House dust mite sublingual tablet is effective and safe in patients with allergic rhinitis.

BACKGROUND: House dust mite (HDM) is the major indoor allergen for allergic diseases such as allergic rhinitis (AR) and asthma. Although sublingual immunotherapy is a curative treatment for HDM-induced AR, data from large-scale studies are limited. We evaluated the efficacy and safety of HDM tablets in adolescent and adult patients (aged 12-64 years) with HDM-induced AR with or without intermittent asthma. METHODS: In a double-blind trial in Japan, 968 subjects were randomized 1 : 1 : 1 to 300 index of reactivity (IR), 500 IR, or placebo groups. The primary endpoint was the Average Adjusted Symptom Score (AASS) in the last eight weeks of the 52-week treatment. Secondary endpoints included individual nasal and ocular symptom scores, rescue medication use, and the Japanese Rhinoconjunctivitis Quality of Life Questionnaire (JRQLQ) scores. RESULTS: The AASS in the last eight weeks of treatment significantly improved in both the 300 IR and the 500 IR groups compared to that in the placebo group (P < 0.001). In the 300 IR group, the onset of action occurred at week 8-10. All four nasal symptoms significantly improved in both active treatment groups; rescue medication use and JRQLQ outcome improved in the 300 IR group. Most adverse events (AEs) were mild, and 16 serious AEs (SAEs) were reported; however, none of them were drug-related. CONCLUSIONS: One-year treatment with 300 IR and 500 IR HDM tablets was effective without major safety concerns. The recommended therapeutic dose for AR is 300 IR.

Variants in the 17q21 asthma susceptibility locus are associated with allergic rhinitis in the Japanese population.

BACKGROUND: Allergic rhinitis (AR) is a very common disorder peaking in the teenage years that is mediated by hypersensitivity responses to environmental allergens. Although it is well established that the ORMDL3 locus at chromosome 17q21 is associated with susceptibility to bronchial asthma, the genetic influences of the polymorphisms of the locus in allergic rhinitis are unclear. OBJECTIVE: To examine whether the polymorphisms in the 17q21 asthma susceptibility locus are associated with allergic rhinitis in the Japanese population. METHODS: We performed linkage disequilibrium (LD) mapping of the locus using the HapMap database and conducted an association study of the locus with a total of 15 tag SNPs in two independent populations. We further evaluated correlations of genotypes with changes in expression of genes at the region in lymphoblastoid cell lines in the Japanese population and assessed the expression levels of the genes in nasal epithelium and various human tissues. RESULTS: We found a significant association between a total of five polymorphisms in the 17q21 asthma susceptibility locus, rs9303277, rs7216389, rs7224129, rs3744246, and rs4794820, and AR (minimum P(combined)  = 0.00074, rs4794820). The expression level of the ORMDL3 transcript was significantly correlated with the genotype of rs12150079, rs7216389, rs3744246, and rs4794820 with P < 0.01 (minimum P = 0.0058, rs7216389), and ORMDL3 mRNA was highly expressed in nasal epithelium. CONCLUSION: Genetic variants in the 17q21 asthma susceptibility locus are significantly associated with AR in the Japanese population.

Drug delivery observation of hydrophobe ferrofluid and magnetite nanoparticals by SPring-8 synchrotron radiation.

In this study, the composite magnetic nanoparticles of coated SiO nano film with about 8 nm size and high saturation magnetization value, were synthesized by liquid phase precipitation method. The magnetic nanoparticles can be dispersed in various liquid media, widely known as magnetic fluids or ferrofluids with both magnetic and liquid properties. The materials been collected great interests and more and more attentions to focus into Drug Delivery System (DDS) as a new technology in this paper. We use the composite nanoparticles to disperse H2O and inject the solutions into rat's in-vivo organs. And, in the experiments by using a strong photon beam of SPring-8 Synchrotron Radiation facility, the distribution stat and the effects of magnetic field as well as drug delivery behaviour of nanoparticles in the rat' kidney are verified by the in-vivo observations.

Transforming growth factor-beta(2) polymorphisms are associated with childhood atopic asthma.

