 Expression in Peripheral
Blood Mononuclear Cells of Infants with Severe
Respiratory Syncytial Virus Disease
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INTRODUCTION
METHODS
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We examined the in vivo cell-mediated immune response in infants with respiratory syncytial virus
(RSV) infection in order to gain information about the pathogenesis of severe RSV disease in infancy.
Semiquantitative reverse transcription-polymerase chain reaction and three-color flow cytometry
were used to determine the levels of messenger RNA (mRNA) for interferon (IFN)-
in peripheral blood mononuclear cells, and the distribution of lymphocyte subsets in infants with acute RSV infection. The findings were correlated with the severity of the patients' illness and the production of
RSV-specific IgE antibodies (RSV-IgE). Significantly lower IFN- levels and T-lymphocyte counts in the
acute phase of illness were observed in infants with severe RSV disease than in those with a milder
clinical course of illness. The induction of RSV-IgE was not related to IFN- levels in the acute phase
of illness, but rather correlated with IFN- expression during convalescence. The data indicate that
reduced IFN- expression may be an important factor in the pathogenesis of severe RSV disease in infancy.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Respiratory syncytial virus (RSV) is a major cause of acute
respiratory illness in infants and adults. The clinical presentation of such illness can be that of a common cold, but progression to lower respiratory symptoms frequently occurs in infancy, with bronchiolitis being the most common clinical
presentation (1). This is particularly true for infants infected
with RSV during the first 6 mo of life. In addition, several authors have described long-term sequelae in infants with RSV
bronchiolitis (2). In prospective studies, as many as 75% to
90% of infants with a clinical diagnosis of bronchiolitis subsequently develop recurrent episodes of wheezing suggestive of
childhood asthma (3). Why some of these infants become so
severely ill remains poorly understood, but it appears likely
that an inadequate immune response is involved in determining the severity of illness in RSV infection (4, 5). Clinical and
experimental evidence suggests an important role for cell-
mediated immunity in RSV infection (6). Besides its importance in the resolution of infection, the immune response has
been implicated in the pathogenesis of severe RSV disease. Proposed mechanisms for this include IgE-mediated type 1 hypersensitivity, which initiates mechanisms such as mast-cell
activation and the release of histamine or other soluble factors
that promote inflammation and bronchospasm (7, 8). The regulation of IgE production in antigen-specific immune responses
is ultimately determined by the relative balance between antigen-specific T-helper (Th)-cell-derived cytokines. Interleukin
(IL)-4 (produced by Th2 cells) activates B cells to produce IgE
(9), whereas interferon (IFN)- (produced by Th1 cells) downregulates this effect (10).
In addition to its role in the regulation of IgE antibody production, IFN- is a key endogenous cytokine in initiating the defense against viral infection. This cytokine has a direct antiviral effect and is particularly important in stimulating the cytolytic activity of natural killer (NK) cells and CD8+ cytotoxic
T lymphocytes (CTL). The latter have been shown to be of
great importance in the control of RSV infection in a murine model (11), but may cause illness themselves if present in
excess (14). Apparently, it is important that CTL effectively
clear the virus before the antigen load becomes too high (15).
It is therefore conceivable that the level of IFN- produced
early in the course of RSV infection may affect the clinical expression of illness. The aim of the present study was to compare both the levels of IFN- messenger RNA (mRNA) expression in peripheral blood mononuclear cells (PBMC) and
the distribution of lymphocyte subpopulations in infants with
severe RSV bronchiolitis and those with a milder clinical
course of illness. The results were that severe RSV disease was
associated with reduced levels of IFN- expression and with
significantly lower T lymphocyte counts in the acute phase of
illness. These findings suggest that weak IFN- production by
PBMC early in the course of RSV infection may be associated
with the development of severe RSV disease, and may also
help to explain at least some of the unique features seen in infants with RSV bronchiolitis and its sequelae.
