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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Atopic asthma is characterized by chronic inflammation of the bronchial mucosa in which eosinophil-
and immunoglobulin E (IgE)-dependent mechanisms are believed to be prominent. Therefore, specific proeosinophilic mediators such as interleukin (IL)-5 and essential cofactors for IgE switching in
B-lymphocytes such as IL-4 could play a pivotal role in asthma. However, the exact role that individual
inflammatory mediators play in the development of the disease in humans is still unknown. Using
semiquantitative reverse transcriptase-polymerase chain reaction amplification in bronchial biopsies
from 10 atopic asthmatics, we have tested the hypothesis that IL-4 and IL-5 mRNA expression relative
to 
-actin mRNA correlates with validated indicators of disease severity. IL-4 and IL-5 mRNA copies
relative to -actin mRNA were detected in bronchial biopsies from atopic asthmatics. The numbers
of IL-5 mRNA copies relative to -actin mRNA correlated with disease severity assessed by the Aas
asthma score (r = 0.70, p = 0.01), baseline FEV1 (r = 
0.94, p = 0.001), baseline peak expiratory flow
rate (r = 0.77, p = 0.01), peak expiratory flow rate variability over 2 wk (r = 0.69, p = 0.028), and
the histamine PC20 (r = 0.72, p = 0.018). Conversely, the numbers of IL-4 mRNA copies relative to
-actin mRNA did not correlate with asthma severity, but they positively correlated with total serum
IgE concentrations (r = 0.90, p = 0.001). Our present results support the concept that IL-5 may determine asthma clinical expression and severity, and by inference they support the development of IL-5 targeted therapies.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Atopic asthma is accompanied by chronic eosinophil-rich bronchial mucosal inflammation, the pathogenesis of which is still poorly understood (1, 2). There is evidence, however, for a critical role for interleukin-5 (IL-5) and other factors orchestrating specific eosinophil accumulation and activation (3). As a result, tissue damage is believed to be due, at least in part, to the release of toxic granule proteins from activated infiltrating eosinophils (9). Because IL-5 selectively activates human eosinophils for proinflammatory effector functions (10, 11), it is reasonable to hypothesize that this cytokine plays a prominent role in asthma. On the other hand, immunoglobulin E (IgE)-mediated release of mediators in sensitized atopic asthmatics after allergen exposure is also thought to be important in directly causing symptoms since allergen exposure provokes acute exacerbations of asthma, whereas removal from exposure ameliorates the disease (12, 13). IL-4 may also promote tissue eosinophilia by selectively increasing VCAM-1 (the counterligand for VLA-4 on eosinophils) (14) and is an essential cofactor for IgE switching (15) in B-lymphocytes. The relative contributions of IL-5 (in promoting tissue eosinophilia) and IL-4 (additionally regulating IgE) to the pathogenesis of atopic asthma remains unclear (16).
In the present study, we chose to focus on the expression of
IL-5 and IL-4 mRNA in the bronchial mucosa of a group of
asthmatics in whom we had stringently documented disease
severity and atopic status. We reasoned that if both cell-mediated and IgE-mediated mechanisms are important in pathogenesis, then disease severity should reflect local expression of
both IL-5 and IL-4, pivotal cytokines regulating cell-mediated
eosinophil recruitment and IgE synthesis, respectively. We
and others (17) have previously shown that both IL-5 and
IL-4 mRNA expression within the bronchial mucosa is elevated in atopic asthmatics as compared with atopic and nonatopic nonasthmatic control subjects. In order further to analyze the possible role of these cytokines in atopic asthma, we
hypothesized that the copy numbers of both IL-5 and IL-4
mRNA (relative to those of the "housekeeping" gene -actin)
within the bronchial mucosa correlates with validated indicators of disease severity.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Patient Population
Bronchial biopsies were obtained at fiberoptic bronchoscopy from 10 atopic asthmatics. Asthma was defined as (1) a clear clinical history
with current symptoms, (2) evidence of more than 20% reversibility of
the FEV1, either spontaneously or after inhaled beta-2 agonists and/or
a histamine PC20 provocation test of 
4 mg/ml during the previous
2 wk. Full clinical examination, lung function testing (FVC, FEV1, and
FEV1/FVC ratio), histamine PC20, skin prick testing, chest radiograph,
full blood count, total serum IgE, and RAST were obtained in all patients prior to bronchoscopy. Morning and evening PEFR (L/min)
were measured before bronchodilator inhalation for 14 d, and the ratio (R) = PEFRmax PEFRmin/PEFRmax was calculated (PEFR variability over 2 wk). Asthma severity was also assessed according to the
five-point scale scoring system of Aas (20).
