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Infect Immun, January 1998, p. 176-180, Vol. 66, No. 1
Department of Microbiology, National
Institute of Respiratory Diseases, Mexico D.F.,
Mexico,1 and
Department of Medicine,
Case Western Reserve University, Cleveland, Ohio2
Received 25 April 1997/Returned for modification 6 June
1997/Accepted 16 October 1997
Patients with active tuberculosis (TB) have a stronger humoral but
a poorer cellular immune response to the secreted 30-kDa antigen
(Ag) of Mycobacterium tuberculosis than do healthy
household contacts (HHC), who presumably are more protected
against disease. The basis for this observation was studied by
examining the Th1 (interleukin 2 [IL-2] and gamma interferon
[IFN- Tuberculosis (TB) remains an
important world health problem. Each year, approximately 8 million
people worldwide develop active TB and 3 million die from this disease
(20). Despite the severity of this medical problem, however,
mechanisms of protective immunity against Mycobacterium
tuberculosis in humans have not been clarified. In animal models,
a protective immune response against M. tuberculosis depends on the emergence of CD4 T lymphocytes that produce
cytokines which activate macrophages to kill intracellular mycobacteria (25). Gamma interferon (IFN- In mice, live, but not killed, mycobacteria and culture filtrates of
growing mycobacteria induce a protective immune response (37). Thus, secreted Ags are of particular interest as
potential targets of the human protective immune response in TB. The Ag 85 complex is a group of three major extracellular Ags of M. tuberculosis encoded by separate genes and secreted by actively
proliferating cultures (37). Each of these three proteins is
a major secreted product of growing bacilli. Ag 85B is identical to the
previously described Ag 6 or We found that the 30-kDa Ag induces lymphocyte proliferation in cells
from healthy household contacts (HHC), who presumably have a protective
immune response, but does not stimulate blastogenic responses in
lymphocytes from patients with active TB (36). Patients with
TB, however, have a greater serological response to this Ag than HHC do
(36). The 30-kDa Ag and certain of its epitopes directly
stimulate IFN- Study groups.
Seven patients with active pulmonary TB in
radiographically advanced stages were studied. Each of the patients had
acid-fast bacilli in his sputum and a positive sputum culture for
M. tuberculosis. A PPD skin test was positive for six of the
seven patients before treatment. All of the TB patients had a negative
human immunodeficiency virus serologic test. Patients were studied
before treatment and at 4 months after initiation of treatment. Twelve
PPD-skin-test-positive HHC of TB patients were also studied. Each of
these PPD-positive HHC had received Mycobacterium bovis BCG
vaccination as a child. Active pulmonary TB was excluded from these HHC
by chest roentgenogram and sputum smears for acid-fast bacilli. Seven
PPD-skin-test-negative HHC who had received BCG as children were also
studied.
Preparation of 30-kDa Ag.
The 30-kDa Ag was purified from
the H37Ra strain of M. tuberculosis as described previously
(31) and was a gift of Thomas Daniel (Case Western Reserve
University, Cleveland, Ohio). In brief, culture filtrates of H37Ra were
fractionated by DEAE-cellulose ion-exchange chromatography. The
isolated product was identified as a single band by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunoblotting with the
30-kDa-Ag-specific monoclonal antibody TB-C-27 (31).
DNA synthesis (blastogenesis) assay.
PBMC were separated on
Ficoll-Hypaque (Nycomed Pharma, Oslo, Norway) and suspended in growth
medium which consisted of RPMI 1640 (Sigma, St. Louis, Mo.), 10% fetal
calf serum (Gibco BRL, Grand Island, N.Y.), 4 mM
L-glutamine, 25 mM HEPES buffer, and 100 U of penicillin
per ml. A total of 2 × 105 cells/well were cultured
in triplicate with the 30-kDa Ag (2 µg/ml) at 37°C with 5%
CO2. This concentration of 30-kDa Ag was used because
preliminary studies showed that this level stimulated peak responses by
PBMC. After 5 days, the cells were pulsed with [methyl-3H]thymidine (1 µCi/well; specific
activity of 3H, 185 GBq/mmol); 16 h later, the cells
were harvested and [3H]thymidine incorporation was
measured by liquid scintillation spectroscopy. Incorporation of
[3H]thymidine into DNA was expressed as the following
stimulation index (SI): (counts per min [cpm] of triplicate wells
with antigen)/(cpm of the triplicate wells without antigen). An index
of >3 was considered a positive response. The baseline count without
antigen was <1,500 cpm.
