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Respiratory syncytial virus (RSV) and adenovirus (Advs) serotype 3 (Adv3) and 7h (Adv7h) are associated with mild to severe respiratory infection and are indistinguishable during the acute phases of
the illnesses. However, outcome and long-term prognosis are different with both infections. RSV infection is associated with later development of asthma, and Adv, mainly Adv7h, with severe lung
damage, bronchiectasis, and hyperlucent lung. We hypothesized that this difference could be partly
due to different immune responses induced by these viruses. To test this hypothesis we quantified TCD4+, TCD8+, and BCD19+ expressing the interleukin-2 receptor-alpha chain (CD25) and interferon-
(IFN-), interleukin (IL)-10, and IL-4 in the supernatant of peripheral blood mononuclear cells (PBMC) from school children infected in vitro with and without RSV, Adv7h, and Adv3 and after phytohemagglutinin (PHA) stimulation in the presence or absence of these viruses at a multiplicity of infection (MOI) of 1. PBMC from every child produced more IL-10 (p 
0.05) when infected with RSV
than with Advs and noninfected control, and Adv induced more (p 0.05) IFN- than did RSV and control. The IL-10/IFN- ratio was significantly higher (p 0.05) in RSV-infected and significantly
lower (p 0.05) in Adv-infected PBMC, than in noninfected cells. PHA-stimulated BCD19+ RSV-
infected cells expressed more (p 0.05) IL-2R than did Adv-infected cells. These results suggest that
Advs induce a Th-1-type immune response that is not seen with RSV. These patterns persist despite
intersubject variation in the absolute quantity of cytokine produced.
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Respiratory tract infections are a common cause of morbidity and mortality in children, mainly in infants. Respiratory syncytial virus (RSV) and adenovirus (Adv) are the most common causes of respiratory infection during the first year of life (1). Respiratory symptoms caused by RSV or Adv vary from an acute upper airways infection, usually in the older children and adults, to a lower respiratory tract disease, wheezing, and/ or bronchopneumonia, more often in the youngest. In young asthmatics, RSV has been shown to have a negative impact on asthma clinical outcomes (2). Respiratory symptoms during infection can be clinically indistinguishable between viruses. However, the outcome and long-term prognosis may be different with both infections. RSV infection is associated with later development of allergic asthma (3) and Adv, particularly Adv7, with chronic lung disease, severe lung damage, bronchiectasis, and hyperlucent lung (4).
The pathogenesis of RSV and Adv and their related immunology-induced diseases are incompletely understood. Host
defense for viruses and intracellular pathogens is produced
by cellular immunity. The combination of cytokines dictates
the development of cellular or humoral immune responses.
T-lymphocytes can be divided into two subsets, Th-1 and Th-2,
on the basis of cytokine profile (8). Th-1 cells predominantly
produce interferon- (IFN-) and interleukin-2 (IL-2) and
promote cell-mediated immunity, whereas Th-2 cells are associated with interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6), all cytokines related to humoral immunity. Interleukin-10 (IL-10), first described as a cytokine produced by Th-2-type cells, now is recognized as an immunosuppressive cytokine for the Th-1 and Th-2 types of immune responses and
produced by T-lymphocytes, monocytes, and other cells. We
recently described a predominant Th-2-type immune response
in RSV-infected infants that could explain the inflammatory
response (9). Patients with Adv infection show lung damage
and deposits of immune complexes containing adenovirus antigen (10). Pathogenicity of adenoviruses varies according to
group and serotypes. From the 42 known serotypes, Adv3 and
Adv 7, belonging to subgenus B, one of the serotypes most frequently isolated in association with respiratory diseases in
children, will induce mild to severe respiratory infection (11).