Atopy was defined by a positive skin prick test to extracts of one or more aeroallergens (including house dust mite, mixed grass pollen, mixed tree pollen, mixed moulds, cat fur, and dog dander) (Soluprick; ALK-Abello, Horsholm, Denmark). Using the Phadebas system (Pharmacia Diagnostics, Uppsala, Sweden) all atopics demonstrated elevated serum allergen-specific IgE concentrations > 0.70 IU/ml to one or more of these allergens. All subjects were nonsmokers and had not taken oral corticosteroids for the previous 2 mo, or inhaled corticosteroids for the previous 2 wk. Exclusion criteria included age < 18 yr or > 65 yr, FEV1 < 50% of the predicted value on the proposed bronchoscopy day, evidence of acute or chronic infection, pregnancy, breast-feeding, or any chronic medical illness other than asthma. The study was approved by the Ethics Committee of the Royal Brompton Hospital, London, and all patients and volunteers gave written informed consent.
Fiberoptic Bronchoscopy
All subjects (asthmatics and controls) underwent bronchoscopy with
bronchial biopsies as previously described (21). Bronchial biopsies
were snap-frozen and stored at 80° C to be used later for RT-PCR.
Reverse Transcriptase-Polymerase Chain Reaction
Semiquantitative RT-PCR reactions were performed as previously
described (19). All reagents were from Sigma Chemicals (Poole, UK),
unless otherwise indicated. Bronchial biopsies were thawed and homogenized (Omni 1000 homogenizer; Camlab, Cambridge, UK) in
TRI Reagent (1 ml) containing Microcarrier Gel (4 µl) (both from
Molecular Research Center Inc., Oxford, UK), and total RNA was
extracted according to the manufacturer's instructions. The RNA was
reverse-transcribed (total volume, 40 µl) using oligo-dT12-18 primers
(Pharmacia) and M-MLV reverse transcriptase (Gibco BRL, Paisley,
UK) as previously described (19). Reverse transcribed mRNA was
stored at 80° C until used for PCR amplification.
For PCR amplification, 5-µl aliquots of 1:20 diluted reverse transcription mixture from all subjects were made up to 40 µl in reverse
transcription PCR buffer containing 1.25 mM primers (Clontech, Palo
Alto, CA) and 2.5 U of Taq polymerase (BRL). Samples were overlaid with mineral oil and transferred to a thermal cycler (Hybaid, Teddington, UK). After denaturing (at 94° C for 5 min), samples were
subjected to 40 (IL-4, IL-5) or 36 (-actin) cycles of denaturation (at
94° C for 1 min), annealing (at 60° C for 2 min), and extension (at 72° C
for 3 min), followed by a final extension step (at 72° C for 5 min). The
primers used were as follows: PCR 5' primers: AAG GCC AAC CGC
GAG AAG ATG (-actin), CGG CAA CTT TGA CCA CGG ACA
CAA GTG CGA TA (IL-4), and GCT TCT GCA TTT GAG TTT
GCT AGC T (IL-5); PCR 3' primers: ACA GGA CTC CAT GCC
CAG GAA (-actin), ACG TAC TCT GGT TGG CTT CCT TCA
CAG GAC AG (IL-4), and TGG CCG TCA ATG TAT TTC TTT
ATT AAG (IL-5). All primers spanned at least one genomic intron.
