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Infection and Immunity, November 1999, p. 5597-5603, Vol. 67, No. 11
Department of Biotechnology and Department of
Medicine,
Received 1 February 1999/Returned for modification 12 March
1999/Accepted 13 July 1999
To better understand the role of cytokines in susceptible and
resistant subjects exposed to Mycobacterium tuberculosis
infection, intracellular gamma interferon (IFN- With the advent of AIDS and
multidrug resistance (MDR), tuberculosis has emerged as a disease of
public health importance both in developed and developing countries,
with annual associated death rate of 3 million (23, 31). The
lack of the availability of an effective protective vaccine has further
aggravated the situation (30). Elucidation of the immune
response of immunocompetent healthy individuals after exposure to
tubercle bacilli may provide strategies for effective immunotherapeutic
and prophylactic regimens (43). The mode of activation of
effector cells in humans is different from that of experimental models,
thereby limiting the extrapolation of data (12, 13, 16, 26, 35,
46).
Cell-mediated immunity is the major component of host defense against
tuberculosis. Antigen-specific T cells secrete cytokines that activate
natural effector cells (6). Classically, the CD4+ T cells have been considered to play the most
important role in antimycobacterial immunity (24, 40, 45).
Ever since Mosmann and Coffmann proposed the Th1 and Th2 paradigm, the
study of their roles in various diseases in order to develop
prophylactic vaccines and therapeutic regimens has become a major focus
of immunological studies (29, 34). The degree of Th1 and Th2
polarization increases with the severity and the chronicity of the
immune response (22, 33, 51). Thus, the Th0 cytokine pattern
is most noticeable early after lymphocyte activation, and the clearest
demonstrations of Th1 and Th2 cytokine profiles have been made in
chronic disease states, in which antigens are persistent and cannot be
eliminated (1).
Hence, the present study was undertaken to assess by flow cytometry the
interleukin-4-positive (IL-4+) and
gamma-interferon-positive (IFN- Subjects.
Twenty untreated pulmonary-tuberculosis patients
in an age group ranging between 22 and 50 years (X-ray and sputum
acid-fast-bacillus positive) attending the outpatient clinic at the LRS
Tuberculosis and Allied Diseases Hospital and the All India Institute
of Medical Sciences (AIIMS), New Delhi, were included in the study. All
patients were human immunodeficiency virus HIV negative. The
institutional review board approved the study, and the subjects gave
informed consent to participate in the study. Seventeen healthy
volunteers who were closely associated with the patients were included
in the study as high-risk healthy contacts. The contacts were
radiologically screened for clinical signs of tuberculosis by chest
X-ray and, when warranted on the basis of symptoms, additional tests
such as sputum examination for acid-fast bacilli and erythrocyte
sedimentation rate were undertaken. Tuberculin (1 tuberculin unit
[TU]) (BCG Vaccine Laboratory, Guindy, Chennai, India) was injected
intradermally into all individuals (patients and contacts) included in
the study. After 48 h, an induration of Cell preparation and in vitro culture.
PBMC were isolated
from heparinized (GIBCO-BRL, Grand Island, N.Y.) blood by density
gradient centrifugation on Ficoll-Hypaque (Sigma Aldrich, St. Louis,
MO.) (5). PBMC were suspended at a concentration of
106 cells/ml in RPMI 1640 (GIBCO-BRL) supplemented with 20 mM HEPES (GIBCO-BRL), 2 mM glutamine (GIBCO-BRL), 0.1 mM sodium
pyruvate (GIBCO-BRL), and 10% heat-inactivated human AB serum. A
100-µl portion of the PBMC suspension was distributed into 96-well
round-bottom plates (Linbro) and then incubated with 20 µl of
integral M. tuberculosis organisms/well (Tuberculosis
Research Materials, National Institute of Allergy and Infectious
Diseases, National Institutes of Health). Four different concentrations
of bacilli (0.005, 0.05, 0.5, and 5 × 106 bacilli/ml
[M1 to M4, respectively]) were used in the study. These cultures were
incubated for 40 h at 37°C in a 5% CO2-95% air
atmosphere. With positive controls, cells were cultured in the presence
of 1% phytohemagglutinin (PHA; GIBCO-BRL) for 24 h, followed by
restimulation with PHA, phorbol myristate acetate (Sigma), and
ionomycin (Sigma) for 12 h. Cultures incubated with medium alone
served as negative controls.