Recent molecular biologic techniques have identified a genomic variant of serotype 7 (type 7h), which has been the
cause of severe chronic pulmonary disease that is sometimes
fatal. This subtype has been associated with childhood disease
in patients of the south cone of South America (Chile, Argentina, and Uruguay) (12). The immune system has several
mechanisms for counteracting virus infections. However, viruses have in turn a variety of mechanisms that help them
evade the antiviral defenses of the host (15). The final response to viral infections will depend on the type of virus, infected target cells, previous immune condition of the host, and
their genetic immune response. The outcome will be total or
partial immunity with or without damage and with or without
sequelae. We hypothesized that when cells are infected, they
respond in a manner determined by the genetic pattern and the infectious agent. The response will induce immunity or be associated with disease. As previously shown, RSV and Adv
can infect lymphocytes and monocytes from peripheral blood
(16, 17). We used an in vitro model of RSV and Adv infection
of peripheral blood mononuclear cells (PBMC) in order to
compare the response of the mononuclear cells to infection
with both viruses. The aim of this work was to study the IL-4,
IL-10, and IFN- production, the expression of cell activation
markers, and susceptibility to infection of different lymphocyte subpopulations when cells from the same donors were infected in vitro with RSV and two adenovirus serotypes, Adv7h
and Adv3, with the same multiplicity of infection (MOI). This
study focused on the PBMC response to viral infections, leaving for another report the question of PBMC response with different immune conditions of the host.
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Reagents
Sodium metrizoate (Lymphoprep; Nycomed Pharma AS, Oslo, Norway), AIM-V culture medium (Gibco, BRL, New York, NY), and phytohemagglutinin (PHA) were purchased from Sigma (St. Louis, MO). Mouse monoclonal antibody to respiratory syncytial virus and to adenovirus were obtained from the Centers for Disease Control (Atlanta, GA). Monoclonal antibodies (fluorescein isothiocyanate [FITC] or phycoerythrin [PE] conjugated) against CD19, CD4, CD8, CD25, and goat antimouse IgG directly conjugated to fluorescein isothiocyanate were purchased from Becton-Dickinson (Immunocytometry Systems, San Jose, CA). Dynabeads, with affinity for CD4, CD8, and CD19, and detachabeads (Dynal Inc., New York, NY) were used to purify specific lymphocyte populations.
Virus Preparation
RSV long strain and Adv3 and Adv7h were propagated in Hep-2
cells. Titration was carried out inoculating serial dilution of virus
stock on Hep-2 cells and was stored in aliquots at 
70° C until used
(18).
Collection of Blood
Heparinized blood (5 to 10 ml) was collected under sterile conditions from 23 children 5 to 14 yr of age. Patients who presented for routine follow-up were selected from the outpatient clinics at the Hospital Roberto del Río, and they were healthy at the time of entry. Mononuclear cells were separated by a density gradient (sodium metrizoate, Lymphoprep) and counted in a Neubauer chamber, and viability was assessed by trypan blue dye exclusion.
The study was approved by the hospital and the Comisión Nacional de Investigación Científica y Tecnológica (Conicyt) ethical committees.
Culture of Cells and Cytokine Production
PBMC were cultured in flat-bottomed 24-well culture plates (NUNC)
at a concentration of 1 × 106 cells per well in 600 µl of AIM-V medium in the presence or absence of RSV, Adv7h, and Adv3 at an MOI
of 1 for 48 h. Then cells were activated with PHA (5 µg/ml) for a further 24 h at 37° C with 5% CO2. The supernatant obtained at 48 and at
72 h were kept at 20° C prior to IL-4, IFN-, and IL-10 assay.
IFN-, IL-4, and IL-10 Assay
The concentrations of these cytokines were determined in neat mononuclear cell supernatants using the ELISA kit from Endogen. The limit of detectability of these assays was about 2 pg/ml. Samples that registered above the standard curve were diluted and reanalyzed. When values were not detectable, the minimum detectable level was used for analysis.
Activation Markers of Blood Mononuclear Cells
Using a panel of monoclonal reagents to CD3/CD25, CD4/CD25, CD8/CD25, and CD19/CD25, phenotyping was performed before and after viral infection and mitogen activation. The samples were analyzed on the FACScan flow cytometer (Becton-Dickinson) using Lysis software. From each sample 20,000 cells were acquired and the nonspecific staining, assessed by an isotype control, was adjusted to less than 1%. Mononuclear subsets were expressed as a percentage.
Susceptibility of Lymphocyte Subpopulations to Viral Infection
Monocytes were separated by adherence to petri dishes by incubating mononuclear cells 1 h at 37° C. Then lymphocyte subpopulations CD4, CD8, and CD19 were separated by incubating with dynabeads for 1 h at 37° C and then separated by incubating them with detachabeads for 1 h at room temperature. Expression of markers exceeded 95% for CD4 and CD8 and was above 90% for CD19. Cell viability assessment by trypan blue dye exclusion was above 90% for all cell preparation. The different subpopulations were incubated with medium alone or medium containing RSV, Adv3, or Adv7h at an MOI of 1 in 24-well plates at a concentration of 1 × 106 in 0.6 ml of AIM-V medium for 48 h. At the end of the incubation cells were counted in a Neubauer chamber, and viability was assessed by trypan blue dye exclusion.