IgG assay.
For detection of antibodies against the 30-kDa
Ag, an enzyme-linked immunosorbent assay (ELISA) technique was used as
published previously (30). In brief, round-bottom ELISA
plates (Falcon, Oxnard, Calif.) were coated with the 30-kDa Ag (0.25 µg/well). Serum (diluted 1:75) from study subjects was added in
triplicate to the wells. A second anti-IgG antibody conjugated to
alkaline phosphatase was then added, followed by
p-nitrophenyl as a substrate. The optical density of the
plates was read at 410 nm. For characterization of the different
subclasses of IgG, the plates were coated with the 30-kDa Ag and serum
was added (diluted 1:75). A 1:2,000 dilution of a mouse monoclonal
antibody against either human IgG1, IgG2, IgG3, or IgG4 (Calbiochem, La
Jolla, Calif.) was then added, followed by anti-mouse IgG (1:10,000)
conjugated to biotin (Sigma). Streptavidin peroxidase and the substrate
were then added to each well, and the optical density of the plates was
read at 492 nm.
Immunoassays for cytokines.
PBMC were cultured at
106 cells/well in 24-well plates with or without the 30-kDa
Ag (2 µg/ml). Preliminary studies indicated that the peak production
of IFN- Reverse transcription (RT)-PCR for cytokines.
PBMC
(106 cells/well) were stimulated for 48 h with or
without the 30-kDa Ag. The cells were lysed, and mRNA was extracted by
using RNAzol (Biotecx Laboratories, Houston, Tex.) and chloroform as
described by Chirgwin et al. (5). Any residual DNA was
removed by treatment with DNase 1 (Gibco BRL). Reverse transcriptase
reactions of total RNA were performed with 200 U of the enzyme Moloney
murine leukemia virus reverse transcriptase and the
oligo(dT)12-18 primer (Gibco BRL). Samples were incubated
for 50 min at 42°C in the presence of 25 mM MgCl2 and 2 mM deoxynucleoside triphosphate (dNTP) (Pharmacia, Piscataway, N.J.).
Amplification of cDNA by PCR was performed by using specific primers
for Statistical analysis.
Differences in the responses between
groups were calculated by using analysis of variance (ANOVA) for the
DNA synthesis and antibody assays. For differences between levels of
DNA synthesis before and after treatment in the group of patients with
TB, a nonparametric Kruskal-Wallis ANOVA and a Fridman two-way ANOVA were used. All of the statistical analyses were performed with a
statistical packet for personal computers (SYSTAT: the System for
Statistics, 1990; SYSTAT, Inc., Evanston, Ill.).
Cellular and humoral responses to the 30-kDa Ag.
First, DNA
synthesis was determined in 30-kDa-Ag-stimulated PBMC from patients
with active TB and from PPD skin test-positive HHC without TB (Fig.
1A). Similar to our previous findings
(36), the mean response to the 30-kDa Ag of TB patients was
significantly lower than that of HHC (P < 0.05). In
addition, PBMC from only 1 of 7 TB patients showed a significant
proliferative response to this Ag (SI, >3.0), whereas PBMC from 7 of
12 HHC responded significantly. The response of PBMC from PPD-negative
HHC was minimal, 2,601 ± 814 cpm (mean ± standard error
[SE]; data not shown). Figure 1B also shows the results of the ELISA
used for detection of antibodies against the 30-kDa Ag. An OD of >0.2
was used as the positive cutoff value based on the positive predictive value for this assay determined in previous studies of patients with TB
and other diseases (30). The OD for 30-kDa-Ag-specific antibodies in the sera of PPD-negative HHC was 0.2 (SE, 0.17) (data not
shown). Each of the seven patients with active TB had antibodies
against the 30-kDa Ag. In addition, the mean 30-kDa-Ag-specific antibody level in sera from TB patients was significantly higher than
that in sera from HHC (P < 0.001). Thus, in these
subjects, consistent with previous results, the serologic response to
the 30-kDa Ag, but not the blastogenic response of PBMC, was higher in
TB patients than in HHC (36).
