Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.

In this paper we used a multiparametric approach to analyze extensively the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure and function are occurring in early stages of apoptosis. A multiparametric quantitative analysis was performed on normal or apoptotic thymocytes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scattering properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apoptotic cells) allowed a precise analysis of the preapoptotic and apoptotic stages. Moreover a thorough study of mitochondrial transmembrane potential (delta psi m) assessed following in a time course study the uptake by apoptotic cells of the cationic lipophilic dye DiOC6(3) or the J-aggregate-forming cation JC-1, indicates that a drop in delta psi m occurs very early in thymocyte apoptosis, before DNA fragmentation. This is associated with alteration in mitochondrial structure assessed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function occurring in early stages of apoptosis were confirmed by confocal and electron microscopy analysis.

SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells.

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

Apoptosis is antagonized by large T antigens in the pathway to immortalization by polyomaviruses.

The viability of rat embryo cells immortalized by thermosensitive mutants of SV40 or polyoma Large T antigen is impaired at the non-permissive temperature thus demonstrating that the immortal phenotype is dominantly maintained by Large T antigens. We have observed that exposing these cells to the restrictive temperature not only induces growth arrest but also causes apoptotic cell death. We present evidence supporting the model that polyomaviruses may indeed establish immortality by antagonizing the lethal effects of tumor suppressor genes via physical interactions between their products and Large T antigens. In the case of SV40-immortalized cells REtsAF, shift-up to 39.5 degrees C dissociates Large T antigen/p53 complexes releasing wild-type p53 molecules capable of inducing apoptotic cell death. In polyomavirus-immortalized cells, apoptosis may result from an alternative pathway mediated by other unidentified negatively acting molecules.

p53-independent apoptotic effects of the hepatitis B virus HBx protein in vivo and in vitro.

The hepatitis B virus protein HBx is a promiscuous transactivator implicated in both cell growth and death and in the development of hepatocellular carcinoma. We recently reported that HBx can potentiate c-myc-induced liver oncogenesis in a transgenic model where low level expression of HBx induces no pathology. To assess if HBx could affect the hepatocyte turnover, we investigated the HBx-elicited apoptotic responses in transgenic livers and in primary hepatocyte cultures. Here we show that transgenic expression of HBx is associated with a twofold increase of spontaneous cell death in the mouse liver. The finding that apoptosis was enhanced to similar extents in HBx mice carrying homozygous p53 null mutations implied that functionally intact p53 was not required to transduce the death signal. A direct, dose-dependent apoptotic function of HBx was demonstrated in transient transfections of liver-derived cell lines. We further show that stable expression of HBx at low, presumably physiological levels in primary hepatocytes, induced cellular susceptibility to diverse apoptotic insults, including growth factor deprivation, treatment with anti-Fas antibodies or doxorubicine and oxidative stress. HBx expression, but not p53 status profoundly affected the commitment of cells to die upon apoptotic stimuli. These data strengthen the notion that HBX may contribute to HBV pathogenesis by enhancing apoptotic death in the chronically infected liver.

Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis.

We have used the Hepatitis B Virus DNA genome as a probe to identify genes clonally mutated in vivo, in human liver cancers. In a tumor, HBV-DNA was found to be integrated into the gene encoding Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA), which pumps calcium, an important intracellular messenger for cell viability and growth, from the cytosol to the endoplasmic reticulum. The HBV X gene promoter cis-activates chimeric HBV X/SERCA1 transcripts, with splicing of SERCA1 exon 11, encoding C-terminally truncated SERCA1 proteins. Two chimeric HBV X/SERCA1 proteins accumulate in the tumor and form dimers. In vitro analyses have demonstrated that these proteins localize to the ER, determine its calcium depletion and induce cell death. We have also shown that these biological effects are related to expression of the SERCA, rather than of the viral moiety. This report involves for the first time the expression of mutated SERCA proteins in vivo in a tumor cell proliferation and in vitro in the control of cell viability. Oncogene (2000).

The early intracellular production of a reactive oxygen intermediate mediates apoptosis in dexamethasone-treated thymocytes.

Many conditions that induce an oxidative stress are capable of evoking apoptosis. This has lead to the proposal of oxidative stress as a mediator of apoptosis. We show that, in murine thymocytes, oxidative stress and apoptosis occur in the same cell. We identified four distinct apoptotic subpopulations that appeared sequentially in time. Catalase protected from dex-amethasone-induced death in the initial stages of apoptosis, while iron chelators and Vitamin E did not. Further studies provided evidence supporting the early production of an intracellular oxidative intermediate as an obligatory step for the efficient induction of apoptosis. We propose that at least one of the molecules capable of filling this role is hydrogen peroxide.

Comparative analysis of flow cytometric methods for apoptosis quantitation in murine thymocytes and human peripheral lymphocytes from controls and HIV-infected persons. Evidence for interference by granulocytes and erythrocytes.

