Altered Cortisol/DHEA Ratio in Tuberculosis Patients and its Relationship with Abnormalities in the Mycobacterial-driven Cytokine Production by Peripheral Blood Mononuclear Cells

Authors


Dr M. L. Bay, Instituto de Inmunología, Facultad de Ciencias Médicas, Santa Fe 3100, Rosario 2000, Argentina. E-mail: bay.mahuad@yahoo.com.ar

Abstract

We have investigated the relationship between cortisol and dehydroepiandrosterone (DHEA) levels and the immune response to mycobacterial antigens in peripheral venous blood, from a male population of active tuberculosis patients and age-matched healthy controls of the same sex (HCo). Peripheral blood mononuclear cells were cultured for 36 or 96 h with whole sonicated Mycobacterium tuberculosis (WSA) for measurement of proliferation, interferon gamma (IFN-γ) and interleukin-10 (IL-10) in culture supernatants. Comparisons on the in vitro mycobacterial-driven immune responses demonstrated that TB patients had a higher IL-10 production, a decreased lymphoproliferation and a trend to reduced IFN-γ synthesis, in relation to HCo. Active disease was also characterized by increases in the plasma levels of glucocorticoids (GC) and reduced concentrations of DHEA which resulted in a higher cortisol/DHEA ratio respect the HCo group. Plasma DHEA levels were positively correlated with IFN-γ values. An inverse correlation was found between the cortisol/DHEA ratio and IFN-γ levels. Novel evidence is provided showing that the balance between cortisol and DHEA is partly responsible for the immune perturbations seen in TB patients.

Introduction

Human infection with Mycobacterium tuberculosis results in distinct clinical outcomes ranging from asymptomatic latent infection, through mild forms of disease, to progressive disease with high bacillary load and important tissue damage [1]. Such clinical spectrum is determined largely by a complex interaction between M. tuberculosis, its aetiologic agent, and the host immune response [2]. Critical components of the antimycobacterial immune response comprise macrophages as primary phagocytic cells of invading bacilli and T lymphocytes, particularly CD4+ cells which are able, among other functions, to stimulate a variety of effector functions through cytokine secretion [3]. In particular, interferon gamma (IFN-γ) that constitutes the type 1 cytokine response involved in the protective immunity against intracellular pathogens like M. tuberculosis [4]. Disturbances in cytokine production have been described in tuberculosis (TB) patients, with the predominant view suggesting adequate cellular immune responses (Th1-type pattern) in patients with the early forms of tuberculosis that is gradually lost as the disease progresses [5–8]. Besides the intrinsic immunomodulatory influences accounting for such a different pattern, factors like steroid hormones are likely to play a role in this regard. Cytokines released during immune responses, in particular those with inflammatory activities, stimulate the production of corticotrophin-releasing hormone in the hypothalamus, leading to the pituitary production of ACTH followed by the adrenal steroid hormone secretion [9, 10]. In turn, glucocorticoids (GC) can facilitate Th2 activity, partly by inhibiting Th1 cells [11, 12], whereas its natural antagonist dehydroepiandrosterone (DHEA) is able to both favour Th1 cytokine production and interfere with Th2 cytokine synthesis [13, 14].

Within this setting, we have recently explored the in vitro effect of cortisol and/or DHEA on the immune response to antigens obtained from M. tuberculosis by peripheral blood mononuclear cells (PBMC) from patients at different stages of lung TB. The results showed that the addition of cortisol within physiological range concentrations inhibited the mycobacterial antigen-driven proliferation of patient cells and the production of IFN-γ. By contrast, DHEA did not reverse the cortisol-mediated inhibitory effects, but suppressed the in vitro transforming growth factor-β production by lymphoid cells from TB patients with advanced disease [15]. Collectively, and as a closer appraisal to the in vivo situation, it was sensible to ascertain to what extent the endogenous levels of adrenal steroids may be accounting for the profile of specific cellular immune responses observed during TB. Accordingly, our aim was to find whether there was a relationship between the in vitro mycobacterial-driven immune response and cortisol and DHEA concentrations in plasma in a population of active TB patients and healthy controls. With that purpose, we performed immunological studies on the specific lymphoproliferation as well as the production of two-well representative cytokines in TB immunopathogenesis, IFN-γ and interleukin-10 (IL-10).