BACKGROUND: Transforming growth factor (TGF)-beta plays an important role in the regulation of airway inflammation and remodelling in asthma. Recent studies suggest that TGF-beta(2) is a predominant isoform expressed in severe asthma and it is also associated with airway remodelling. OBJECTIVE: To determine whether the polymorphisms in TGF-beta(2) are associated with childhood atopic bronchial asthma in a Japanese population. METHODS: We identified a total of eight polymorphisms and characterized the linkage disequilibrium (LD) mapping of the gene. Three variants in the promoter and 3'UTR were genotyped, and we conducted an association study of TGF-beta(2) (childhood atopic asthma n=297, normal controls n=555). An association analysis of these variants and an expression and functional analysis were performed. RESULTS: 3'UTR 94862T >A was found to be significantly associated with the risk of childhood atopic asthma (P=0.00041). The -109-->ACAA ins promoter variant was also associated with the risk of childhood atopic asthma (P=0.0037). TGF-beta(2) expression was observed in both the normal and asthmatic bronchial epithelium, and both real-time PCR and an ELISA showed a significant basal and TGF-beta(1)-induced TGF-beta(2) expression in the bronchial epithelial cell line BEAS2B. Furthermore, the promoter variant -109-->ACAA ins increased the TGF-beta(2) promoter-reporter activity in BEAS2B cells. CONCLUSIONS: Our data suggest that TGF-beta(2) may therefore be involved in the development of childhood atopic asthma by means of functional genetic polymorphism. The polymorphisms in TGF-beta(2) may become important information for asthma susceptibility in children.

Increases in collagen type I synthesis in asthma: the role of eosinophils and transforming growth factor-beta.

BACKGROUND: Collagen type I is one of the major deposits in thickening of the reticular basement membrane of asthma. OBJECTIVE AND METHODS: In this study, we assessed turnover of collagen type I in asthma by measuring procollagen type I C-terminal peptide (PICP) and collagen type I C-terminal telopeptide (ICTP) in induced sputum. RESULTS: PICP but not ICTP was found to be significantly higher in asthma subjects than in normal volunteers (P < 0.05). In asthma, PICP was inversely correlated with %FEV(1.0) (r = -0.539), and its levels significantly increased upon exacerbation (P < 0.05), indicating that collagen synthesis increases during asthma exacerbation. Additionally, PICP was found to significantly correlate with eosinophil counts in sputum (r = 0.539), indicating that eosinophils stimulate collagen turnover. Because eosinophils can produce TGF-beta, a potent stimulator of collagen synthesis, we immunocytochemically examined TGF-beta-positive cells in sputum. TGF-beta-positive cells significantly correlated with eosinophil counts (r = 0.811) and PICP (r = 0.569), suggesting that TGF-beta released from eosinophils is involved in collagen synthesis. CONCLUSIONS: The results of the present study suggest that collagen synthesis is stimulated in asthmatic airways by eosinophils through TGF-beta, while collagen degradation is not, and that PICP in sputum can act as a new marker for airway inflammation in asthma.

Exposure to diesel exhaust exacerbates allergen-induced airway responses in guinea pigs.

Diesel exhaust (DE) is a major air pollutant in urban areas. To clarify the effects of DE on the exacerbation of asthma, guinea pigs were exposed 12 h daily to 3 mg/m(3) DE or air for 8 wk with or without sensitization to ovalbumin (OVA). In the DE-exposed sensitized animals, both immediate (IAR) and late (LAR) airway responses were enhanced after the inhalation challenge by OVA, compared with the DE-unexposed sensitized animals. Mucus was greatly accumulated in the airways of DE-exposed sensitized animals during IAR. The number of eosinophils and level of sialic acid concentration in bronchial lavage fluids were also significantly higher in the DE-exposed sensitized animals than in the DE-unexposed control animals. During LAR, intercellular spaces of the bronchial epithelium became enlarged in the DE-exposed sensitized animals, showing infiltration by numerous eosinophils. Albumin concentration was significantly higher in the bronchial lavage fluids from the DE-exposed sensitized animals than in those from the DE-unexposed control animals. These results suggest that exposure to DE enhances mucus hypersecretion and eosinophilic inflammation during IAR. DE exposure also increases airway permeability and airway inflammation during LAR. Thus, DE exposure exacerbates allergen-induced airway responses in guinea pigs.

Maintenance of the differentiated type II cell characteristics by culture on an acellular human amnion membrane.