Detection of Viral Antigens in Infected Cells
Intracellular expression of viral antigens in infected cells was detected
by indirect immunofluorescence test (IFAT). Briefly, mononuclear
cells were permeabilized with 70% ethanol at 20° C, incubated at
least 15 min, and washed twice in phosphate-buffered saline (PBS).
Infected and noninfected cells were incubated with anti-RSV and
anti-Adv antibodies for 1 h at 4° C, washed twice in PBS 2% fetal calf
serum (FCS), and incubated with goat antimouse IgG-FITC for 1 h at
4° C. The cells were washed twice in PBS 2% FCS, centrifuged, and
resuspended at a concentration of 1 × 106/ml in filtered PBS 2% FCS.
A minimum of 20,000 cells were analyzed on a FACScan flow cytometer using Lysys software. Nonspecific staining was assessed by incubating the cells with the second antibody (19). Samples were also analyzed in a fluorescence microscope (Labophot 2; Nikon, Japan).
Statistical Analysis
Data were expressed as medians and 25 to 75 percentile ranges, and
were analyzed by Friedman's repeated measures one-way analysis of
variance (ANOVA) on Ranks and Student-Newman-Keuls multiple comparison testing. Correlations were analyzed by Pearson's product moment correlation test. The results of cell receptors expressed as
percentages were converted to arcsin for statistical analysis. A value
of p 0.05 was accepted as statistically significant for all analyses.
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Cytokine production by RSV-, Adv7h-, and Adv3-infected mononuclear cells and by PHA-stimulated mononuclear cells in the presence or absence of Adv7h, Adv3, and RSV is decribed below.
IL-10 Production
IL-10 (pg/ml) production (expressed as individual values and medians) by mononuclear cells infected with Adv7h, Adv3, and RSV for 48 h is shown in Figure 1 with and without PHA stimulation for 24 h after infection, compared with uninfected cells.
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p 
p 
p The production of IL-10 was significantly higher (p 0.05 each comparison) when cells were infected with RSV (median: 579.5, p25-p75: 295.6 to 1,085.0) compared with Adv7h,
Adv3, and noninfected cells (median: 237.4, p25-p75: 186.6 to
575.9; median: 223.5, p25-p75: 165.0 to 331.8; and median:
138.3, p25-p75: 115.1 to 218.7, respectively). The production
of IL-10 by cells infected with Adv7h and Adv3 were also significantly higher (p 0.05) than that in noninfected cells.
PHA-stimulated cells showed significantly increased (p 0.05) IL-10 production by RSV-infected cells (median: 727.6, p25-p75: 402.4 to 1,005.6) compared with Adv7h, Adv3, and noninfected cells (median: 378.6, p25-p75: 233.0 to 614.9; median: 369.0, p25-p75: 203.4 to 478.0; and median: 219.9, p25-
p75: 164.1 to 298.8, respectively). PHA-stimulated cells infected with Adv7h and Adv3 had a significant increase (p 0.05) in IL-10 production compared with that in noninfected
cells. IL-10 production was not significantly different in PHA-stimulated cells from that in unstimulated cells when they
were infected with RSV and Adv7h and in noninfected cells.
PBMC infected with Adv3 and stimulated with PHA showed a
significantly increased (p 0.05) IL-10 production compared
with that in unstimulated PBMC.
IFN- Production
IFN- production (expressed as individual values and medians) by PBMC infected with Adv7h, Adv3, and RSV, noninfected, and PHA-stimulated for 24 h after infection, is shown
in Figure 2.
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The production of IFN- in cells infected with Adv7h (median: 116.8, p25-p75: 32.7 to 254.5) was significantly higher
(p < 0.05 each comparison) than that in cells infected with
Adv3 (median: 22.9, p25-p75: 4.1 to 53.5), RSV (median: 8.4, p25-p75: 2.4 to 14.6), and noninfected cells (median: 3.5, p25-
p75: 2.5 to 10.8). Adv3-infected cells also had a higher (p 0.05) production of IFN- than did RSV or noninfected cells.
RSV did not induce a significant amount of IFN- when compared with noninfected cells.