Patients and methods

Sample population.  Twenty-five newly diagnosed active pulmonary TB patients were enrolled in this study. All individuals were HIV negative and were not receiving anti-mycobacterial therapy at the time of collection of the blood. All patients were males, and aged 49.9 ± 18.9 years (mean ± SD). Clinical presentation and chest radiographs were compatible with pulmonary TB and sputum was positive for acid-fast bacilli. Disease severity was determined through the X-ray pattern [6] and it was classified into two categories: moderate (unilateral involvement of two or more lobes with cavities, if present, reaching a total diameter no greater than 4 cm, 17 cases) and severe (bilateral disease and multiple cavities, eight cases). Fourteen volunteers, age-matched males without close contact with TB patients (healthy contacts, HCo) constituted the control population. All individuals gave informed consent for participating in the study and the protocol was approved by the ethics committee at the Facultad de Ciencias Médicas. As adrenal steroid hormones show physiological circadian variations, blood samples from all individuals were taken before 08:00 hours. Exclusion criteria comprised: pathologies affecting the hypothalamus–pituitary–adrenal axis (i.e. tumour and vascular), direct compromise of the adrenal gland, age under 18 years or any disorder requiring treatment with corticosteroids, immunosuppressors or immunomodulators.

Hormone assessments.  Blood samples were anti-coagulated with ethylenediamine tetraacetic acid (EDTA). After centrifugation the plasma was treated with Trasylol® (100 U/ml of plasma) and preserved at −20 °C. Cortisol and DHEA levels were measured in plasma with commercially available ELISA methods according to the manufacturer's instructions (DRG Systems, Berlin, Germany). Detection limits were as follows: cortisol = 2.5 ng/ml and DHEA ≤0.1 ng/ml.

Cell isolation and antigen stimulation.  Peripheral blood mononuclear cells (PBMC) were isolated from whole blood anticoagulated with EDTA by Ficoll separation (Ficoll-Paque plus: Amersham Biosciences Inc., Piscataway, NJ, USA). Cells were resuspended in RPMI-1640 (PAA Laboratories GmbH, Linz, Austria) with standard concentrations of l-glutamine, penicillin and streptomycin, containing 10% heat-inactivated pooled normal AB human serum (CMS, PAA Laboratories GmbH) and centrifuged at 400 g for 30 min at room temperature. Cells were cultured in quadruplicate in flat-bottomed microtiter plates (2 × 105 cells per well in 200 μl) with or without the addition of whole sonicate antigen of M. tuberculosis (WSA, 10 μg/ml) kindly provided by Dr J.L. Stanford, London. Cultures were incubated for 5 days at 37 °C, in a 5% CO2-humidified atmosphere and pulsed with 3H-thymidine for 18 h before cell harvesting. Results of proliferation were expressed as the mean counts per minute (cpm) of the quadruplicate cultures. A four-dose titration experiment performed with PBMC from healthy persons was previously carried out to establish antigen concentrations and time point evaluations for immunological studies.

Cytokine measurements.  To evaluate cytokine production, 1 × 106 cells/ml were cultured with or without the addition of 10 μg/ml of WSA. Culture supernatants collected 36 or 96 h later were stored at −20 °C until use. Supernatants from unstimulated cultures, or from those stimulated with WSA were assayed in duplicate for IFN-γ and IL-10 assessment using commercially available ELISA kits (OptEIA Set, Pharmingen, San Diego, CA, USA), according to manufacturer's instructions. Detection limits were as follows: IFN-γ 4.7 pg/ml, IL-10 7.8 pg/ml. Results were expressed as the average of two determinations (pg/ml) in an ELISA microplate reader at 450 nm. Cytokines were quantified using reference standard curves generated with human recombinant cytokines.

In vitro effects of cortisol and DHEA on lymphoproliferation.  Peripheral blood mononuclear cells were cultured as described above with the addition of different physiological concentrations of cortisol (1 and 0.1 μm) and/or DHEA (0.1, 0.01 and 0.001 μm). Lymphoproliferation was analysed as described above. Because hormones were dissolved in alcohol, parallel cultures exposed to the same final alcohol concentration required to dissolve the hormones, were also performed. Such treatment did not affect cell viability and proliferation (data not shown). Cultures carried out throughout experiments were as follows: neither stimulation nor treatments (baseline); and WSA stimulation, alone or plus treatment with DHEA and/or cortisol.