The present article reports a multiparametric cytofluorimetric analysis of apoptosis in murine thymocytes and human PBMC from healthy donors or HIV-infected patients. We have evaluated four previously described cytofluorimetric methods of apoptosis quantification, each of them detecting distinct cellular alterations of the apoptosis process. Reduced DNA stainability was detected with the PI assay on nuclei and the AO/EB dual staining method was evaluated on entire and non-fixed cells. DNA strand breaks were detected following in situ nick translation, and alterations in membrane integrity were evaluated following 7-AAD incorporation. When apoptosis was quantified in murine thymocytes under various conditions of induction, the combined analysis of FSC/SSC criteria and 7-AAD or AO/EB staining on the same samples permitted the identification of distinct steps in the apoptosis process. Moreover these four methods proved to be reliable and gave statistically similar results both on murine thymocytes and PBMC from healthy donors. However, in HIV-infected persons, some discordant apoptosis determinations were observed with PI and 7-AAD staining assays. We found that after Ficoll isolation, PBMC from AIDS patients were enriched in erythrocytes and granulocytes. On the one hand, granulocytes were found to be responsible for a poor apoptosis estimation with the PI assay whereas erythrocytes were responsible for an underestimation rate of apoptosis in the 7-AAD assay. To prevent such interference, we propose some modifications which render these methods more suitable for application to PBMC from HIV-infected patients. Taken together these observations indicate that it is essential to assess critically the apoptosis quantification methods with respect to their applicability to complex lymphoid populations such as those from AIDS patients.

A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.

The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.

Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods.

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.

A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

Apoptosis and the CD95 system in HIV disease: impact of highly active anti-retroviral therapy (HAART).

Apoptosis markers and the rate of lymphocyte apoptosis were followed in the peripheral blood of HIV infected persons at various stages of disease. Our study suggests that the early increase in memory cells following therapy may also be due to a significant decrease in apoptosis in this subset. The intrinsic resistance to apoptosis in the naive subset appears to be maintained following HIV infection and is not modified following highly active anti-retroviral treatment (HAART).

Comparative analysis of apoptosis in HIV-infected humans and chimpanzees: relation with lymphocyte activation.

Programmed-cell death (apoptosis) is a physiological cell death process which appears exacerbated in peripheral lymphocytes from HIV-infected persons. On the contrary, a barely detectable level of apoptosis is found in peripheral lymphocytes from HIV-infected chimpanzees, which support long-term productive infection without developing AIDS. In the present study, we analyzed the relationship between apoptosis and the general state of immune activation in PBMC from HIV-infected humans and chimpanzees. In addition, apoptosis control in the CD8 subset by the bcl-2 proto-oncogene was compared in both human and chimpanzees. Taken together, the results indicate that the degree of apoptosis correlates with the state of activation of the immune system and this observation together with the finding that apoptosis concerns all lymphocyte subsets indicates that the low level of apoptosis in HIV-infected chimpanzees is related to the lack of immune activation in this nonpathogenic model.

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

Programmed cell death in AIDS-related HIV and SIV infections.

One of the difficulties in understanding the complex pathology of human immunodeficiency virus (HIV) infection is to explain the progressive depletion of the CD4 helper T cell population and consequently the destruction of the immune system. Although cytopathic effects of HIV are observed in vitro, they cannot in vivo account for CD4 T cell depletion because relatively few cells are productively infected. Thus immunological mechanisms must be envisaged. We have found that peripheral blood lymphocytes (PBLs) from asymptomatic HIV-infected individuals are primed for a suicide process known as apoptosis or programmed cell death (PCD). DNA fragmentation characteristic of apoptosis was enhanced by stimulation of lymphocytes with ionomycin, a known inducer of apoptosis in suitably primed cells. Identification of the T cell subpopulations programmed for apoptosis indicated that both CD4+ and CD8+ cells died when cultured without stimulation or when polyclonally stimulated with ionomycin. Activation-induced cell death was also observed after stimulation with self-MHC class II-dependent superantigens, namely bacterial toxins from Staphylococcus (SEB), Streptococcus (ETA), and Myocoplasma (MAM) and under these conditions the CD4+ T cells were preferentially affected. To explore whether new macromolecular synthesis were required for apoptosis, various known inhibitors of apoptosis such as cycloheximide, cyclosporin A, Zn2+, or EGTA were tested. Activation-induced apoptosis was found sensitive to these inhibitors, indicating an active mechanism, but apoptosis observed in nonstimulated cultures was not, suggesting that these cells already contained the complete machinery for death. Prevention of apoptosis could be obtained in the presence of a mixture of cytokines and the minimal signal necessary for this prevention was IL-1 alpha and IL-2. Finally, a correlation between PCD and AIDS-pathogenesis was suggested by the comparison of lymphocytes from lentivirus-infected primates suceptible (SIV-infected macaques) and resistant (HIV-infected chimpanzees) to AIDS. Altogether our results suggest that, during HIV or SIV infection, PCD may contribute in vivo to the deletion of reactive T cells after antigenic stimulation.

Clinical evaluation of the management of community-acquired pneumonia by general practitioners in France.