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cytokine Profiles for Peripheral Blood Lymphocytes from
Patients with Active Pulmonary Tuberculosis and Healthy
Household Contacts in Response to the 30-Kilodalton Antigen of
Mycobacterium tuberculosis
![]()
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
])- and Th2 (IL-10 and IL-4)-type cytokines produced in
response to the 30-kDa Ag by peripheral blood mononuclear cells (PBMC)
from patients with active pulmonary TB (n = 7) and
from HHC who were tuberculin (purified protein derivative) skin test
positive (n = 12). Thirty-kilodalton-Ag-stimulated PBMC from TB patients produced significantly lower levels of IFN-
(none detectable) than did those from HHC (212 ± 73 pg/ml,
mean ± standard error) (P < 0.001). Likewise,
30-kDa-Ag-stimulated PBMC from TB patients failed to express IFN-
mRNA by reverse transcription-PCR, whereas cells from HHC expressed the
IFN-
gene. In contrast, 30-kDa-Ag-stimulated PBMC from TB patients produced significantly higher levels of IL-10 (403 ± 80 pg/ml) than did those from HHC (187 ± 66 pg/ml) (P < 0.013), although cells from both groups expressed the IL-10 gene. IL-2
and IL-4 were not consistently produced, and their genes were not
expressed by 30-kDa-Ag-stimulated cells from either TB patients or HHC. After treatment with antituberculous drugs, lymphocytes from four of
the seven TB patients proliferated and three of them expressed IFN-
mRNA in response to the 30-kDa Ag and produced decreased levels of
IL-10.
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) is an essential protective
cytokine in mice (1, 6, 10). IFN-
peaks when
protective immunity is maximally expressed and is produced by CD4
lymphocytes when these cells are in contact with macrophages previously
infected by live mycobacteria or primed with secreted mycobacterial
antigens (Ags) (1). Mice which fail to produce IFN-
because of disruption of its gene develop a fatal tuberculous infection
upon intravenous or aerogenic challenge (6, 10).
Ag and is now designated the 30-kDa Ag
of M. tuberculosis. The 30-kDa Ag induces protective
immunity against TB in guinea pigs (13).
production by T cells from tuberculin purified
protein derivative (PPD)-positive donors (35). Together, these results suggest that the 30-kDa Ag may stimulate Th1-type protective cytokine responses in HHC but not in TB patients. The pattern of cytokines produced by 30-kDa-Ag-stimulated lymphocytes from
HHC as compared with that produced by patients with active TB, however,
is not known. In this study, selected Th1 (IFN-
and interleukin 2 [IL-2])- and Th2 (IL-4 and IL-10)-type cytokine responses
(22) to the 30-kDa Ag were examined in peripheral blood
mononuclear cells (PBMC) from patients with active pulmonary TB and
from HHC. The distribution of immunoglobulin G (IgG) subclasses in
antibodies to the 30-kDa Ag in patients with TB also was studied because IgG1 is associated with a Th2 response and IgG2 is associated with a Th1 response in other systems. Results show no differences between levels of IL-2 and IL-4 production by stimulated cells from TB
patients and from HHC. No predominant IgG1 or IgG2 subclass was found
in the sera of TB patients. Lymphocytes from TB patients, however,
showed decreased 30-kDa-Ag-stimulated blastogenesis and production of
IFN-
and increased IL-10 compared to cells from HHC. After 4 months
of antituberculous therapy, production and expression of these
cytokines were also evaluated.
