Contact investigations in congregate settings, New York City.

SETTING: Large urban tuberculosis control program. OBJECTIVE: To evaluate results of procedures implemented for systematic investigation of tuberculosis exposures in congregate settings. DESIGN: Between October 1995 and December 2000, a unit consisting of epidemiologists, health educators and tuberculin screening staff investigated exposures in sites with >15 persons. Transmission at the site was defined as likely, possible, unlikely or unknown. RESULTS: Among 100 investigations, 12 were tuberculosis case clusters, five were source case investigations, and 83 were exposures to single infectious cases. Transmission was likely in 24 (21%), possible in eight (8%), unlikely in 62 (62%), and could not be assessed in four (4%). Among the 83 exposures to single infectious cases, 2740 contacts were tested; 502 (18%) were infected. Among 1202 close contacts, 996 (82%) were tested, 197 (20%) were infected and started treatment of latent tuberculosis infection (LTBI) and 102/197 (52%) completed treatment. Sites with likely transmission had index patients with longer duration of cough (13 vs. 6 weeks, P = 0.01) and cavitary lesions (84% vs. 44%, P = 0.01) compared to sites with unlikely transmission. CONCLUSION: A systematic approach for conducting contact investigations in congregate settings is useful for assessing transmission. As such investigations are resource intensive and transmission is not common, performing tuberculin skin testing after most persons would have converted should be considered in low-risk groups. Additional efforts are needed to increase completion of treatment for LTBI in contacts identified in these settings.

Macrophage apoptosis in mycobacterial infections.

Mycobacterial diseases are a major public health concern. In the case of tuberculosis, the problem has been acerbated due to the emergence of drug-resistant strains of Mycobacterium tuberculosis, and Mycobacterium avium is the major opportunistic pathogen in HIV-1 infection in the United States. M. tuberculosis and M. avium replicate in human macrophages and induce apoptosis. Incubation of freshly added uninfected autologous macrophages with apoptotic M. avium-infected macrophages results in 90% inhibition of bacterial growth. Apoptosis also prevents the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. Consistent with the model that host cell apoptosis is a defense mechanism against mycobacteria is the finding that the virulent M. tuberculosis strain H37Rv induces substantially less macrophage apoptosis than the attenuated strain H37Ra. Evasion of apoptosis by this pathogen is achieved by enhanced release of sTNFR2 by H37Rv-infected macrophages and subsequent formation of inactive TNF-alpha-TNFR2 complexes. These observations contribute to the hypothesis that apoptosis of the host macrophage is an important defense mechanism in mycobacterial infections, which prevents the spread of the infection.

Interleukin 10 produced by macrophages inoculated with Mycobacterium avium attenuates mycobacteria-induced apoptosis by reduction of TNF-alpha activity.

Normal human macrophages respond to infection with Mycobacterium avium, serovar 4, by producing tumor necrosis factor (TNF)-alpha, which mediates apoptosis, and by elaborating interleukin (IL)-10, a TNF-alpha antagonist. We show that IL-10 down-regulates apoptosis by inhibiting the TNF-alpha production of the inoculated macrophages and by inducing the release of soluble TNF receptor type 2 from the macrophages, which leads to inactivation of TNF-alpha. These experiments suggest that induction of IL-10 production is a virulence factor that creates an intracellular sanctuary for the bacteria that is inaccessible to the defense mechanisms of the host.

Pathogenic Mycobacterium tuberculosis evades apoptosis of host macrophages by release of TNF-R2, resulting in inactivation of TNF-alpha.

Infection by Mycobacterium tuberculosis (MTB) induces human alveolar macrophage (AMphi) apoptosis by a TNF-alpha-dependent mechanism. The apoptotic response is postulated to be a defense mechanism, limiting the growth of this intracellular pathogen. Consistent with that model, recent studies showed that the virulent MTB strain H37Rv induces substantially less AMphi apoptosis than the attenuated strain H37Ra. We now report that AMphi infection with either H37Rv or H37Ra induces comparable levels of TNF-alpha measured by ELISA but that TNF-alpha bioactivity is reduced in supernatants of H37Rv-infected AMphi. Differential release of soluble TNFR2 (sTNFR2), with formation of inactive TNF-alpha-TNFR2 complexes accounted for the difference in TNF-alpha bioactivity in these cultures. Release of sTNFR2 by H37Rv-infected AMphi was IL-10 dependent since it was inhibited by neutralizing anti-IL-10 Ab. Thus, the effect of TNF-alpha produced by AMphi following infection can be modulated by virulent MTB, using IL-10 as an upstream mediator.

Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis.

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.

Opposing regulatory effects of thioredoxin and eosinophil cytotoxicity-enhancing factor on the development of human immunodeficiency virus 1.