The expected product sizes were 480 bp (-actin), 344 bp (IL-4), and
294 bp (IL-5). Serial 10-fold dilutions of control cDNA, starting from a maximal number of copies of 1.5 × 108 (2.5 × 10
16 mole) (Clontech) were amplified along with samples from the subjects.
The size and purity of PCR amplified products were verified by
preliminary agarose gel electrophoresis. To semiquantify each PCR-amplified cDNA, 10 µl of PCR amplification mixture from all subjects
were denatured, Southern blotted, and probed with internal oligonucleotides. The probes were end-labeled (specific activities, 9.6 to
15.5 × 106 cpm/ng) with 
32P-ATP (Amersham, Buckinghamshire,
UK) using T4 polynucleotide kinase then purified on Sepharose G20
(Pharmacia) centrifuge columns. The probe sequences were as follows:
GGC TGG GGT GTT GAA GGT CTC AAA CAT GAT (-actin),
GTC CTT CTC ATG GTG GCT GTA GAA CTG CCG (IL-4), and
ATT TTT ATG TAC AGG AAC AGG AAT CCT CAG (IL-5). After a high-stringency wash the membranes were exposed for a time
period sufficient to allow visualization of the blots but not to saturate the autoradiographs.
Autoradiographs were scanned using a flat bed scanner and associated software (Quantity One Software; PDI Inc., New York, NY) to
measure the areas under the densitometry peaks. Standard curves relating the areas of the blots produced by amplification of the serial 10-fold dilutions of the cDNA standards (IL-4, IL-5, -actin) to their
starting concentrations were constructed. In each case, the logarithms
of the blot areas were approximately linearly related to the starting
concentrations of the cDNA standards within the ranges of concentrations measured in the samples from the subjects, as predicted by the
exponential nature of PCR amplification under nonsaturating conditions. No signal was obtained from blotted irrelevant cDNA controls.
The areas of the blots from each amplified cDNA sample from the
subjects were then expressed in terms of the numbers of starting copies of standard cDNA required to produce a blot of the same area. Finally, for each subject sample the numbers of starting copies of IL-4
and IL-5 cDNA were expressed as percentages of the numbers of
starting copies of -actin cDNA.
Statistical Analysis
Data were analyzed nonparametrically using a statistical package (Minitab Release 7; Minitab Inc., State College, PA). Median values and ranges are indicated in RESULTS. The Mann-Whitney U test was then used for intergroup comparison. Correlation coefficients were obtained by Spearman's rank-order method.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IL-5 mRNA was detected by semiquantitative RT-PCR in
bronchial biopsies from atopic asthmatics (median, 154% -actin
mRNA; range, 0 to 443). In these patients, IL-5 mRNA expression (relative to -actin mRNA) was higher in patients
with the more severe symptoms (Aas asthma score 4 versus
Aas asthma score 1 to 3, p = 0.01) (Figure 1). Moreover, IL-5
mRNA expression (relative to -actin mRNA) positively correlated with the Aas asthma score (r = 0.70, p = 0.01) and
peak expiratory flow rate variability over 2 wk (r = 0.69, p = 0.028), and inversely correlated with the patients' bronchial
responsiveness as measured by histamine PC20 (r = 0.72, p = 0.018), baseline FEV1 (r = 0.94, p = 0.001), FEV1/FVC ratio (r = 0.72, p = 0.018), and baseline peak expiratory flow rate (r = 0.77, p = 0.001) (Figures 2 and 3). Conversely, IL-5
mRNA expression (relative to -actin mRNA) did not correlate with serum IgE concentrations (p > 0.25).