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Patients
We studied 20 infants (aged 3 to 44 wk), hospitalized with acute lower
respiratory tract illness due to RSV. None of these infants had suspected congenital heart disease or bronchopulmonary dysplasia. A
summary of the subject characteristics is given in Table 1. The patients
were divided into two groups according to the severity of their illness.
Oxygen saturation (SaO2) was monitored continuously through 24-h
percutaneous oximetry, as part of the routine care of these patients.
The lowest values (minimal oxygen saturation, MOS) measured with
the patient breathing room air were used as an objective indication of
the severity of illness. Group 1 consisted of infants who did not require supplemental oxygen (MOS 
93%), whereas all infants in
Group 2 required an oxygen supply, had bronchiolitis with MOS 
92% for at least 30 min or MOS 90% for 15 min, and were therefore
considered severely ill. Infants in Group 2 also differed from those of
Group 1 with respect to the total number of days of their hospital stay
(mean duration of hospital stay for Groups 2 and 1: 7.7 d versus 4.0 d,
respectively).
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Heparinized blood, drawn as part of routine serologic investigation, and a nasopharyngeal aspirate were obtained from each infant within 24 h after admission to the hospital (mean day of illness: 4.6; range: Days 3 to 8), and a second blood sample (convalescent phase sample) was drawn within 11 d thereafter (mean: 10.3 d, range: 6 to 14 d), when the patient was considered healthy enough to be discharged from the hospital. A complete blood count, including an automated differential count, was done on each patient sample. Because of the small amount of blood that was obtained, we were unable to perform all of the tests for every child.
Heparinized blood samples obtained from six healthy infants
(mean age: 12.5 ± 3 wk; range: 1 to 22 wk), hospitalized for minor surgery, were included as controls to evaluate the constitutive expression
of IFN- mRNA in PBMC.
The study was approved by the Ethics Committee of the St. Anna Kinderspital, and informed consent was obtained from the parents of all participating children.
Confirmation of RSV Infection
Nasopharyngeal aspirates were diluted 1:3 in RPMI 1640 medium (GIBCO, Grand Island, NY) and were screened for the presence of RSV by virus isolation from tissue culture and enzyme-linked immunosorbent assay, as previously described in detail (16, 17).
Lymphocyte Subset Identification by Flow Cytometry and Quantification
PBMC were separated by density gradient centrifugation in Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden) and were washed twice in phosphate-buffered saline containing 2% fetal calf serum (GIBCO). Three-color flow-cytometric immunophenotyping of PBMC was done with matched combinations of murine monoclonal antibodies (MAb) directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or R-phycoerythrin-cyanine-5 (PE-CY5). For immunofluorescence labeling, 1 × 105 cells per analysis were incubated for 30 min in the dark at 4° C with the conjugated antibodies. We used the following antibodies to human lymphocyte cell-surface antigens: anti-CD19 (HD37-PE), anti-CD16 (DJ130c-FITC), anti-CD4 (MT310-PE), anti-CD8 (DK25-FITC), anti-CD45R0 (UCHL1-FITC), anti-CD45RA (4KB5-PE), all from Dako (Carpinteria, CA); anti-CD3 (UCHT-1-PE-CY5) and anti-CD4 (13B8.2-PE-CY5), all from Coulter Immunology (Krefeld, Germany); and anti-CD25 (a-IL-2R-PE) from Becton Dickinson (Heidelberg, Germany). To exclude false positivity, each experiment involved FITC-conjugated IgG1 and PE-conjugated IgG2a (Dako) for the determination of nonspecific binding. Cell fluorescence was analyzed with a FACScan flow cytometer, using Paint-a-Gate software (Becton Dickinson, Mountain View, CA).
T lymphocytes were identified through their CD3 expression, and
were further subdivided according to the presence of CD4 and CD8.
B cells were identified through CD19, and NK cells were defined by
the presence of CD16 on CD3
lymphoid cells. The sum of the percentages (on an event basis) of T, B, and NK cells was equated to
100%. On this basis, we recalculated the absolute cell counts as the
product of the white blood cell count and the lymphocyte percentage
derived from the hematology count, and the percentages of individual
lymphocyte types obtained by flow cytometry (18).