When PBMC infected with Adv7h were stimulated with
PHA, they showed significantly increased (p 0.05) IFN-
production (median: 490.2, p25-p75: 165.5 to 705.1) compared
with that produced by Adv3 (median: 168.2, p25-p75: 83.9 to
331.3), RSV (median: 126.7, p25-p75: 48.1 to 306.9), and noninfected cells (median: 168.3, p25-p75: 74.2 to 369.4). No statistically significant differences were found between PHA-stimulated cells infected with Adv3 and noninfected cells. A
significantly (p 0.05) decreased IFN- production was found
after PHA stimulation by RSV-infected cells compared with
noninfected cells and Adv3-infected, PHA-stimulated cells.
IL-4 Production
IL-4 production by RSV, Adv7h, and Adv3 infected and noninfected and PHA-stimulated cells is shown in Table 1. No significant IL-4 production was observed with any of the viruses
when compared with noninfected cells. PHA-stimulated, uninfected cells showed significantly increased (p 0.05) IL-4
production compared with that produced by PHA-stimulated
cells infected by RSV, Adv7h, and Adv3, and by infected and
noninfected unstimulated cells.
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Ratio of IL-10/IFN- Production
The ratio of IL-10/IFN- measurements of cells infected by
RSV, Adv7h, and Adv3 and control cells is shown in Figure 3.
The median of IL-10/IFN- ratio in noninfected cells was 43.3 (p25-75: 22.4 to 63.3). This value went down significantly (p 0.05) to 2.2 (p25-p75: 1.2 to 15.2) and 14.9 (p25-p75: 3.2 to
61.4) in cells infected with Adv7h and Adv3, respectively, and
went up significantly (p 0.05) to 85.3 (p25-p75: 54.7 to
119.8) in RSV-infected cells.
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The IL-10/IFN- ratio in PHA-stimulated cells infected by
Adv7h (median: 1.0, p25-p75: 0.4 to 3.0), Adv3 (median: 1.6, p25-p75: 0.9 to 2.8), RSV (median: 4.0, p25-p75: 2.6 to 20.8),
and noninfected control (median: 1.6, p25-p75: 0.5 to 2.6)
were significantly lower (p 0.05 each comparison) than in
unstimulated cells. The ratios of IL-10/IFN- in PHA-stimulated cells infected by Adv7h and Adv3 were not significantly
different from those in noninfected cells, whereas in RSV-
infected cells the ratio was significantly higher (p 0.05).
Ratio of IL-4/IFN- Production
The ratio of IL-4/IFN- measurements of cells infected by
RSV, Adv7h, and Adv3 and control cells is shown in Figure 4.
The IL-4/IFN- ratio was significantly lower (p 0.05) when
cells were infected with Adv7h (median: 0.036, p25-75: 0.01 to
0.076) and Adv3 (median: 0.18, p25-75: 0.07 to 0.77) than in
noninfected cells (median: 0.71, p25-75: 0.23 to 1.0). The ratio
of IL-4/IFN- in RSV-infected cells (median: 0.43, p25-75:
0.19 to 1.1) was significantly higher (p 0.05) than in Adv7h-infected cells (0.43 versus 0.036). The IL-4/IFN- ratio in
PHA-stimulated cells infected by Adv7h (median: 0.009, p25-
75: 0.006 to 0.02), Adv3 (median: 0.02, p25-75: 0.009 to 0.065),
RSV (median: 0.03, p25-75: 0.009 to 0.067), and noninfected
cells (median: 0.047, p25-75: 0.016 to 0.19) were significantly
lower (p 0.05 each comparison) than in unstimulated cells.
The ratio of IL-4/IFN- in PHA-stimulated cells infected by
Adv7h and Adv3 was significantly lower (p 0.05) than that
from noninfected cells, whereas in RSV-infected cells this ratio was not significantly different. The ratio of IL-4/IFN- in
PHA-stimulated, RSV-infected cells was significantly higher
(p 0.05) than that in PHA-stimulated, Adv7h-infected cells
(0.03 versus 0.009).
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Correlation of IFN- and IL-10 Production in
Cells Infected by Adv7h, Adv3, and RSV
In order to determine whether or not the amount of IFN-
and IL-10 produced by each subject was similar with every
stimuli, we correlated the cytokine responses induced by the
viruses. As shown in Figure 5, a positive correlation (p 0.03)
of IFN- production was seen when cells were infected with
Adv7h and Adv3. No correlation was found when cells were
incubated with RSV and either of the adenoviruses. The production of IL-10 by infected cells was broadly correlated
(Adv7h and Adv3, p 0.001; RSV and Adv7h, p 0.001; and
RSV and Adv3, p 0.002).