Intracellular analysis of cytokine production in CD4+
T cells.
Intracellular cytokine staining was used to determine the
cytokine expression in CD4+ T cells by flow cytometry
(3, 20, 37). A total of 20 µl of brefeldin A, a potent
nontoxic inhibitor of protein secretion (10 µM; Sigma) was added
4 h prior to termination of the culture. Cells were harvested and
stained for the surface expression of CD4+ antigen in T
cells by using fluorescein isothiocyanate (FITC)-conjugated anti-CD4
antibody (Becton Dickinson). After a washing with Dulbecco phosphate-buffered saline (PBS) containing 1% fetal calf serum (DPBS-FCS; pH 7.2 to 7.4; GIBCO-BRL), the cells were fixed with 4%
paraformaldehyde in PBS (0.05 M, pH 7.4) for 15 min on ice. Cells were
washed with DPBS-1% FCS and permeabilized with 0.2% saponin (Sigma)
in PBS for 30 min at room temperature. Phycoerythrin (PE)-conjugated
anti-IFN- Staining specificity.
Both cold antibody competition and
recombinant cytokine blocking established the staining specificity. In
the former assay, an excess of nonconjugated antibody (Genzyme,
Cambridge, Mass.) was allowed to react with permeabilized cells,
followed by further incubation with PE-conjugated anti-cytokine
antibody (anti-IFN-
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Dichotomy of Cytokine Profiles in Patients and
High-Risk Healthy Subjects Exposed to Tuberculosis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) and interleukin-4
(IL-4) in ex vivo peripheral blood-derived CD4+ T cells
were examined by flow cytometry. Of the 37 individuals examined, 20 had
clinical evidence of pulmonary tuberculosis and showed acid-fast
bacilli in the sputum. Other individuals in close contact with these
patients showed no evidence of disease. Patients had a higher number of
CD4+ T cells expressing IFN- and IL-4 in unstimulated
cultures compared to healthy subjects. Despite this, the ratio of
IFN-+ to IL-4+ CD4+ T cells was
similar in both groups. The Th1 response seen in CD4+ T
cells in patients was also observed in the overall pattern of IFN-
and IL-4 detected in control culture supernatants by enzyme-linked
immunosorbent assay (ELISA). However, after in vitro stimulation of
PBMC with heat-killed M. tuberculosis there was a
significant reduction in the percentage of IFN-+
CD4+ T cells (P < 0.001) in patients.
This trend was reflected in the IFN- ELISA assay with supernatants
derived from stimulated cultures. However, the accumulated levels of
IFN- were higher than those for IL-4. The reduction of
IFN-+ CD4+ T cells resulted in the dominance
of IL-4+ CD4+ T cells in 13 patients
(P < 0.05). The elevated levels of IL-4+
CD4+ T cells seen in patients may contribute to the
downregulation of IFN- expression and the crucial effector function
of CD4 T cells, leading to the persistence of disease and the
immunopathology characteristically seen in patients. Preliminary data
on the indicators of apoptosis in antigen-stimulated cultures in PBMC
derived from patients are presented. Of the 17 high-risk healthy
individuals examined, 11 differed in that, after mycobacterial-antigen
stimulation, there was an enhancement in IFN-+
CD4+ T cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
+) CD4+ T
cells derived from the peripheral blood mononuclear cells (PBMC) of
patients and healthy contacts following coculture with various concentrations of integral heat-killed tubercle bacilli. Our results show the preferential decrease in IFN-+ CD4+
T cells, along with the sustained maintenance of IL-4-producing CD4+ T cells in patients. The predominance of
IL-4-producing CD4+ T cells in patients may be one of the
principal factors leading to an ineffective immune response against the
tubercle bacilli (1).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
5 mm was considered a
positive reaction. Of the 20 patients, 12 were tuberculin reactive
(mean diameter of induration, 10.7 ± 1.7 mm) and 8 were negative
(<5 mm). Eight contacts were tuberculin reactive (15.0 ± 2.5 mm), and 9 were nonreactive (<5 mm, CNR). Six tuberculin-negative
volunteers from Europe were included in the study as nonendemic-area controls.