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IL-4 mRNA was detected by semiquantitative RT-PCR in
bronchial biopsies from atopic asthmatics (median, 153% -actin
mRNA; range, 0 to 893). In the atopic asthmatics, IL-4 mRNA
expression (relative to -actin mRNA) positively correlated
with serum IgE concentrations (r = 0.90, p = 0.001) (Figure 4).
Conversely, IL-4 mRNA expression (relative to -actin mRNA)
was not greater in patients with the more severe symptoms,
and IL-4 mRNA expression did not correlate with the patients' baseline FEV1, baseline peak expiratory flow rate, peak
expiratory flow rate variability over 2 wk, and bronchial responsiveness as measured by histamine PC20 (p > 0.25).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study we have shown that bronchial mucosal IL-5
mRNA expression (relative to -actin mRNA) in atopic asthmatics correlated with several objective measures of asthma
severity. In contrast, IL-4 mRNA expression (relative to -actin
mRNA) did not correlate with any of these asthma variables,
although there was a significant association with total serum
IgE concentrations.
As discussed above, we hypothesized that IL-5 mRNA expression reflects a T-cell-mediated hypersensitivity reaction leading to eosinophil recruitment, whereas IL-4 mRNA expression reflects IgE synthesis. As our PCR technique required homogenization of biopsy material, we were unable to correlate IL-5 mRNA expression and bronchial mucosal eosinophil counts. However, previous reports have shown that IL-5 mRNA expression in the asthmatic bronchial mucosa correlated with the numbers of eosinophils within the mucosa (2, 17). Moreover, the numbers of eosinophils in the asthmatic bronchial mucosa have been shown in many other studies to correlate with disease severity (1, 2). Taken together with the recognized eosinophil-specific biologic activities of IL-5 (3, 10, 11), these observations provide strong support for the hypothesis that IL-5 regulates asthma severity through eosinophil recruitment.
IL-4 is one of two cytokines known to cause IgE-switching in B-cells, a prerequisite for elevated IgE synthesis (15), and by inference in the pathogenesis of atopy. Our data do not support the hypothesis that increased IL-4 synthesis regulates disease severity in atopic asthma. Our data do not confirm an initial report that did show a significant association between the numbers of bronchoalveolar lavage cells expressing mRNA for IL-4 (using in situ hybridization) and disease severity in atopic asthma (18). It is, however, important to emphasize that although in situ hybridization can identify gene expression in discrete cells, the numbers of mRNA+ cells only gives an estimate of the proportion of cells within the sample that transcribe cytokine genes, not the total number of copies of mRNA per cell.
We have recently demonstrated that RT-PCR was a more sensitive method than in situ hybridization for detecting bronchial mucosal cytokine mRNA expression (19). Therefore, a validated semiquantitative RT-PCR technique provides a more accurate method of analyzing IL-4 and IL-5 mRNA expression in asthma. It is important to note that our data do not preclude the possibility that asthma may be exacerbated acutely by IgE-mediated release of mast cell mediators after acute allergen exposure since this results in local release of bronchoconstrictor substances in hyperresponsive airways. On the other hand, this does not necessitate an obligatory role for IL-4/IgE in the pathogenesis of chronic asthma.
Implicit in our arguments is the assumption that IL-4 and IL-5 mRNA expression within the asthmatic bronchial mucosa is translated into active protein. Although this cannot be assumed, we have recently demonstrated expression of IL-4 and IL-5 protein in the bronchial mucosa of both atopic and nonatopic asthmatics (19), which strongly suggests mRNA translation, although the translation rate and efficiency for individual cytokines may vary. Recently, an alternatively spliced transcript of human IL-4, which lacks exon 2 and functions as an IL-4 antagonist (22, 23), has been described. Such a variant, if present in our samples, would have been detected by our PCR protocol as an amplification product of shorter length (296 bp) than that produced by amplification of full-length IL-4 mRNA. Although we could detect no such products on preliminary sizing gels, the amplification product from this variant would have been expected to hybridize with our IL-4 probe in the Southern blotting procedure, and we cannot rule out the possibility that it may have been present at low copy numbers.