Statistical analysis.  Unpaired statistical comparisons were performed by the Kruskall–Wallis and Mann–Whitney U tests, whereas the Wilcoxon and Friedman tests were used to compare paired data. Correlations between hormone levels and the in vitro mycobacterial-driven immune response (lymphoproliferation and cytokine production) were analysed by parametric and non-parametric methods. Statistical significance was inferred for values of P < 0.05. Medians are given with interquartile ranges, and means are given with ± standard error of the mean (SEM).

Results

Comparisons between patients and HCo

Cortisol and DHEA concentrations in plasma. Mean (±SEM) plasma cortisol showed elevated levels (ng/ml) in the whole series of TB patients (233.1 ± 15.1), yielding a statistically significant difference when compared with HCo (182 ± 14.9; P < 0.05). The same was true when the latter group values were compared with those obtained in each patient group, with no differences being found among patient group comparisons. Plasma DHEA levels (ng/ml) were reduced in all TB cases (3.54 ± 0.36) markedly below the values seen in HCo (10.9 ± 1.2; P < 0.0001). Analyses among TB patients revealed that DHEA levels diminished with increasing disease severity (moderate = 4.18 ± 0.45, severe 2.26 ± 0.18; P <0.0001 vs. HCo, for all patient groups). Further analyses of the cortisol/DHEA ratio revealed that TB patients had a much higher ratio (78.7 ± 6.9) than HCo (20.37 ±3.6, P < 0.0001), with ratios among TB patients increasing with respect to disease progression. In the present sample, levels of cortisol and DHEA were not age-correlated (data not shown).

In vitro responses. Confirming our earlier observations [15], comparisons on the in vitro mycobacterial-driven immune responses demonstrated that the whole series of TB patients had a higher 36-h IL-10 production (152.7 ± 22.2), decreased lymphoproliferation (cpm = 7331.3 ± 1789.7, n = 24) and reduced 36-h IFN-γ synthesis (72.7 ± 14.9 n = 21), in relation to HCo (IL-10, 71.3 ± 14.2, n = 13, P < 0.025; cpm = 21,067.4 ±6178.2 n = 14, P < 0.01; IFN-γ, 314.5 ± 107, n = 13, P < 0.05). Comparisons among TB patients groups showed a lower, but statistically insignificant, lymphoproliferation and IFN-γ production in severe cases (data not shown).

Relationship between circulating steroid levels and the mycobacterial-driven immune response

Based on these findings, analyses on the relationship between steroid levels and lymphoproliferation and cytokine production were carried out by separating both groups under study. In a first step, pairwise correlations between cytokine levels and hormonal findings (cortisol, DHEA and the cortisol/DHEA ratio) were performed with correlation coefficients being virtually identical, irrespective of the method employed. Results are referred to values detected in 36-h culture supernatants. As seen in Table 1, plasma DHEA levels showed a significantly positive association with the amount of IFN-γ present in culture supernatants from TB patients (r = 0.44, r2 = 0.19, P < 0.05). Comparisons between DHEA and IL-10 showed negative but statistically insignificant correlations in both subject groups. Further analyses using the cortisol/DHEA ratio revealed an inverse correlation with IFN-γ levels from cultures of TB patients (r = −0.65, r2 = 0.42, P < 0.01). Analyses of 96-h culture supernatants showed that IFN-γ and IL-10 levels from HCo were positively correlated with their DHEA values and cortisol/DHEA ratio respectively (P < 0.05, in both cases, data not shown).

Table 1.   Correlations between hormonal results and in vitro 36-h cytokine levels in TB patients and HCo.
Pairwise correlationsHCoTB
rnP valuernP value
  1. HCo, healthy controls; TB, tuberculosis; r, correlation coefficient.

DHEA and IFN-γ0.2413ns0.4420<0.05
DHEA and IL-10−0.5013ns−0.3224ns
Cortisol/DHEA ratio and IFN-γ−0.1313ns−0.6520<0.01
Cortisol/DHEA ratio and IL-100.4313ns0.3724ns

There were no significant correlations when comparing either cortisol levels with in vitro cytokine production or hormonal profiles with lymphoproliferation.

Based on the median levels of cortisol, DHEA and the cortisol/DHEA ratio seen in all TB patients and HCo, both groups were next classified into two categories, i.e. those whose hormonal results were below or above their respective median values. Therefore, TB patients and HCo were compared with respect to these categories. Results of the HCo analysis are presented in Table 2. As shown in panel a, subjects with DHEA values above the median level had a significantly increased proliferation when compared with controls showing DHEA levels under the median value. No significant differences in cytokine concentrations and lymphoproliferation were found when HCo were analysed with respect to cortisol levels or cortisol/DHEA ratio below or above the median values (panels b and c).

