TNF-dependent BALB/c murine macrophage apoptosis following Mycobacterium tuberculosis infection inhibits bacillary growth in an IFN-gamma independent manner.

SETTING: In vitro model of murine macrophage M. tuberculosis infection. OBJECTIVE: To evaluate the association and cytokine control of host cell apoptosis and bacillary killing in M. tuberculosis -infected murine peritoneal macrophage (PM). DESIGN: Murine PM from different strains of mice were infected with H37Ra. Bacillary growth and macrophage apoptosis were evaluated under different cytokine conditions. RESULTS: Like human alveolar macrophages, PM from BALB/c mice were found to undergo apoptosis after infection with M. tuberculosis in a TNF-dependent manner. Neutralizing TNF with anti-TNF antibody inhibited PM apoptosis following infection, and resulted in increased bacillary growth. Pre-treatment of PM with interferon (IFN-gamma) resulted in significant killing of the infecting bacilli, which was not dependent on TNF or apoptosis of the cells. In contrast to BALB/c mice, PM from C3H/HeJ mice did not undergo apoptosis following infection and did not undergo TNF- and apoptosis-dependent inhibition of bacillary growth. CONCLUSION: These findings suggest that TNF contributes to macrophage inhibition of M. tuberculosis growth by a mechanism that is dependent on apoptosis and independent of IFN-gamma activity. This protective phenotype was not seen in all strains of mice and merits investigation as a marker of mycobacterial host susceptibility.

Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses.

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.

Type 2 diabetes: evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs.

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, chi = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (chi = 57 cM, recurrence risk,lambda(s) = 1. 25, P = 0.009). Weighted logarithm of odds scores of 2.00 (chi = 69.5 cM, P = 0.010) and 1.92 (chi = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2. 12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4alpha) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Phytoremediation of trichloroethylene with hybrid poplars.

Axenic tumor cultures of poplar cells, clone H11-11, were grown in the presence of [14C]-trichloroethylene (TCE) (uniformly labeled). The cells were capable of metabolizing TCE to produce trichloroethanol, di- and trichloroacetic acid. Some of the carbon from TCE was found in insoluble, nonextractable cell residue, and small amounts were mineralized to [14C]CO2. Poplar cuttings grown in soil and exposed to TCE produced the same metabolites. In field trials, trees were planted in soil in test cells and exposed to TCE via underground water injection during the growing season. During the growing season, at least 95% of the TCE was removed from the influent water stream in cells containing trees. Mass balance studies conducted in the laboratory indicated that 70 to 90% of the TCE was transpired; however, greenhouse and field study results showed that less than 5% of the total TCE taken up by the plants is transpired. These results show that significant TCE uptake and degradation occur in poplars. Poplars appear to be useful for in situ remediation of TCE-contaminated sites under proper conditions.