Determination of RSV-specific IgE Antibodies
RSV-IgE antibodies were quantitated by immunoblotting as described previously (17). In brief, a semipurified RSV (strain Long) grown in Hep-2 cells was used as antigen. Sera diluted 1:10 were tested simultanously on strips with virus-specific proteins and on strips prepared from mock-infected Hep-2 cells. All incubation steps took place at room temperature. Biotinylated mouse monoclonal antibodies (mAbs) against IgE (Southern Biotechnology Associates, Birmingham, AL) were used as conjugate. Membrane-bound biotinylated antibodies were visualized with a blotting detection kit (Amersham International, Amersham, UK). Molecular weights of the proteins recognized by antibodies were determined by densitometric comparison with coelectrophoresed biotinylated standard proteins of known molecular weights. Rabbit pre- and RSV-immune sera, a human convalescent serum (RSV complement-fixing titer 1:128), and a mouse mAb directed against the 35-kD protein of RSV (provided by C. Orwell, of the Department of Virology, Karolinska Institute, Stockholm, Sweden) were included as controls.
Analysis of IFN- mRNA Expression with Reverse
Transcription-Polymerase Chain Reaction
RNA was extracted from 2 × 105 cryopreserved PBMC with RNAzol B (Molecular Research Center, Inc., Cincinnati, OH) and phenol/chloroform extraction, with subsequent isopropanol precipitation, as described previously (19). The RNA pellet was washed once in 70% ethanol, was dried, and was resuspended in ribonuclease-free double-distilled water.
Reverse transcription (RT) of mRNA was done in the presence of 4 µl polydeoxythymidine12-18 as a primer (1 mg/ml; Pharmacia Biotechnology), 400 U Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD), 20 U RNAse inhibitor, 4 µl 10 mM deoxynucleotide triphosphates (dNTPs), 8 µl 5× RT buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2) at 37° C for 60 min, after which an additional 200 U of reverse transcriptase was added and incubated for another 40 min at 37° C. This was followed by a 5-min incubation at 90° C to inactivate the reverse transcriptase.
To minimize variability in the efficiency of RT-polymerase
chain reaction (PCR), we normalized the amount of IFN- mRNA to the amount expressed of mRNA for the internal housekeeping gene 
-actin. Serial 4-fold dilutions of each complementary DNA (cDNA) product were amplified and tested for IFN- mRNA and -actin
mRNA. The final reaction volume was 50 µl, and contained 8 µl 10×
PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2 mM MgCl2), 200 µM dNTPs, 2.5 U of AmpliTaq polymerase (Perkin Elmer-Cetus, Norwalk, CT), and 50 pmol each of the following specific primers, as described previously (7): 5'-AGTTATATCTTGGCTTTTCA-3' (IFN-1);
5'-ACCGAATAATTAGTCAGCTT-3' (IFN-2); and 5'-TGAAGTCTGACGTGGACATC-3', and 5'-ACTCGTCATACTCCTGCTTG-3' (-actin). To prevent erroneous results from cross-contamination, several negative controls were included in each run. Seminested PCR
was performed by adding 2 µl of the amplicon to 8 µl of sterile water
and 40 µl of the reaction mixture described earlier, except that the
appropriate oligonucleotide primers, which were 5'-CCAGAGCATCCAAAAGAGTG-3' (IFN-3) and the IFN-1 primer, were included. The thermocycling procedure for the first-step (seminested)
PCR consisted of 40 amplification cycles (denaturation at 95° C for
1 min, primer annealing at 50° C for 2 min, and extension at 72° C for
2 min). Ten microliters of PCR-amplified products were run on a 3%
Nusieve (FMC BioProducts, Rockland, ME) gel and identified by
ethidium bromide staining. The identity of the obtained fragment was
confirmed by automated direct nucleotide sequence analysis of the
PCR fragments (373A Sequencer; Perkin Elmer).