Our observed correlation between bronchial mucosal IL-4 mRNA expression and serum IgE concentrations in atopic asthmatics is compatible with the hypothesis that the maintenance of elevated IgE synthesis in these patients is at least partly IL-4-dependent. This cannot be assumed a priori because of the potential longevity of activated B-cell clones, which might continue (perhaps for years) to produce IgE after IL-4-dependent switching in an IL-4-independent manner. The data are also compatible with the hypothesis that ongoing IgE synthesis in atopic asthmatics, if present, occurs actually within the bronchial mucosa itself, but on the other hand do not exclude the possibility that cytokines may exert their activities at least partially in immune compartments not accessible to bronchial biopsy such as regional lymph nodes. These important questions should be addressed in future studies.
Our data do not support a role for IL-4 in regulating disease symptoms and severity in atopic asthma. Nevertheless local IL-4 synthesis may still play some role in directing local differentiation of T-cells towards a "Th2-type" (IL-5 producing) phenotype (24). On the other hand IL-4 expression in this situation may simply reflect a surrogate marker of "Th2-type" T-cell activation, although recent studies suggest that coexpression of IL-5 and IL-4 by individual T-cells is not always obligatory (25).
In summary our data strongly implicate IL-5 as a major molecular target in atopic asthma, whereas the role of IL-4 is less clear. The effects of specific IL-5 and IL-4 antagonists in ameliorating human asthma will give more specific answers to these questions (26). Indirect approaches, based on studies of "models" of asthma in cytokine-deficient mice sensitized and challenged with aerosolized proteins have provided interesting information regarding lung eosinophilic inflammatory responses (15, 27). A problem with such "models" is that since the precise mechanism of bronchial hyperreactivity in human asthma is unknown, it is difficult to assume that experimental manipulations that produce bronchial hyperreactivity in animals, although arguably physiologically similar to that observed in humans, are also pathogenetically similar. Therefore, studies designed to dissect the respective roles of putatively important cytokines in human asthma are still critical to improve our knowledge of the disease.
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Footnotes |
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Correspondence and requests for reprints should be addressed to A. B. Kay, Professor and Head, Allergy and Clinical Immunology, Imperial College School of Medicine at the National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, UK.
(Received in original form October 10, 1996 and in revised form May 20, 1997).
Dr. Humbert is the recipient of grants from the Institut Electricité Santé, the Société de Pneumologie de Langue Française, and the Wellcome Trust (UK).Dr. Corrigan is the recipient of a Medical Research Council (UK) Clinician Scientist Fellowship.
Acknowledgments: The writers acknowledge Mr. D. Cramer (Royal Brompton Hospital Lung Function Unit), Dr. D. S. Robinson, Dr. B. Assoufi, and the staff of Lind ward, Royal Brompton National Heart and Lung Hospital.
Supported by a programme grant from the Medical Research Council (UK).
