents tested. The MIC50 and MIC90 values obtained from the Para SL/C assay and from serial broth microtiter dilutions correlated well for ciprofloxacin and amikacin. However, results of the MIC50 and MIC90 for azithromycin did not correlate.

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.