Table 2.   Analysis of the in vitro cell responses from healthy controls with respect to the plasma levels of cortisol and dehydroepiandrosterone (DHEA) and the cortisol/DHEA ratio.
VariablesBelow the median valueAbove the median valueP value
  1. *Counts per minute (median and rank) crude values.

  2. PBMC were stimulated with whole sonicate antigen of M. tuberculosis. Cytokine concentrations (36-h supernatants) are given as median and rank (pg/ml). Statistical comparisons were made by the Mann–Whitney U-test.

(a) Classification with respect to the median level of DHEA (10.88 ng/ml)
 IFN-γ 36 h135.8 (33.4–263.0), n = 790.9 (11.8–916.0), n = 6ns
 IL-10 36 h63.8 (32.9–153.1), n = 745.7 (30.9–90.6), n = 6ns
 Proliferation*12,744.2 (7839.3–12,861.1), n = 720,834.1 (9359.9–67,189.3), n = 7<0.05
(b) Classification with respect to the median level of cortisol (181.332 ng/ml)
 IFN-γ 36 h90.3 (33.4–160.6), n = 7199.4 (12.8–916.0), n = 6ns
 IL-10 36 h71.6 (59.0–120.1), n = 632.4 (20.2–90.6), n = 7ns
 Proliferation*12,861.1 (9359.9–20,244.5), n = 712,748.0 (1777.4–67,189.3), n = 7ns
(c) Classification with respect to the median level of cortisol/DHEA ratio (16.377)
 IFN-γ 36 h99.9 (11.8–892.3), n = 6135.8 (33.4–263.0), n = 7ns
 IL-10 36 h42.6 (30.9–59.0), n = 679.5 (32.9–153.1), n = 7ns
 Proliferation*20,244.5 (9359.9–24,231.7), n = 712,744.2 (7839.3–12,861.0), n = 7ns

When analysing TB patients, those whose DHEA values above the median level had significantly higher levels of IFN-γ with compared with patients with lower-than-the-median DHEA concentrations (Table 3, panel a). Patients showing cortisol/DHEA ratios below the median value had a significantly increased production of IFN-γ (panel c). Comparisons with respect to cortisol levels revealed no significant differences in lymphoproliferation and cytokine production (panel b). Comparisons in relation to 96-h cultures were in the same direction. That is, our results showed significantly augmented IFN-γ concentrations in cases with DHEA values above the median level, and a trend to show less IFN-γ in cases with a higher cortisol/DHEA index (data not shown).

Table 3.   Analysis of the in vitro cell responses from tuberculosis patients with respect to the plasma levels of cortisol and DHEA and the cortisol/DHEA ratio.
VariablesBelow the median valueAbove the median valueP value
  1. *Counts per minute (median, rank) crude values.

  2. PBMC were stimulated with whole sonicate antigen of M. tuberculosis. Cytokine concentrations (36-h supernatants) are given as median and rank (pg/ml). Statistical comparisons were made by the Mann–Whitney U-test.

(a) Classification with respect to the median level of DHEA (3.144 ng/ml)
 IFN-γ 36 h26.2 (17.2–32.0), n = 1189.1 (48.0–180.9), n = 10<0.006
 IL-10 36 h154.0 (104.2–306.2), n = 1387.7 (45.3–210.9), n = 12ns
 Proliferation*2461.6 (1389.3–6440.9), n = 127134.0 (1938.6–16,439.3), n = 12ns
(b) Classification with respect to the median level of cortisol (204.805 ng/ml)
 IFN-γ 36 h80.9 (25.6–184.7), n = 1130.9 (26.2–48.6), n = 10ns
 IL-10 36 h104.2 (86.6–180.3), n = 13174.1 (63.1–268.5), n = 12ns
 Proliferation*5430 (1598.8–15,804.7), n = 132173 (990.8–7356.5), n = 11ns
(c) Classification with respect to the median cortisol/DHEA ratio (86.163)
 IFN-γ 36 h80.9 (25.6–180.8), n = 1129.4 (26.2–48.6), n = 10<0.05
 IL-10 36 h104.2 (54.2–195.6), n = 13151.0 (83.3–308.2.), n = 12ns
 Proliferation*5430.3 (1598.8–15,804.7), n = 132173.2 (990.8–7356.5), n = 11ns