Endpoint titers of -actin mRNA and IFN- mRNA were compared, and IFN- values were expressed as the ratio of the IFN-
mRNA titer to the -actin mRNA titer ×1,000. The validity of quantitation of mRNA with RT-PCR was tested by amplifying 4-fold serial
dilutions of RNA, obtained from PBMC of normal healthy adults,
with IFN- and -actin-specific primers. Interassay variation was < 10% in three PCR reactions run with PBMC obtained on three different days.
Statistical Analysis
The Mann-Whitney U test was used for intergroup comparison of cell
counts and for comparison of IFN- mRNA levels in patient groups
and controls. Spearman's correlation coefficient was computed to determine the degree of correlation between IFN- mRNA levels and
cell counts, between IFN- mRNA levels and patient age, and between IFN- mRNA levels and the production of RSV-IgE, respectively. Wilcoxon's signed ranks test was used to compare values for
the same group of infants tested during the acute phase of illness with
those in the convalescent phase.
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METHODS
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DISCUSSION
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IFN- mRNA Expression
Infants with confirmed RSV infection were divided into two
groups according to the severity of their illness (Table 1), as described in METHODS. When the levels of IFN- mRNA in
PBMC from infants with moderate illness (Group 1) were
compared with those of infants with severe disease (Group 2),
we found that moderate illness was associated with significantly higher IFN- mRNA expression in the acute phase of
illness (p = 0.01) (Figure 1). This difference was not due to a
difference in the patients' ages, because the mean age of both
groups was quite similar (age: 16.1 ± 2.8 wk [mean ± SEM]
[Group 1] versus 17.3 ± 5.1 wk [Group 2]), and because no
correlation was observed between IFN- mRNA levels and
patient age (r = 0.07, p = 0.7). In contrast to the findings obtained in the acute phase of illness, no difference in levels of
IFN- mRNA expression of infants in Group 1 and 2 was observed during convalescence (Figure 1). IFN- mRNA levels
of individual patients are shown in Table 2. When the IFN-
levels of the two patient groups were compared with the levels
of IFN- constitutively expressed in PBMC of six healthy, age-matched controls (Figure 1), a specific increase in IFN- levels
was observed only in infants in Group 1 in the acute phase of
illness (p < 0.008); levels of IFN- in infants of Group 2 did
not differ significantly from those of the healthy controls.
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Reduced IFN- expression may derive from a lower proportion of previously primed "memory" (CD45R0+) Th cells,
which, in contrast to "naive" (CD45RA+) Th cells, produce
large amounts of IFN- upon activation (20). To address this
question, we determined the number of Th memory (CD4+
CD45R0+/CD45RA) cells in the acute phase of RSV disease
(Table 2). Numbers of Th memory cells/mm3 did not differ significantly between infants with moderate and those with severe illness (mean ± SEM: 294 ± 31.8 [Group 1] versus 220 ± 26.5 [Group 2]). In addition, no correlation was observed between the number of T memory cells and levels of IFN-
mRNA (data not shown).
Lymphocyte Subset Response
Because PBMC of infants with severe RSV disease expressed
significantly lower levels of IFN- mRNA than did those of infants with moderate illness, we next examined whether there
was a specific pattern of lymphocyte subset response related
to disease severity. Although no significant difference between the two groups was observed in B (CD19+) cells and
NK (CD16+) cells (data not shown), infants with severe RSV
disease (Group 2) exhibited significantly lower T (CD3+) lymphocyte counts in the acute phase of illness than did those in
Group 1 (mean ± SEM: 4,495 ± 444/mm3, Group 1, versus
2,297 ± 253/mm3, Group 2; p < 0.008). The lower frequency of
T (CD3+) cells in severely affected infants was related both to
lower CD4+ Th cell counts and to somewhat lower CD8+ T
cell counts (CD4+, p = 0.01, Group 1 versus Group 2; CD8+,
p = 0.05, Group 1 versus Group 2) (Figures 2A and 2B). Since both the CD4+ and CD8+ subsets of T (CD3+) lymphocytes
were reduced in infants with severe disease, no difference between the two RSV groups of infants was observed when comparing their ratios of CD4+ to CD8+ T cells (data not shown).
