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note
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Six-week-old male A/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were housed under laminar flow hoods in an environmentally controlled specific pathogen-free animal facility for the duration of experiments (there were 4 to 10 mice per experimental group). The studies reported here conformed to the principles for laboratory animal research, as outlined by the Animal Welfare Act and the Department of Health, Education, and Welfare (NIH) guidelines for the experimental use of animals. Mice were immunized by an intraperitoneal injection of OVA (10 μg; crude grade IV) (Sigma) in PBS (0.2 ml) or of PBS alone. Fourteen days after immunization, mice were anesthetized with a mixture of ketamine and xylazine [45 and 8 mg per kilogram of body weight (mg/kg), respectively] and challenged intratracheally with 50 μl of a 1.5% solution of OVA or an equivalent volume of PBS as a control. Ten days after this first antigen challenge, mice were challenged again intratracheally with either OVA or PBS. Characterization of the allergic phenotype was performed 96 hours after the second antigen challenge.
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0032167603
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For soluble expression of the murine IL-13Rα2, a pED expression vector containing DNA encoding the murine slL-13Rα2 extracellular domain, fused in-frame with the heavy chain constant domains 2 and 3 of human IgG1, was transfected into CHO cells [D. D. Donaldson et al., J. Immunol. 161, 2317 (1998)]. The slL-13Rα2-Fc was purified with protein A-Sepharose (33). The in vitro median inhibitory dose, as determined by its ability to neutralize 3 ng/ml of murine IL-13 in the B9 proliferation assay was ∼10 ng/ml. Human Ig, used as a control for slL-13Rα2-Fc, was similarly purified by protein A-Sepharose chromatography from a 10% solution of human immune globulin that is commercially available for intravenous administration (Miles, Berkeley, CA) (33). Mice were given slL-13Rα2-Fc (400 μg) or an equivalent amount of the control human Ig by intraperitoneal injection on days -1,0, +1, and +3 of the secondary antigen challenge.
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note
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Airway reactivity to the intravenous administration of acetylcholine was measured (12). Three days after the final intratracheal challenge, mice were anesthetized with sodium pentobarbital (90 mg/kg), intubated, ventilated at a rate of 120 breaths/min with a constant tidal volume of air (0.2 ml), and paralyzed with decamethonium bromide (25 mg/kg). After a stable airway pressure was established, acetylcholine was injected intravenously (50 μg/kg), and the dynamic airway pressure was measured for 5 min.
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35
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Pulmonary eosinophilia was assessed by BAL (12)
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Pulmonary eosinophilia was assessed by BAL (12).
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36
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20644446605
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note
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A kidney was excised, and pooled blood was collected for antibody analysis (12). Serum was separated by centrifugation and stored at -80°C until analysis. Serum OVA-specific IgE concentrations were determined by sandwich enzyme-linked immunosorbent assay (ELISA). Sample wells were coated with a 0.01% OVA solution in PBS, blocked with 10% fetal bovine serum (FBS) in PBS, and washed with 0.05% Tween-20 in PBS. Serum samples were diluted 1:10 and 1:100 with 10% FBS in PBS. After an overnight incubation, plates were washed with 0.05% Tween-20 in PBS, and biotin-conjugated anti-mouse IgE (Pharmingen, San Diego, CA) was added. After a wash, avidin peroxidase (0.0025 mg/ml) (Sigma) in 10% FBS/PBS was added, and plates were developed with 2.2′-azino-di(3-ethyl-benzthiazone sulfonate) (Kirkegaard and Perry, Gaithersburg, MD). Plates were read at 405 nm within 30 min. Reported optical density (OD) values are from serum samples that were diluted 1:10, because these values were proven to be below the saturation point of the assay by comparison of OD values of serum samples diluted 1:100 with 10% FBS/PBS.
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A murine IgE-specific ELISA was used to quantitate total IgE concentrations in serum with complementary antibody pairs for mouse IgE (R35-72 and R35-92) (Pharmingen), according to the manufacturer's instructions. Duplicate samples (of a 1:10 dilution in 10% FBS in PBS) were examined from each animal. OD readings of the samples were converted to picograms per milliliter with the values that were obtained from standard curves generated with known concentrations of recombinant mouse IgE (5 to 2000 pg/ml).
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note
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To examine the effects of rlL-13 on the mucus cell content of the airway epithelium, lungs were excised and fixed in 10% formalin. They were then washed in 70% ethanol; dehydrated; embedded in glycol methacrylate; cut into 10-μM sections; mounted on slides; and stained with hematoxylin, eosin, and periodic acid-Schiff. Four sections were examined per animal, and four fields were scored per lung section. Sections were scored on a scale from 1 to 4, with 1 representing no mucus cell content.
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This work was supported by grants from NIH (HL58527) and the Center for Indoor Air Research to M.W.-K. We thank B. Annis and Z. Lu at Genetics Institute for murine rlL-13 and the Research Support Team at Genetics Institute for murine s13Rα2-Fc.
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