Ex vivo studies commented so far suggested that the hormonal environment was likely to modify the features of a specific immune response. To gain a better insight on this issue, culture experiments were carried out where hormones were added before antigen stimulation, in an attempt to approximate to the in vivo situation. Studies were performed in PBMC from tuberculin-responder healthy blood donors (n = 10, age matched). Cells were incubated with cortisol and/or DHEA for 24 h prior to WSA stimulation. Parallel cultures in which hormones were added simultaneously with the antigen were also performed for comparison purposes.

Analyses on the blastogenic response revealed that DHEA-treated cultures (0.001 μm) showed a significantly higher lymphoproliferation, when compared with their untreated counterparts or cultures receiving greater DHEA doses (0.1 or 0.01 μm), irrespective of whether DHEA was added before or simultaneously with the antigen stimulation (Fig. 1). All cultures incubated with DHEA 24 h before stimulus, had an increased cell proliferation in relation to those receiving DHEA at the time of WSA stimulation, but the trend did not reach statistical significance (Fig. 1).

Figure 1.

 Effect of dehydroepiandrosterone (DHEA) incubation on antigen-driven lymphoproliferation in PBMC from the responder control group. DHEA was added 24 h previous or simultaneously with antigen stimulation (whole sonicate antigen –WSA- of M. tuberculosis). cpm, counts per minute. *Statistically different from WSA, p < 0.01. **Statistically different from DHEA 0.001 μM, p < 0.01.

Cells cultured in the presence of cortisol (0.1 μm) showed a significantly decreased proliferation regardless of whether cultures were treated with cortisol before or simultaneously with WSA stimulation, or not (Fig. 2). The same was true when analysing cultures exposed to DHEA plus cortisol (Fig. 2). Compared with simultaneously cortisol-plus-antigen-exposed cells, cells pretreated with cortisol for 24 h had an even further and statistically significantly decreased proliferation, independently of whether DHEA was present in cultures, or not (Fig. 2). Similar results were obtained in experiments using a higher cortisol concentration (1 μm, data not shown).

Figure 2.

 Effect of Cortisol (GC 0.1 μM) incubation alone or combined with different dehydroepiandrosterone (DHEA) concentrations on antigen-driven lymphoproliferation in PBMC from the responder control group. Cortisol (alone or combined with DHEA) was added 24 h previous or simultaneously with antigen stimulation (whole sonicate antigen –WSA- of M. tuberculosis). cpm, counts per minute. *Statistically different from the remaining groups, p < 0.02. **Significantly different from their simultaneously-treated counterparts, p < 0.02.

Discussion

The bilateral communication between the neuroendocrine and immune systems is a critical element in assuring a finely tuned regulatory mechanism addressed to both protect host and prevent from tissue damage [9, 10]. Therefore, disturbances in this homeostatic system may lead to substantial changes in the mechanisms by which the host deals with an infectious process. As the progression of TB is associated with impaired cell-mediated immunity [6, 16, 17] and adrenal steroids were found to modify the in vitro immune response during TB [15], the relationship between serum steroid levels and the specific ex vivo immune response was worth exploring. Present findings indicate that the active disease is characterized by increases in the plasma levels of GC and reduced concentrations of DHEA. Accompanying these results, variations in the circulating levels of adrenal steroids were associated with a different pattern of immune response by antigen-stimulated PBMC. In fact, the higher the DHEA plasma levels, the greater the IFN-γ production in TB patients. Following the same trend, HCo (96 h) also depicted a higher lymphoproliferative response with increased DHEA concentrations. Comparisons with respect to cortisol levels revealed no substantial associations likely because plasma cortisol fluctuations were less remarkable than DHEA changes. The cortisol/DHEA ratio, which reflects plasma adrenal steroid profile, was also associated with immune changes. In fact, this ratio correlated negatively with IFN-γ levels from TB patients, while presenting a positive correlation with IL-10 values from HCo (96 h cultures). Separation with respect to categories revealed that patients with a lower cortisol/DHEA ratio also showed higher IFN-γ levels.