and anti-IL-4 (2.5 µg/ml; Pharmingen, San Diego, Calif.)
were added to separate aliquots of membrane stained and permeabilized
cells and were incubated for 45 min. Cells were washed thrice with
DPBS-0.2% saponin and finally with DPBS-1% FCS. Samples were
acquired in a Bio-Rad flow cytometer (Bio-Rad; flow cytometer was
provided courtesy The National Institute of Immunology (NII), New
Delhi, India), and data were analyzed by using WIN MDI and WIN Bryte
software. Control samples were incubated with irrelevant,
isotype-matched monoclonal antibodies (Becton Dickinson) in parallel.
and anti-IL-4 antibody), whereas in the latter
experiment preincubation of a 500-fold molar excess of recombinant
cytokine (GIBCO-BRL) with anti-cytokine antibody was undertaken for
1 h before it was added to the sample.
ELISA for IFN- and IL-4.
Supernatants were collected at
48 h from cultures of PBMC stimulated with various concentrations
of heat-killed M. tuberculosis bacilli (M1, M2, M3, and M4).
Triplicate wells were pooled. IFN- and IL-4 concentrations in
supernatants were assessed by use of enzyme-linked immunosorbent assay
(ELISA) kits (Genzyme) according to the manufacturer's instructions.
Apoptosis (DNA fragmentation).
PBMC cultured with or without
M. tuberculosis (0.005 × 106 bacilli/ml)
were harvested and washed twice with PBS. The cell pellet was lysed in
400 µl of lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM EDTA, pH 8.0;
0.1% sodium dodecyl sulfate; 0.2% Triton X-100; proteinase K at 0.1 mg/ml) at 50°C for 16 h. This was followed by incubation with 50 µg of RNase per ml for an additional hour at 68°C. DNA was
extracted twice with phenol-chloroform-isoamyl alcohol (25:24:1
[vol/vol/vol]) and twice with chloroform-isoamyl alcohol (24:1
[vol/vol]). DNA in the aqueous phase was precipitated overnight at
20°C with twice the volume of 100% ethanol, washed with 70%
ethanol, air dried, and dissolved in TE buffer (10 mM Tris and 1 mM
EDTA) and subjected to electrophoresis through a 1.8% agarose gel. DNA
fragments were stained with ethidium bromide and visualized under UV light.
Statistical analysis. All statistical calculations were done by using MICROSTAT software. All values in text and figures have been expressed as the mean ± the standard error of the mean (SEM). Since the variables under study were not normally distributed, nonparametric statistical tests were used for data analysis. The Wilcoxon matched-pair test was used to analyze paired values in the same group, the Wilcoxon rank sum test was used to analyze paired values in different groups, the Friedman test was used to compare multiple values within the same group, and the Kruskal-Wallis test was used to compare multiple groups (38).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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The in vitro immune response to M. tuberculosis as
determined by flow cytometry was assessed in 20 pulmonary tuberculosis patients and 17 high-risk healthy subjects. PBMC isolated from these
individuals were incubated with wide-ranging concentrations of
heat-killed tubercle bacilli (0.005 × 106 to 5 × 106/ml) for 40 h. The cells were assessed for the
intracellular presence of IL-4 and IFN- in CD4+ T cells.
The specificity of the staining technique used for the detection of
intracellular IFN- and IL-4 in the CD4+ subset of T
cells was established by including the following controls: (i) positive
control, (ii) nonspecific isotype-matched control, (iii) neutralization
of IFN- and IL-4 staining with appropriate antibodies, and (iv)
competitive inhibition of staining with recombinant IFN- and IL-4
(Fig. 1A and B). The 2D overlays of a
representative European control establish the efficacy of the CD4
staining (44.2% CD4+ T cells; Fig. 1C). Reports of
lymphopenia and absolute low CD4 numbers in HIV seronegative patients
with advanced tuberculosis and pulmonary cavitation could account for
the low percentage of the CD4+ population seen in patients
(47). Studies reveal a lower percentage of CD4 T cells in
the Indian population (4). Similarly, there have been
reports in healthy adult Thais and Malaysians of low CD3 and CD4 cell
numbers and the CD4/CD8 ratio appears to be significantly less than
those reported in Caucasians (7, 49, 50). The low proportion
of CD4 T cells seen in the subjects included in our study fall into the
general observation made regarding the normal range of lymphocyte
subpopulations in Asians.