Exogenous recombinant human thioredoxin (rTRX, > or = 500 nM), a dithiol reductase enzyme, inhibited the expression of human immunodeficiency virus (HIV) 1BaL in human macrophages (M phi) by 71% (range, 26-100%), as evaluated by p24 antigen production and the integration of provirus at 14 d after infection. The stoichiometric reducing agent N-acetylcysteine (NAC) also inhibited HIV production, but to a lesser degree, and only at 30,000-fold higher concentrations. Exogenous rTRX is cleaved by M phi to generate the inflammatory cytokine, eosinophil cytotoxicity-enhancing factor (ECEF). In contrast to rTRX, rECEF (concentrations from 50 pM to 2 microM) enhanced the production of HIV by 67% (range, 33-92%). Thus, whereas TRX is a potent inhibitor of the expression of HIV in human M phi, cleavage of TRX to ECEF creates a mediator with the opposite effect. TRX also inhibited the expression of integrated provirus in the chronically infected OM 10.1 cell line, showing that it can act at a step subsequent to viral infection and integration.

Human eosinophil cytotoxicity-enhancing factor. Eosinophil-stimulating and dithiol reductase activities of biosynthetic (recombinant) species with COOH-terminal deletions.

U937 cells produce eosinophil cytotoxicity-enhancing factor (ECEF) polypeptides of 14 and 10 kDa that have identical NH2-terminal amino acid sequences. The 10-kDa form has greater eosinophil-stimulating activity (half-maximal at > 20-fold lower concentration). We considered the hypothesis that there is a precursor-product relationship between the 14- and 10-kDa species. Recombinant 14-kDa 104-amino acid ECEF (rECEF-104) had a slight stimulatory effect on eosinophil cytotoxic function at concentrations of 160 nM and above. In contrast, two species, rECEF-80 and rECEF-84, representing cleavage products of approximately 10 kDa had substantial statistically significant cytotoxicity-enhancing activity at concentrations as low as 10 pM. This evidence demonstrates the potential to generate the high-activity ECEF species by proteolytic cleavage of the 104-amino acid species. Another feature of this cytokine is the sequence from amino acids 31 to 34, which constitutes the conserved and active site of the enzyme thioredoxin. When tested for dithiol reductase enzymatic activity, rECEF-104 was active in a dose- and time-dependent manner, whereas the truncated forms of the molecule had no dithiol reductase activity. Thus the eosinophil-stimulating functions of the molecule do not correlate with its enzymatic activity. The evidence shows that the enzymatic activity is not essential for the initial interaction of ECEF with the eosinophil, and it suggests that the ECEF molecule functions by means of two discrete mechanisms.

Human eosinophil cytotoxicity-enhancing factor. II. Multiple forms synthesized by U937 cells and their relationship to thioredoxin/adult T cell leukemia-derived factor.

Recently, our laboratory reported the purification and partial amino acid sequence of a 10-kDa eosinophil cytotoxicity-enhancing factor (ECEF) polypeptide from the U937 cell source. This cytokine enhanced human eosinophil antibody-dependent cytotoxic function by greater than 200% and was half-maximally effective at a concentration of approximately 1 ng/ml. In this study, we describe the conditions required for ECEF synthesis and the use of rabbit antibody raised to 10-kDa ECEF to investigate the existence of related polypeptide species. Unstimulated U937 cells released an immunoreactive 14-kDA species. Cells stimulated with 7.5 micrograms/ml of LPS also released a 13-kDa species. Cells stimulated with 400 ng/ml of PMA also synthesized a 10-kDa species (equivalent in size to the form we had purified). This 10-kDa species remained primarily cell associated, but detectable amounts were released into the supernatant by 48 h of culture. In washed cell pellets, the location of the 10-kDa species was found to be in the plasma membrane, externally oriented, as determined by FACS analysis, iodination with the membrane impermeable reagent 125I-sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate, and by its removal with brief trypsin treatment. Partial amino acid sequence data suggested that the 14-, 13-, and 10-kDa species all share the same N-terminal. The 14- and 10-kDa ECEF species were recovered by electroelution from SDS-PAGE gels and tested for activity in the assay of eosinophil cytotoxic function. Because of the amino acid sequence similarities between the ECEF species and thioredoxin (TRX), rTRX (synthesized in Escherichia coli and purified) was also tested for activity. The 14-kDa ECEF and rTRX induced a slight, but consistent and statistically significant enhancement of eosinophil cytotoxic function. By comparison, lower doses of the 10-kDa ECEF induced a major increase in cytotoxic function. Thus the forms of ECEF differ in size, conditions required for synthesis, trafficking by the U937 cell after synthesis, and biologic activity. It is likely that these considerations bear on the involvement of ECEF in the pathophysiology of eosinophilia in vivo.