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The number of CD4+ and CD8+ T lymphocytes expressing early activation antigen (CD25: IL-2 receptor pp 55 antigen), a marker typical of lymphokine-secreting cells, was also determined. As illustrated in Figures 2C and 2D, both, CD4+ and CD8+ T cells expressing the activation marker CD25 were significantly fewer in number in infants with severe RSV disease (CD4+/CD25+, p < 0.001; CD8+/CD25+, p < 0.04; Group 1 versus Group 2, respectively). Again, no difference between the two groups was observed in the convalescent phase.
To investigate whether the differences in IFN- mRNA
levels in the acute and convalescent phases of illness resulted
from differences in the number of certain cell subsets (e.g.,
CD4+ T cells, CD8+ T cells, or NK cells), in the PBMC samples we investigated, we correlated the proportions of T cells
and NK cells with IFN- mRNA levels; no correlation was observed between IFN- levels and the values of any of the lymphocyte subsets (r = 0.3, p = 0.3, CD4+ T cells; r = 0.17, p = 0.6, CD8+ T cells; r = 0.19, p = 0.5, NK cells).
Detection of RSV-IgE Antibodies
In order to investigate whether reduced IFN- mRNA expression had physiologic consequences, we measured RSV-IgE in
the convalescent phase plasma samples of 12 infants (Table 2).
RSV-IgE was detected in one of six samples from infants in
Group 1 and in three out of six samples from infants in Group
2. The presence of detectable RSV-IgE was not related to the
amount of IFN- in the acute phase of illness (r = 
0.289, p = 0.4), but was correlated with the IFN- mRNA levels in the
convalescent phase (r = 0.693, p = 0.04).
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Cytokine production by T cells is one of the key factors determining the outcome of RSV infection in the murine model (5, 12, 21). In the present study we analyzed the in vivo expression of IFN- in infants with primary RSV infections. We
chose to measure IFN- mRNA rather than the corresponding
translated protein products because the technique for doing so
is more sensitive, and also because stimulus-induced cytokine
responses obtained after in vitro exposure to antigen or mitogen may not accurately reflect those occurring in vivo (22). Our
finding that primary RSV infection is associated with IFN-
expression would suggest a Th1 type of response, which confirms earlier findings in mice (5, 23) and humans (19), obtained after in vitro stimulation of PBMC. However, the levels
of IFN- expression in infants with severe RSV disease differed considerably from those of infants with a milder clinical
course of illness. These differences were not due to the presence of different proportions of certain lymphocyte subsets in
the PBMC from which RNA was isolated for IFN- mRNA measurement. This suggests that the differences in IFN-
mRNA levels observed in infants with mild and severe RSV
disease may be at least in part due to differences in the amount
of IFN- mRNA expressed by certain cells. High levels of
IFN- in the acute phase of illness were commonly found in
infants with moderate illness, whereas IFN- levels were low
in those with the most severe disease. These findings can be
explained in several ways.
IFN- designates a Th1-type of response, which is usually
accompanied by strong CD8+ CTL activity. Elimination of
RSV seems to involve an effective CTL response, both in animal models (11) and in humans (24). An ineffective IFN-
and/or CD8+ CTL response may result in delayed virus clearance, leading in turn to an increased inflammatory response
and increased illness. Indeed, infants with a severe clinical
course of RSV disease exhibited significantly lower CD8+ T
cell and CD8+/CD25+ (activated) T cell counts in the acute
phase of illness. This is in accord with previous findings of
RSV-specific cellular cytotoxic immune responses in some infants with mild illness but not in those with the most severe
disease (25), and with the observation that numbers of CD8+
T cells in the peripheral blood of infants with severe disease are significantly reduced as compared with those of infants
with milder forms of illness caused by RSV (26) or with those
of a healthy control group (4).