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IL-4+ or IFN-+ T cells were not detected in
European samples in control or antigen-stimulated cultures where
M. tuberculosis is not endemic and thus the data from these
cultures was not considered for further analysis.
Representative dot plots of IFN-+ and
IL-4+ CD4 T cells in patients and contacts.
Representative dot plots of CD4+ T cells showing
intracellular IFN- and IL-4 in a healthy contact and in a
tuberculosis patient are depicted in Fig.
2. The healthy contacts showed negligible levels of CD4+ T cells expressing IFN- and IL-4 in basal
unstimulated cultures (0.3 and 0.2%, respectively). After stimulation
with killed M. tuberculosis (0.005 × 106/ml; M1), selective increases in the frequency of
IFN-+ CD4+ T cells were detected (3.5%). A
large number of non-CD4+ T cells (15.5%) expressing
IFN- in healthy contacts at M1, the lowest antigen concentration,
was detected. Further studies have shown this population to be a
non-T-cell population, since neither CD8 nor 
antibodies (data
not shown) are able to stain it. IL-4-containing CD4+ T
cells also increased from 0.2 to 0.7% with stimulation, although the
frequency was lower compared to the percentage of IFN-+
CD4+ T cells.
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+ CD4+ T cells was detected in
stimulated cultures (0.4%) compared to the control cultures (2.0%).
Similar percentages of IL-4-positive cells were present in both control
and stimulated cultures (1.3%).
Cytokine profile in contacts and patients in control and stimulated
cultures.
Figure 3 and Table
1 show the individual and mean data on
the percentages of IFN-+ and IL-4+
CD4+ T cells of patients and contacts, respectively. In
control cultures only one contact showed >5% of CD4+ T
cells expressing IFN- and IL-4, respectively (Fig. 3).
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(Fig. 3). The percentage of
IL-4 expression was also increased in five healthy contacts. This
percentage was lower than in the patients.
A shift in the baseline cytokine profile was seen upon antigen
stimulation (Table 1). The stimulated cultures of contacts showed a
gradual and sustained increase in IFN--producing cells compared to
the baseline levels. However, the predominance of the ratio of
IFN--containing CD4+ T cells (Th1 response) was confined
to cultures stimulated with 0.005 × 106 (M1) and
0.05 × 106 (M2) bacilli/ml.
In contrast, the patients examined had higher mean percentages of
IFN- (P < 0.001), along with IL-4+
CD4+ T cells, in unstimulated cultures compared to contacts
(Table 1, P < 0.05). The average percentage of cells
expressing IFN- was 8.3 ± 0.6% compared to contacts (2.1 ± 0.5 [Table 1], P < 0.001). Similarly, elevated
levels of IL-4+ CD4+ T cells were detected in
patients (5.9 ± 1.7) compared to contacts (1.5 ± 0.5)
(Table 1, P < 0.05).
Of the 20 patients, 14 showed significant reduction of
IFN-+ CD4+ T cells after mycobacterial
stimulation (P < 0.001). No concomitant reduction was
detected in the percentage of CD4+ T cells expressing IL-4
upon mycobacterial stimulation; in fact, an increase was seen in few
patients (Fig. 3 and Table 1). The reduction in IFN-+
CD4+ T cells resulted in the dominance of IL-4+
CD4+ T cells in 13 patients (P < 0.05).
The shift in cytokine profile, namely, from Th1 to Th2, was clearly
evident in the CD4+ subset of T cells in patients (Table
1).
Cytokine ELISA.
IFN- and IL-4 present in the supernatants
of PBMC were estimated by cytokine-specific ELISA for the groups of
subjects mentioned above. IFN- levels (mean ± SEM) in
supernatants of stimulated cultures were significantly higher in
contacts compared to patients (Fig. 4,
P < 0.05). Consistent with the data obtained by flow cytometry and whereas the healthy subjects showed an increase in
IFN- upon stimulation with various doses of antigen, the PBMC of
patients showed a decrease in IFN- from the second concentration (M2) onwards. Of interest was the absence of a detectable amount of
IL-4 in the healthy group compared to the pulmonary tuberculosis patients. IL-4 was detected in the control cultures with a significant increase after antigen stimulation in patients. The relative levels of
IFN- produced remained higher than the IL-4 at all antigen concentrations in patients. Hence, no differences in the type of immune
response (Th1) were seen between patients and healthy contacts, as
evaluated by cytokine-specific ELISA for IFN- and IL-4.