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References |
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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P. T. Flood-Page, A. N. Menzies-Gow, A. B. Kay, and D. S. Robinson Eosinophil's Role Remains Uncertain as Anti-Interleukin-5 only Partially Depletes Numbers in Asthmatic Airway Am. J. Respir. Crit. Care Med., January 15, 2003; 167(2): 199 - 204. [Abstract] [Full Text] [PDF]
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 W. M. Abraham Of Mice and Men Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 1 - 4. [Full Text] [PDF]
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E L J van Rensen, R G Stirling, J Scheerens, K Staples, P J Sterk, P J Barnes, and K F Chung Evidence for systemic rather than pulmonary effects of interleukin-5 administration in asthma Thorax, December 1, 2001; 56(12): 935 - 940. [Abstract] [Full Text] [PDF]
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R. G. STIRLING, E. L. J. VAN RENSEN, P. J. BARNES, and K. FAN CHUNG Interleukin-5 Induces CD34+ Eosinophil Progenitor Mobilization and Eosinophil CCR3 Expression in Asthma Am. J. Respir. Crit. Care Med., October 15, 2001; 164(8): 1403 - 1409. [Abstract] [Full Text] [PDF]
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 K. J. Staples, M. Bergmann, K. Tomita, M. D. Houslay, I. McPhee, P. J. Barnes, M. A. Giembycz, and R. Newton Adenosine 3',5'-Cyclic Monophosphate (cAMP)-Dependent Inhibition of IL-5 from Human T Lymphocytes Is Not Mediated by the cAMP-Dependent Protein Kinase A J. Immunol., August 15, 2001; 167(4): 2074 - 2080. [Abstract] [Full Text] [PDF]
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K. OSHIKAWA, K. KUROIWA, K. TAGO, H. IWAHANA, K. YANAGISAWA, S. OHNO, S.-I. TOMINAGA, and Y. SUGIYAMA Elevated Soluble ST2 Protein Levels in Sera of Patients with Asthma with an Acute Exacerbation Am. J. Respir. Crit. Care Med., July 15, 2001; 164(2): 277 - 281. [Abstract] [Full Text] [PDF]
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J. C. LAPORTE, P. E. MOORE, S. BARALDO, M.-H. JOUVIN, T. L. CHURCH, I. N. SCHWARTZMAN, R. A. PANETTIERI Jr, J.-P. KINET, S. A. SHORE, and S. A. SHORE Direct Effects of Interleukin-13 on Signaling Pathways for Physiological Responses in Cultured Human Airway Smooth Muscle Cells Am. J. Respir. Crit. Care Med., July 1, 2001; 164(1): 141 - 148. [Abstract] [Full Text] [PDF]
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E M Glare, M Divjak, M J Bailey, and E H Walters The usefulness of competitive PCR: airway gene expression of IL-5, IL-4, IL-4{delta}2, IL-2, and IFN{gamma} in asthma Thorax, July 1, 2001; 56(7): 541 - 548. [Abstract] [Full Text] [PDF]
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 J. Van Wauwe, F. Aerts, M. Cools, F. Deroose, E. Freyne, J. Goossens, B. Hermans, J. Lacrampe, H. Van Genechten, F. Van Gerven, et al. Identification of R146225 as a Novel, Orally Active Inhibitor of Interleukin-5 Biosynthesis J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 655 - 661. [Abstract] [Full Text]
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 J. Tavernier, J. Van der Heyden, A. Verhee, G. Brusselle, X. Van Ostade, J. Vandekerckhove, J. North, S. M. Rankin, A. B. Kay, and D. S. Robinson Interleukin 5 regulates the isoform expression of its own receptor alpha -subunit Blood, March 1, 2000; 95(5): 1600 - 1607. [Abstract] [Full Text] [PDF]
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A. Mori, O. Kaminuma, K. Miyazawa, K. Ogawa, H. Okudaira, and K. Akiyama p38 Mitogen-Activated Protein Kinase Regulates Human T Cell IL-5 Synthesis J. Immunol., November 1, 1999; 163(9): 4763 - 4771. [Abstract] [Full Text] [PDF]
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M. P. McLane, A. Haczku, M. van de Rijn, C. Weiss, V. Ferrante, D. MacDonald, J.-C. Renauld, N. C. Nicolaides, K. J. Holroyd, and R. C. Levitt Interleukin-9 Promotes Allergen-Induced Eosinophilic Inflammation and Airway Hyperresponsiveness in Transgenic Mice Am. J. Respir. Cell Mol. Biol., November 1, 1998; 19(5): 713 - 720. [Abstract] [Full Text]
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J. Temelkovski, S. P Hogan, D. P Shepherd, P. S Foster, and R. K Kumar An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen Thorax, October 1, 1998; 53(10): 849 - 856. [Abstract] [Full Text]
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