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Expression of the Activation Marker
The expression of the IL-2R (CD25) in TCD4+, TCD8+, and
BCD19+ PHA-stimulated lymphocytes expressed as a percentage of the total subpopulation is shown in Figure 6. When
cells were stimulated with PHA in the presence of Adv7h,
Adv3, and RSV, there was a significantly decreased (p < 0.05)
expression of CD25 in TCD4+ and TCD8+ cells. BCD19+
lymphocytes showed significantly increased (p 0.05) expression of CD25 only after incubation with RSV.
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Susceptibility of Lymphocyte Subpopulations
TCD4+, TCD8+, and BCD19+ subpopulations were evaluated for susceptibility to viral infection with Adv7h, Adv3, and
RSV in vitro. All T cells showed equivalent susceptibility to
infection with all three viruses. However, a significantly (p 0.05) increased proportion of BCD19+ cells were infected
with RSV compared with that in Adv3 or Adv7h (Table 2).
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This study demonstrated that when PBMC from the same donor are infected in vitro with three different respiratory viruses, the response of the cells depends on the infectious agent
and on the host characteristics of the donor. This effect was
demonstrated by the differences in cytokine profiles induced
by RSV and Advs, the effect of RSV predominantly on BCD19+
cells, and the individual variability in the amount of cytokine
produced. To infect mononuclear cells from the same donor
with three different viruses, and with the same multiplicity of
infection, allowed us to compare the response of the cells with
each virus. The production of IFN-, IL-4, and IL-10, markers
of the immunologic responses Type-1 and Type-2, were analyzed in the supernatant of cultured cells with each virus. Although intersubject variation was seen in the amount of cytokine production, there was also a clear effect of virus and virus
type. RSV induces predominantly IL-10 and no IFN-, whereas
Advs induce predominantly IFN-. IFN- mediates cellular immunity, which has been classically described as the defense mechanism for viral infections and other intracellular pathogens. We did not find IFN- production in the supernatant of
RSV-infected cells. Although IFN- message has been detected (20, 21) the quantity of translated protein is minimal.
In agreement with other investigators (22), we found that
RSV was a potent inducer of IL-10 from PBMC, but not IL-4,
in this in vitro model of infection. We are not aware of a report on IL-10 or IL-4 induced by Adv infection. It was interesting to find a significant positive correlation in the amount of IL-10 induced by Adv7h, Adv3, and RSV and in the amount of IFN-
induced by Adv7h and Adv3. This observation suggests that
cytokine production was virus-dependent and host-dependent.
We did not find correlation either negative or positive among
IFN- and IL-10 induced by Advs.
IFN- production was greater from cells infected with
Adv7h than from cells infected with Adv3. A number of potential explanations are possible, including transcriptional control, translational control, and secretory control. The genomic
variation of Adv7h from Adv3 is at the level of the E3 region
protein (23), which could be the cause of this difference. The
clinical picture of Adv7h infection is also more severe and sometimes fatal. The higher IFN- production could be related to
severity since it can activate macrophages and monocytes and
induce tumor necrosis factor (TNF)-
secretion, a cytokine
with antiviral function and activities. Further, TNF- can alter
myocardial contractility, induce arterial hypotension, and alter
blood coagulability (disseminated coagulation syndrome with
progressive shock syndrome), as has been described in severe
cases of adenovirus infection (24, 25). When PBMC were
PHA-stimulated, IFN- production was upregulated in noninfected and Adv-infected cells. However, the increase was significantly higher in Adv7h-infected cells and not significant for
Adv3-infected cells when compared with uninfected cells.