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, IL-4, and IL-10 was at 48 h after stimulation. IL-2
production peaked at 24 h and was comparable to this peak level at
48 h. Therefore, supernatants were collected routinely at 48 h after initiation of culture. IFN-
, IL-2, IL-4, and IL-10 levels in
culture supernatants were determined by ELISA with commercial kits for
human cytokines (Endogen [Cambridge, Mass.] for IFN-
, IL-4, and
IL-2; R&D [Minneapolis, Minn.] for IL-10). The cytokine sensitivities
for these assays were as follows: IFN-
, 2 pg/ml; IL-2, 31.3 pg/ml;
IL-4, 7.8 pg/ml; IL-10, 3 pg/ml.
actin, IL-4, IFN-
, and IL-10 deduced from published
sequences (39) and with the following conditions: 2.5 mM
MgCl2, 0.2 mM dNTP, 200 nM 5' and 3' primers, and 1 U of
Taq DNA polymerase (Gibco BRL). A DNA thermocycler 480 (Perkin-Elmer Cetus, Norwalk, Conn.) was used to amplify DNA for 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 1.5 min for both IFN-
and IL-10.
The same conditions were used for IL-4 except that annealing was
performed at 65°C. The PCR products were electrophoresed on 2%
agarose gels and detected by ethidium bromide staining.
![]()
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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FIG. 1.
Blastogenic (A) and serologic (B) responses to the
30-kDa Ag in TB patients and HHC. (A) PBMC from patients with active TB
and HHC that were PPD skin test positive were stimulated with the
30-kDa Ag (2 µg/ml) for 6 days, and incorporation of
[3H]thymidine was measured. Results are expressed as SIs.
An SI of >3 was considered a positive response. The baseline counts
without Ag were 660 ± 398 cpm for TB patients and 678 ± 271 cpm for HHC. (B) ELISA was performed on serum samples from TB patients
and PPD-positive HHC for reactivity to the 30-kDa Ag. Results are
expressed in OD units. An OD of >0.2 was considered a positive
result.
|
Induction of cytokines by the 30-kDa Ag in TB patients and
HHC.
Production of IFN-
, IL-2, IL-4, and IL-10 by PBMC in
response to the 30-kDa Ag was determined by ELISA. Neither unstimulated cells from TB patients nor those from PPD-positive HHC produced detectable levels of any of the cytokines measured (data not shown). IL-4 was produced by 30-kDa-Ag-stimulated PBMC from only one of the
patients with active TB (15 pg/ml) and was not detectable in cell
supernatants from any of the HHC. IL-2 was produced by 30-kDa-Ag-stimulated PBMC from 3 of 6 TB patients (131 ± 30 pg/ml [mean ± SE]) and from 1 of 12 PPD-positive HHC (198 pg/ml).
Overall, there were no significant differences in the production of
IL-4 and IL-2 by stimulated cells from TB patients and HHC. The results for IFN-
and IL-10 production are shown in Fig.
3. The mean concentration of IFN-
in
supernatants of 30-kDa-Ag-stimulated PBMC from TB patients was
significantly lower than that of HHC (P < 0.01). In
addition, there was no detectable IFN-
produced by stimulated cells
from any of the TB patients, but stimulated cells from 7 of 12 HHC
produced detectable levels (212 ± 73 pg/ml; range, 6 to 535 pg/ml). Stimulated PBMC from PPD-negative HHC produced a low level of
IFN-
(31 ± 6 pg/ml; data not shown). In contrast, PBMC from
all patients with active TB produced IL-10 after stimulation with the
30-kDa Ag (403 ± 80 pg/ml), and mean levels of this group were
significantly higher than those of PPD-positive HHC (P < 0.005), of whom only 4 of 12 produced a detectable level of IL-10 (187 ± 66 pg/ml).
|
Steady-state expression of cytokine mRNA in TB patients and
HHC.
Unstimulated PBMC from the TB patients and HHC did not
express IFN-
or IL-4 mRNA as determined by RT-PCR. The IFN-
gene was not expressed by 30-kDa-Ag-stimulated cells from any of the patients with active TB. In contrast, stimulated cells from each of the
12 HHC expressed IFN-
mRNA. A representative experiment is shown in
Fig. 4. IFN-
mRNA was expressed by
30-kDa-Ag-stimulated cells from two of seven PPD-negative HHC (data not
shown). IL-4 was not expressed by the 30-kDa-Ag-stimulated cells from
any of the HHC and was expressed by cells from only 1 of the TB
patients (data not shown). Thirty-kilodalton-Ag-stimulated cells from
both TB patients and HHC expressed IL-10 mRNA.
|
Production of cytokines after antituberculous therapy.