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) and contacts (
) in
the absence or presence of various mycobacterial-antigen concentrations
(M1, 0.005 × 106; M2, 0.05 × 106;
M3, 0.5 × 106; and M4, 5 × 106
M. tuberculosis bacilli/ml). Each point denotes the
mean ± the SEM of the estimated cytokine concentrations.
Apoptosis (DNA fragmentation). Experiments were carried out with PBMC of patients and contacts to check for the DNA laddering characteristic of apoptosis. The unstimulated (Fig. 5B, lanes 9 to 11) and stimulated (Fig. 5B, lanes 12 to 14) cultures of contacts showed no DNA fragmentation. In contrast, unstimulated cultures of two patients (Fig. 5A, lanes 3 and 4), along with the stimulated cultures (Fig. 5A, lanes 5 to 8) of all patients did show the laddering of the extracted DNA that is characteristic of apoptosis.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
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Discussion
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The present study shows that the expression of IFN-- and
IL-4-producing cells in CD4+ T cells varied among healthy
contacts and patients. More importantly, changes in patterns of the
percentage of CD4+ T cells expressing IFN- and IL-4 were
observed after in vitro stimulation of PBMC with mycobacteria. Patients
had the higher number of CD4+ T cells expressing IFN-
and IL-4 in unstimulated cultures compared to healthy subjects,
although the ratio of these two subsets was similar in both groups
(Fig. 3 and Table 1). This elevated response in patients, namely,
larger numbers of CD4 T cells producing IFN-, is indicative of an on
going Th1 response. However, upon stimulation with heat-killed M. tuberculosis IFN-+ CD4+ T cells were
selectively and significantly depleted in patients. The ELISA data
confirms the impaired IFN- production by patients after
mycobacterial stimulation. In contrast, the percentage of IL-4-expressing CD4+ T cells held steady in patients, with
relatively little change in the percentage with antigen stimulation.
This trend was seen at all concentrations of mycobacteria used in the
study. Zhang and his colleagues (52) reported similar
results. This selective loss of CD4 T cells producing IFN- in
mycobacterium-stimulated cultures in patients may be significant
considering the impaired cellular response in patients in specific
areas such as lesions and granulomas, where the antigen concentration
may be high. Nevertheless, the patients had the ability to
systematically generate CD4 T cells producing IFN-, although local
factors at the lesional site appear to critically influence the
migration, sensitization, and activation of these and other
immunocompetent cells. The preferential depletion of CD4 T cells
producing IFN- in mycobacterium-stimulated cultures observed in
patients appears to reflect the essential defect in cellular response
at the local site of the infectious foci.
The reasons for the decrease in the IFN--producing cells as a result
of antigen stimulation in patients are not clear. Perhaps the effect is
due to apoptosis induced by hyperstimulation of the effector population
(8). Preliminary tests with PBMC incubated with
mycobacterial antigen resulted in DNA fragmentation in patients (Fig.
4). However, further experiments are being carried out to define the
cell subsets undergoing apoptosis. Recent reports have shown apoptosis
of Th1-like cells in experimental tuberculosis (11, 48, 53).
Data implicating apoptosis as having a primary role in the suppression
of the immune response in tuberculosis were reported by Ellner in 1997 (14). The rapid death of Th1 cells by Fas/FasL-mediated
apoptosis has been shown as a likely mechanism leading to selective
survival of Th2 cells in leishmaniasis (53).
In contrast, the CD4+ T cells in healthy contacts showed
the induction of IFN- with mycobacterial-antigen stimulation. The enhancement indicates the importance of an IFN- response in
imparting immunity to pathogenic mycobacteria. IFN- has been shown
to be involved in inducing protective type 1 responses against M. avium and M. tuberculosis infections in mice (2,
17).