When RSV-infected cells were PHA-stimulated, the IFN-
production was significantly downregulated compared with
noninfected cells. These findings are in agreement with the
undetectability of IFN- protein in the supernatants of PHA-stimulated mononuclear cell cultures from infants naturally infected with RSV (9). These findings suggest that RSV may have either a direct suppressive effect on Th-1 lymphoid cells or may act through cytokines with antiproliferative activity
such as IL-1 inhibitor (26). This suppressive effect was also observed in the downregulation of CD25 expression in TCD4+
and TCD8+ lymphocytes when RSV-infected cells were stimulated with PHA. The suppression of PHA-stimulated, RSV-infected T-lymphocytes was selective for IFN- since the
amount of IL-10 induced by RSV was significantly higher than
that induced by Adv-infected and control cells. PHA binds to
many or to all TCR complexes and mimics antigen plus major
histocompatibility (MHC)-induced stimulation of cells, imitating the T-cell response that occurs normally in vivo when antigen-MHC complexes on the surface of APCs or target cells
bind to the TCR. Therefore, we could speculate that in a clinical or subclinical infection with RSV or Advs, the immune response to any other antigen will depend on the infecting virus.
IL-4 production significantly increased only in noninfected PHA-stimulated cells. RSV and Adv-infected cells did not induce IL-4 production, and the three viruses showed a downregulating effect when cells were PHA-stimulated. The decrease of IL-4 production was also seen in the supernatant of mononuclear cell cultures from infants naturally infected with RSV (9).
IL-10 is produced by lymphocytes and monocytes-macrophages. In this study we worked with PBMC, but the percentage of monocytes was not greater than 4%. Thus, it is likely that IL-10 was mainly a lymphocyte cell product.
IFN- has direct antiviral activity; it also has an indirect effect by stimulating the cytolitic activity of natural killer cells
and CD8+ T cells. However, Advs have developed mechanisms to evade the antiviral defenses of the host. Gp19K Adv
protein binds to Class I MHC, preventing its transport out of
the endoplasmic reticulum to the cell surface, avoiding in this
way the cell lysis by adenovirus-specific cytotoxic T-lymphocytes (27). In this work we demonstrated that Adv7h, Adv3,
and RSV decrease the expression of CD25 in TCD4+ and
TCD8+ cells, contributing to the immunosuppression observed in many viral infections. The downregulation of CD25 observed on RSV-infected, PHA-stimulated T-lymphocytes
could explain the RSV inhibition of the PHA proliferative response described in earlier reports (26, 28), and attributed to
IL-1 inhibitor and to IFN-alpha (29). No such effect has been
studied in adenovirus infection, and we cannot rule out the presence of this immunosuppressive factor or others since IL-10 has
also been reported to inhibit human T-cell proliferation (30).
It was interesting to find that CD25 expression on BCD19+ cells was upregulated only in RSV-infected, PHA-stimulated cells. The increase in CD19+/CD25+ could explain the increase in the number of B cells detected in RSV-infected infants (9).
The IL-10/IFN- ratio of RSV-infected cells was significantly higher than those of Adv-infected and noninfected
cells, and the IL4/IFN- ratio was significantly higher than
that of Adv7h-infected cells and not different from that of noninfected cells, suggesting that in vitro RSV infection does not
induce a Th-1-type response. The higher IL-10/IFN- ratio of
RSV-infected cells, compared with Adv-infected and control
cells, could explain the suppressive effect on cellular immune
response observed with this virus. Adenovirus infection induces
a predominant Th-1-type response, with a significantly lower
IL-4/IFN- and IL-10/IFN- ratio than noninfected cells. We
could speculate that the different immunologic response with both respiratory viruses observed in this study could help to explain the outcome and long-term prognosis of RSV and Adv
respiratory infections. We have not definitively shown the
pathogenic mechanism of these viral infections, but the data
we present with this in vitro model offer insight into the biology of PBMC viral infection. We observed that "viral infection" has quantitatively different effects on the biology of lymphocytes depending on the nature of the virus.
In summary, in this study we have shown that PBMC from the same donor have different cytokine profiles after in vitro infection with adenoviruses and RSV. The immune response to Advs but not to RSV, corresponds to that classically described as cell-mediated. These data suggest that to study the immune response evolved by every respiratory virus, is essential to understand the pathogenesis and to develop a proper treatment of distinct respiratory viral infections.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Patricia V. Díaz, Programa Patología ICBM, Fac. de Medicina, Universidad de Chile, Avda. Salvador 486, Casilla 16028, Santiago, Chile.
(Received in original form April 13, 1998 and in revised form April 21, 1999).
Acknowledgments: Supported by Proyecto Fondecyt 195/0647.
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References |
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1. Campbell, H.. 1992. Acute respiratory infections are main killer under 5s. Br. Med. J. 304: 35 .
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Kajon, A., and
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Genome analysis of South American
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