The mechanisms by which the course of TB is characterized by a gradual loss of cell-mediated immune protection are not fully understood, and several possibilities have been proposed. Our results suggest that the immune perturbations seen in TB, and some changes occurring in healthy individuals, may be partly influenced by adrenal steroids. Several lines of evidence support the view that adrenal steroids play an important immunoregulatory role. Indeed, GC decrease in vitro T-cell proliferation, whereas DHEA stimulates helper T-cell function by enhancing the capacity of activated T cells to produce IL-2 [18]. In a similar way, in vitro treatment of PBMC with DHEA induces a pattern of cytokine gene expression which is opposed to the profile seen when cells are exposed to GC [19]. Improved mitogenic responses were also reported in aged rats treated with DHEA [20], as seen in elderly normal men administered DHEA orally [21]. Recent studies among Ethiopians infected with Schistosoma mansoni demonstrated a significant negative correlation between serum levels of DHEA sulphate and intensity of infection [22]. Furthermore, a positive association between serum DHEAS levels and the amount of IFN-γ secreted by mitogen-stimulated PBMC or the number of IFN-γ-secreting PBMC was also found in HIV patients or normal women respectively [23, 24]. Taken together, it is worth speculating that fluctuations in the mycobacterial-driven in vitro responses may be partially explained by changes in adrenal steroids concentrations in plasma. Cell treatments with cortisol or DHEA, either before or simultaneously with WSA stimulation, add evidence on the potential repercussion of the hormonal environment, as DHEA improving effects on lymphoproliferation were not time related, while a decreased T-cell proliferation was more profound in cortisol-pretreated cells. It follows that the pre-existing hormonal environment is a relevant component for cell-mediated responses. Translating to our ex vivo experiments, the balance between cortisol and DHEA may be more relevant than single hormones as to the compartmental features from which cells were obtained. Thus, the drop in DHEA (and its metabolites), and the concomitantly elevated cortisol, emerges as a relevant link between adrenal hormones and the immune alterations seen in TB patients.

In considering the intricate immunoendocrine network, variations in diurnal rhythmicity deserve some comments. Studies wherein the ratio between IFN-γ and IL-10 production from LPS- or tetanus-stimulated whole blood peaked in the early morning and correlated negatively with plasma cortisol [25] may have some relevance when the circadian rhythm of hormonal production is altered, i.e. rheumatoid arthritis [26]. Beyond these considerations, our r2 estimation by which 19% and 42% IFN-γ variation was explained by its linear relation with the DHEA and cortisol/DHEA ratio, respectively, lends support to the biological meaning of present results.

How exactly tuberculosis coexists with an unbalanced cortisol–DHEA relationship remains to be established, although products from the immune response may partly be involved in this regard. Whatever the case, present cytokine alterations, in particular IFN-γ, may have unfavourable immunopathological implications. IFN-γ is a central macrophage-activating cytokine involved in the immune protection against M. tuberculosis [27]. Studies in mice and humans revealed that genetic defects in IFN-γ signalling render them highly susceptible to mycobacterial diseases [7, 28, 29]. Furthermore, detection of IFN-γ or IFN-γ-producing cells after antigen stimulation is frequently used as an indicator of cellular effector activity in this mycobacterial infection [30]. On the other hand, IL-10 is produced during active disease and can play a role in the suppressed cellular response in TB [31–33].

Dissociation of DHEA and cortisol secretion was also observed in HIV infection [34] or chronic inflammatory diseases, like rheumatoid arthritis [35]. In HIV-infected individuals, the level of change in the cortisol/DHEA ratio could be predictive of progression to AIDS. Studies in experimental TB showed that BALB/c mice given DHEA and androstenediol had a better course of the experimental disease [36]. Furthermore, a study performed in TB patients demonstrated that an increase in the urinary cortisol/DHEA relationship appears to be associated with increased TB severity [37]. Extending this observation, our study provides new evidence that similar changes are found in plasma and that such changes correlated with immune response abnormalities.

These findings provide a stimulating background for further studies to address the potential role of adrenal steroids as contributory causes in TB immunopathology, not contemplated in the scope of the present study. Similarly, it should be established whether a restored hormonal balance serves to re-establish a proper pattern of cell-mediated responses favouring disease resolution.

Acknowledgment

This research has been funded by FONCYT research grants (BID 1201/OC-AR, 05-08555 and BID 1728/OC-AR 5-25462), Fundacion J. Prats and a Fogarty International Center/NIH grant through the AIDS International Training and Research Program at Mount Sinai School of Medicine-Argentina Program (Grant no. 5D43 TW0010137).

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