However, all these findings were obtained with PBMC, and
may not necessarily parallel immunopathologic changes in the
respiratory tract. We cannot exclude the possibility that the
reduced IFN- production in severely ill infants resulted from
a stronger sequestration of IFN--producing cells in the respiratory tissue. This would make these cells unavailable for
analysis in the peripheral blood. However, previous findings in
the murine model of RSV infection provide strong evidence
that an alteration in the Th1/Th2 balance in favor of a Th2 response may contribute to enhanced pulmonary disease (12,
27). The observation that levels of histamine, RSV-IgE, and
other asthma-related mediators are increased in nasopharyngeal secretions of infants with RSV bronchiolitis (8, 28, 29), as
well as the clinical analogy of this with childhood asthma, argues in favor of a skewing toward a Th2 (IL-4, IL-5)-dominated response rather than a Th1 (IFN-)-dominated immune
response in the respiratory tract of infants with the most severe RSV disease. We were unable to establish a link between
the presence of detectable RSV-IgE and the amount of IFN-
expressed in the acute phase of infection. Nevertheless, the
presence of detectable RSV-IgE correlated significantly with
the amount of IFN- expression in the convalescent phase,
possibly indicating that RSV-IgE is influenced by the constitutive expression of IFN- mRNA.
The low IFN- expression found in infants with severe disease may also be due to an inhibitory effect of RSV, which is
capable of suppressing both nonspecific and RSV-specific
lymphocyte proliferative responses (30, 31). Indeed, complete
suppression of IFN- production by in vitro-stimulated PBMC
obtained from infants with severe lower respiratory tract disease due to RSV has recently been reported (4). In our study,
PBMC of infants with severe RSV disease did express detectable amounts of IFN- mRNA, but the levels were generally
low, and are presumed to represent baseline levels, which are
probably constantly maintained in PBMC, since similar IFN-
levels were detected in PBMC of healthy controls. Constitutive IFN- production in healthy infants has also been recently
reported (32), and has been attributed to previously experienced infections and/or previous vaccinations. The upregulation of IFN- mRNA in infants with moderate RSV illness in
our study was transient, as was the increase in CD8+/CD25+
(activated) T cells: Convalescent levels of these T cells and of
IFN- mRNA were similar to those of controls. In contrast, IFN- levels and CD8+/CD25+ T-cell numbers in severely affected infants appeared to remain nearly unchanged. These
findings suggest that severe RSV disease is associated with an
impaired Th1 response.
A weak IFN- response to RSV may also result from the
immaturity of an infant's immune system. Human neonates
are impaired in the ability to generate cellular and humoral
immune responses (33). Of particular note is the reduced capacity of neonatal T cells to produce IFN- after stimulation
(34). Interestingly, the maturation process of IFN- production by T cells appears to be heterogenous within the normal
population, and is not complete until 3 to 4 yr after birth (35).
Since RSV infection is most common in infants under 1 yr old,
an age when the immune system is still developing, RSV may
have a varied effect, promoting a Th2-type of response in infants with an immature immune system.
In conclusion, we have identified several parameters of the cell-mediated immune response in infants with severe RSV disease that clearly distinguish these infants from those with a milder clinical course of illness. The principal observation in the present study was a reduced cell-mediated response among PBMC of infants with severe RSV disease as compared with those with a milder clinical course of illness. This reduced response could be involved in the aggravation of illness, and may help to explain some of the immunologic abnormalities that accompany severe RSV disease.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Judith H. Aberle, Institute of Virology, Kinderspitalgasse 15, 1095 Vienna, Austria.
(Received in original form December 4, 1998 and in revised form May 12, 1999).
Acknowledgments: The authors thank Steven L. Allison for critical reading of the manuscript, and Ursula Sinzinger and Sylvia Malik for their excellent technical assistance. The authors are also grateful to Annemarie Witzelsberger and Maria Hacker for collection of specimens, and to the team of the Children's Cancer Research Institute, St. Anna Kinderspital, for helpful discussion.
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References |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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