In humans decreased levels of IFN- have been recorded in
tuberculosis patients and in patients with disseminated M. avium complex infection (18, 42, 52). MDR tuberculosis
patients showed an impaired Th1 response (27). Clinical
manifestations of leprosy correlate with host in vivo and in vitro
immune responses to the M. leprae (28). The
recent demonstration of killing of M. tuberculosis by
CD4+ T cells in M. tuberculosis-infected
macrophages in purified protein derivative-positive individuals
(39) and the successful treatment of MDR pulmonary
tuberculosis patients with aerosol IFN- (9) indicates the
critical role played by IFN- in the clearance of pathogenic
mycobacteria. In the murine system, a similar line of protection has
been proved by studies on (i) MHC knockout mice (25), (ii)
the adoptive transfer of immunized CD4+ T cells
(32), (iii) IFN- (10), and (iv) IFN-
receptor knockouts (21).
Though the present study is a one-point study, the data suggest that
suppression of IFN-, along with the maintenance of the steady-state
of IL-4 production, could be responsible for the persistence and
progression of tuberculosis in susceptible individuals. The generation
of inflammatory lesions in IL-4 transgenic mice (44) and the
significant increase in the mean survival time of mice that had been
infected with lethal dose of M. tuberculosis by the
administration of anti-IL-4 monoclonal antibody supports this
possibility (15). Earlier studies based on ELISA and
enzyme-linked immunospot assay also indicated an increase of IL-4
production in patients with pulmonary tuberculosis (36, 41).
If we take into consideration the ELISA data (Fig. 4), the relative
levels of IL-4 production remained less than the total level of
IFN-. However, the dominant biological influence of IL-4 compared to IFN- and other cytokines could influence the initiation of impaired cellular responses seen in patients (19).
In addition to the CD4+ T cells examined in the present
study, IFN- and IL-4 expression has also been evaluated in
CD8+ and T cells. In all categories of individuals
examined the subsets of T cells showed no significant difference
in the percentage of cells expressing IFN- and IL-4. Similar
observations were made with the CD8+ T subset except in the
case of a limited number of patients. In these individuals
CD8+ T cells expressing IFN- were detected (data not
shown). It was seen that a non-T-cell population contributed the bulk
of IFN- only at the lowest antigen concentration in healthy
contacts. The significance of these cells expressing IFN- and their
role in immunity to tuberculosis still needs to be investigated. There have been reports demonstrating the positive role of non-T cells, such
as NK cells, in antimycobacterial host response, (2, 14).
The present study indicates that the preferential loss of
IFN--producing CD4+ T cells occurs in patients upon
coculture with concentrations of M. tuberculosis antigen
exceeding 0.005 × 106/ml. This then results in an
impaired Th1-type response and the maintenance of steady-state of
IL-4-producing T cells. This cellular response does not reflect the
systemic response since the PBMC of patients had a higher percentage of
IFN--producing CD4+ T cells than did healthy contacts.
However, this effect may be of significance in view of the localized
cellular response in granulomatous lesions where the mycobacterial
antigen concentration may be high. The inability to generate a
protective immune response at the lesional site leads to a prolonged
chronic fatal disease in patients. Therefore, study of the
manifestations of cellular responses at the lesional site, such as
factors influencing impairment of Th1-type immune response and the role
of apoptosis in the elimination of immunoreactive cells, could provide
alternate targets for the immunotherapeutic treatment of these individuals.
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ACKNOWLEDGMENTS |
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This work received financial support from the Department of Biotechnology, Government of India.
Satyajit Rath (NII) and S. N. Das (AIIMS) kindly provided flow cytometry facilities. Incisive discussions with S. Rath and J. S. Tyagi, Indira Nath helped us immensely. S. N. Dwivedi and Rajbir Singh of the Department of Biostatistics, AIIMS., helped us in analyzing the data. S.B. is a recipient of a UGC fellowship. Dhanpal Singh provided technical help. Assistance from Biotechnology Information Service and Bhavneet Singh is also acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110 029, India. Phone: 091-011-659-4994. Fax: 091-011-686-2663. E-mail: hkp{at}aiims.ernet.in.
Editor: S. H. E. Kaufmann
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, November 1999, p. 5597-5603, Vol. 67, No. 11
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