Of the
original seven patients with active tuberculosis, follow-up data are
reported for six. Blastogenic responses of 30-kDa-Ag-stimulated PBMC
from TB patients were increased 4.5-fold (range, 1.5- to 7.5-fold;
P < 0.025) after 4 months of treatment as compared to responses observed at initiation of therapy (Table
1). Thirty-kilodalton-Ag-stimulated cells
from three of six patients studied also expressed IFN-
mRNA as
determined by RT-PCR after 4 months of treatment (data not shown). PBMC
from two of these three patients expressing IFN-
mRNA also secreted
IFN-
, although at very low levels (4 and 17 pg/ml); these two
patients did not produce detectable IFN-
before treatment (Fig. 3).
PBMC from three patients with a negative RT-PCR also did not produce
detectable IFN-
in the culture supernatants. IL-10 production by
30-kDa-Ag-stimulated PBMC from patients with active TB was decreased
after 4 months of treatment (403 ± 80 pg/ml before treatment
versus 146 ± 56 pg/ml after treatment; P = 0.013).
|
| |
DISCUSSION |
|---|
|
|
|---|
This study focused on the immune response to the 30-kDa Ag of
M. tuberculosis by PBMC from patients with active TB and by PBMC from HHC. As we found previously (36), patients with
active TB had strong humoral and weak cellular proliferative responses to the 30-kDa Ag, whereas, inversely, HHC had weak humoral and strong
cellular responses. These results are consistent with those of Havlir
et al. (12). To define the specific profile of cytokines produced in response to the 30-kDa Ag, IFN-
and IL-2 production levels were examined as representative of Th1 responses and those of
IL-4 and IL-10 were examined as representative of Th2 responses (33, 39). Results show that 30-kDa-Ag-stimulated PBMC from TB patients fail to produce IFN-
but produce high levels of IL-10. These results contrasted with the cytokine responses of cells from HHC
which produced high levels of IFN-
and low levels of IL-10. IL-4 and
IL-2 were not produced consistently by stimulated PBMC from either TB
patients or HHC, and responses were not different between the two
groups.
There is unequivocal evidence that IFN-
is a protective cytokine in
animal models of TB (6, 10). The role of IFN-
in protection against TB in humans is less certain (7, 29). IFN-
production by PBMC from patients with active pulmonary TB is,
however, clearly decreased in response to PPD or M. tuberculosis, suggesting a relationship between low IFN-
levels
and lack of protection (26, 40). In contrast, lymphocytes
obtained from the pleural fluid of patients with TB pleurisy produce
IFN-
after being cultivated with the Erdman strain of M. tuberculosis (2). Since patients with active pleural TB
have localized disease, it was proposed that IFN-
confers protection
in this clinical situation (2).
Our results further show a failure of PBMC from TB patients to produce
IFN-
in response to the 30-kDa Ag and a strong response to this
antigen by cells from HHC, suggesting a protective role of IFN-
in
these HHC. Others have shown a decreased IFN-
response to the 32-kDa
Ag in TB patients (17), but ours is the first to
concurrently show high IFN-
levels produced by cells from HHC to the
30-kDa Ag. Boesen et al. (3) and Launois et al. (21), however, found that M. tuberculosis Ag
obtained by culture filtrate or the 85A Ag induces production of
IFN-
in TB patients. An explanation for these differences in results
may be the extent of disease: our TB patients had advanced disease and
were studied before receiving treatment, whereas the patients of the
study of Boesen et al. had minimal disease and those of the study of Launois et al. received treatment before the study. In fact, the patients with advanced disease studied by Boesen et al. also had decreased production of IFN-
similar to our results (3).
The decrease in production of IFN-
during TB might be related to
lack of production of IL-12, which induces a Th1 response (32). Another possibility is that IFN-
-producing cells
may be compartmentalized to the lung during TB. Robinson et al.
(28) demonstrated by in situ hybridization that cells
obtained by bronchoalveolar lavage from patients with TB express the
IFN-
gene, which supports the possibility that IFN-
-producing
lymphocytes are sequestered in the lung during disease.