STUDY OBJECTIVES: To evaluate the management of community-acquired pneumonia (CAP) by general practitioners (GPs) in terms of clinical efficiency and adherence to official recommendations. DESIGN: Prospective cohort study. SETTING: Community-based study from 11 French counties. PATIENTS: Adult patients clinically suspected of having CAP who were seen by GPs were included after confirmation of the presence of an infiltrate on chest radiographs. INTERVENTION: The management of the patients was left to the discretion of the GP. MEASUREMENTS AND RESULTS: One hundred thirty patients were included in the study, and 13 patients (10%) were immediately hospitalized because of the severity of the pneumonia. The remaining 117 patients were treated as outpatients: 108 of 117 patients (92%) were cured, and 9 patients were subsequently hospitalized because of the failure of ambulatory treatment. Diagnostic error (n = 6) rather than antibiotic failure (n = 3) was the most frequent cause of the failure of ambulatory treatment. Only 40% of the patients received an initial antibiotic treatment that was in agreement with French recommendations. However, the rate of antibiotic failure leading to hospitalization was low (3 of 117 patients; 2.6%) and similar for patients treated or not according to recommendations (p > 0.5). Overall, five patients (4%) died; all deaths occurred during hospitalization and were related to the severity of the underlying disease but not to the choice of antibiotic treatment. CONCLUSIONS: The management of CAP by GPs was clinically effective despite a poor adherence to official recommendations. Our results suggest that adequate assessment of severity rather than adherence to recommendations for antibiotic treatment had an impact on clinical outcome of CAP managed by GPs.

HIV, cytokines and programmed cell death. A subtle interplay.

HIV infection is marked by the progressive destruction of the CD4 T lymphocyte subset, an essential component of the immune system and a vital source of cytokines required for differentiation of natural killer (NK) and gamma delta T cells, for maturation of B lymphocytes into plasmocytes, and for differentiation of CD8+ T cells into virus-specific cytotoxic T lymphocytes. CD4 T lymphocytes are also a source of chemokines which control migration of lymphocytes to the site of infection and which also inhibit HIV entry into CD4-expressing targets. Continuous production of viral proteins leads to an unbalanced immune activation and to the triggering of apoptotic programs, turning mononuclear cells, including CD4 T cells, CD8 T cells and APC, into effectors of apoptosis, leading to fratricidal destruction of healthy uninfected cells expressing the death receptors. Inappropriate PCD is also responsible for the disappearance of T helper cells primed for type-1 cytokine synthesis, thus contributing to the lack of survival factors which could prevent spontaneous lymphocyte apoptosis. Under potent anti-retroviral therapies, a significant decrease in spontaneous, TCR- and CD95-induced lymphocyte apoptosis is observed, concomitant with a partial quantitative and qualitative restoration of the immune system in treated patients. However, owing to the suppressive effect of anti-retroviral drugs on physiological apoptosis, these therapies are associated with alteration of TNF-alpha-regulated T cell homeostasis, leading to an accumulation in the blood of T cells primed for TNF-alpha synthesis, and contributing to the development of a new syndrome associated with these treatments, the lipodystrophy syndrome.

The ocular penetration of oral sparfloxacin in humans.

The penetration of sparfloxacin into the aqueous humor after oral administration was studied in 28 patients undergoing cataract surgery. Each patient received a single, oral dose of 400 mg of sparfloxacin. In eight other patients scheduled to undergo vitreal surgery, multiple daily oral doses were administered for a total amount of 1,000 mg. The aqueous levels were (mean +/- SEM) 0.127 +/- 0.036 microgram/ml to 0.404 +/- 0.159 microgram/ml from two to 24 hours after ingestion. In the vitreous, the mean drug level was 0.840 microgram/ml (range, 0.480 to 2.060 microgram/ml), from 4.3 to 8.0 hours after the most recent oral dose. Blood samples obtained at the same time as vitreous and aqueous taps were assayed by high-performance liquid chromatography. These data demonstrate that therapeutic levels of sparfloxacin may be achieved in noninflamed, noninfected eyes undergoing cataract or vitreous surgery.

Pro-apoptotic effect of the hepatitis B virus X gene.

The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.

[BCG vaccination in France]. / La vaccination BCG en France.

First used in 1921 and obligatory since 1950, BCG vaccination is a part of the classical arsenal in the struggle against tuberculosis in France. The progressive reduction in the incidence of tuberculosis leads one to wonder what to expect now and in the future, so much so that the degree of protection conferred by BCG is continually discussed. In animal experiments, BCG vaccination is efficacious but there is no absolute protection conferred. In man, the results of 9 prospective studies performed with control groups have thrice shown an 80% protection, thrice a 30% protection and thrice no protection (in the case of studies from Southern India). On the grounds that there were large differences in the methodology of the 9 studies and that the best methodology was found in the 3 studies which showed good protective efficacy of BCG, it is justifiable to consider that the protection conferred against tuberculosis by a correct BCG vaccination is of the order of 80% and lasts 15 years (direct effect of BCG). Equally, a similar protection has been observed in numerous retrospective studies. But it is not accompanied by a reduction in the transmission of tuberculous bacilli in the population vaccinated with BCG. Since one does not observe any reduction in the incidence of tuberculosis in non-vaccinated subjects who live in contact with the vaccinated population (indirect effect of BCG).(ABSTRACT TRUNCATED AT 250 WORDS)