Orme et al. (24) found that in mice infected with M. tuberculosis, IFN-
is produced initially and IL-4 production
follows later. In the current study, IL-4 was produced by stimulated
PBMC from neither HHC nor TB patients. These results are consistent with the human studies of others (2, 40). Therefore, the role of IL-4 in human TB is not clear.
IL-10 is a potent suppressor of IFN-
synthesis by helper T cells
(9) and by NK cells (15) and inhibits antigen
presentation to Th1 cells (14). In leprosy, IL-10 inhibits
T-cell responses as well as release of IFN-
(34). It is
possible, therefore, that the increase in IL-10 production by PBMC from
TB patients in our study might be responsible for both the decreased
blastogenic response to the 30-kDa Ag during active tuberculosis and
the decrease in the production of IFN-
. Our finding of high IL-10
production is of particular interest because, recently, Murray et al.
(23) have shown that in transgenic mice, secretion of IL-10
by T cells induces a negative effect on BCG infection through
antagonism of macrophage function.
It is of interest that treatment of patients with active TB changes the
pattern of the immune response to the 30-kDa Ag in some of them.
Lymphocytes from four of seven subjects unable to proliferate before
treatment proliferated after treatment. In addition, cells from three
of these four patients also expressed the IFN-
gene and two of them
produced low levels of IFN-
after treatment. Our results are in
agreement with those of Carlucci et al. (4), who also
observed that PBMC from some but not all patients with TB proliferate
in response to different mycobacterial antigens after treatment
(4), and those of Wilkinson et al. (38), who
showed that the production of IFN-
increases during treatment of TB
patients in response to the 16- and 38-kDa Ags of M. tuberculosis. Thus, it can be hypothesized that the initial failure to produce IFN-
is a transitory phenomenon in some patients and possibly genetically related in others in whom no change is observed after treatment. We also demonstrated that there was a
decrease in IL-10 production in cells from all of the TB patients after
treatment. Thus, these changes in IFN-
production and decreases in
IL-10 production suggest that there may be a modification of cytokine
expression during different stages of TB. We can speculate that these
changes are related to the antigenic load, which decreases after
treatment.
BCG has not been demonstrated convincingly to prevent TB
(8), and there are no other alternative effective vaccines.
The search for purified mycobacterial Ags useful for vaccines has thus
been extensive (11, 27). In animal models, secreted Ags of
mycobacteria induce a protective immune response (1). The 30-kDa Ag is a major secreted protein of M. tuberculosis
(11). In mice, the 85A Ag and some peptides of this Ag
induce the production of IFN-
(16), and in guinea pigs,
the 30-kDa Ag confers protection against TB (13). Epitope
mapping of the 30-kDa Ag has elucidated distinct peptides of this
protein that stimulate human T cells, suggesting specific sites that
may induce protection (35). A recent study of mice by Huygen
et al. demonstrated that a DNA vaccine encoding the 85A and 85B Ags of
the Ag 85 complex (including the 30-kDa Ag) induced a Th1
response (tumornecrosis factor, IFN-
, and IL-2), cytotoxic activity
against purified native 85 Ag, and protection against challenge with
BCG (18, 19). Our findings that HHC presumably
protected against TB, but not patients with active TB, produce IFN-
in response to the 30-kDa Ag further suggest that the 30-kDa Ag is a
target of the human protective immune response. The basis for such
protection is speculative but likely involves secretion of this protein
by M. tuberculosis early during active intracellular
infection. Lymphocytes previously sensitized against this Ag then may
start to produce IFN-
, which, in turn, may activate macrophages to
kill mycobacteria by various intermediates and efficiently prevent the
development of active TB.
| |
ACKNOWLEDGMENTS |
|---|
We thank Luis Llorente for his advice on the setup of PCR assays and Rogelio Perez Padilla for help in performing the statistical analyses.
This work was supported by grants from CONACYT Mexico (0628-M9108) and the National Institutes of Health, Bethesda, Md. (HL51630).
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Departamento de Microbiologia, Instituto Nacional de Enfermedades Respiratorias, Calzada de Tlalpan 4502, Mexico D.F. 14080, Mexico. Phone: (525) 666-4539, ext. 117. Fax: (525) 666-6172. E-mail: 103144.566{at}compuserve.com.
Editor: S. H. E. Kaufmann
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