We have developed a Culture system for guinea pig alveolar type II cells using an epithelium-denuded human amnion membrane as a substratum. The differentiated morphology was maintained for 3 wk by both air-interface feeding and immersion feeding when type II cells were cultured on the basement membrane side of the amnion with fibroblasts on the opposite side (coculture). Functionally high levels of surfactant protein B (SP-B) and C (SP-C) messenger ribonucleic acids (mRNAs) were expressed even after the 3-wk cultivation and surfactant protein A mRNA was detected on day 10 of the culture. The differentiation was also maintained when fibroblasts were cultured on lower chambers of the culture plates (separate culture). In contrast, culture of type II cells without fibroblasts (monoculture) could not preserve the mature morphology. When the monoculture was supplemented with keratinocyte growth factor or hepatocyte growth factor, a monolayer of rather cuboidal type II cells with apical microvilli was maintained. However, the percent area of lamellar bodies in these cells was significantly less than that in freshly isolated type II cells, and mRNA expressions of SP-B and SP-C were also considerably suppressed. These findings suggest that other growth factors or combinations of these factors are necessary for the maintenance of the differentiated phenotype. As substratum, a permeable collagen membrane or a thin gel layer of Engelbreth-Holm-Swarm mouse sarcoma extracts did not preserve the mature characteristics. This culture system using an acellular human amnion membrane may provide novel models for research in type II cells.

The effects of histamine and its antagonists on the cochlear microphonic and the compound action potential of the guinea pig.

OBJECT: we studied the effects of histamine, the H1 receptor antagonist pyrilamine, and the H2 receptor antagonist cimetidine on the cochlear potential of guinea pigs (cochlear microphonic, CM; compound action potential, CAP). METHODS: histamine was applied into the cochlear perilymph at three different dosages (10 microM, 50 microM or 10 mM). Pyrilamine and cimetidine were applied at 50 microM each. RESULTS: histamine increased the CAP at 10 and 50 microM without any significant effects on the CM. The effects of histamine at 50 microM were suppressed by the 50-microM of pyrilamine and cimetidine. At 10 mM of histamine, CAP and CM amplitudes were significantly decreased. CONCLUSION: in low concentrations, histamine may act as an extracellular signal on inner hair cells (IHCs) or it may stimulate the afferent nerve by binding to their H1 and H2 receptors. A possible explanation for the inhibitory effects of histamine at 10-mM dosage was apparently found in that the effects of the high concentration may be supraphysiological; and furthermore, there is a difference in the mechanism by which histamine exerts its effects mediated by the histamine receptors on the cochlea.

Triggering the induction of myofibroblast and fibrogenesis by airway epithelial shedding.

Myofibroblasts have been thought to participate in subepithelial fibrosis in asthma, but the mechanism of myofibroblast induction has not been fully understood. In this study we investigated injury-related myofibroblast induction in a coculture system of guinea-pig epithelial cells and fibroblasts cocultured in a human amnion chamber. After pseudostratified epithelial cells were mechanically scraped, migrated flat epithelial cells differentiated into cuboidal appearances on Day 4 and then returned to their original shapes on Day 8. During the course of the epithelial redifferentiation, it was found by Northern blot analysis, immunohistochemistry for alpha-smooth muscle actin, and electron microscopic observation that the myofibroblasts were transiently induced on Day 4. The myofibroblast induction was inhibited by the blocking of transforming growth factor (TGF)-beta1 and thrombospondin (TSP)-1, indicating that the activation of TGF-beta1 by TSP-1 would induce myofibroblasts. This finding was also supported by a transient upregulation of TSP immunoreactivity and TSP-1 messenger RNA (mRNA) in fibroblasts. Interestingly, epithelial injury reduced TGF-beta1 immunoreactivity in the amnion membrane but did not affect TGF-beta1 mRNA in epithelial cells and fibroblasts, indicating that TGF-beta1 supplied from the extracellular matrix can participate in myofibroblast induction. Concurrently with myofibroblast induction, procollagen type I and III mRNAs were upregulated in fibroblasts, and obvious collagen deposition was observed ultrastructurally around the myofibroblasts compared with the fibroblasts. These results indicate that induced myofibroblasts can be functionally more active in producing collagen than are resting fibroblasts. The present study suggests that epithelial injury stimulates TGF-beta1 release from the extracellular matrix and its activation via TSP-1 production, causing collagen synthesis through myofibroblast induction.

Tissue inhibitor of metalloproteinases-2 gene polymorphisms in chronic obstructive pulmonary disease.

Proteinase/antiproteinase imbalance is the most widely accepted theory for development of chronic obstructive pulmonary disease (COPD). Mutations of tissue inhibitor of metalloproteinases-2 (TIMP-2) that downregulate its activity may increase the activities of matrix metalloproteinases and result in the degradation of the lung matrix. Polymorphisms of the TIMP-2 gene were investigated in 88 COPD patients and 40 control subjects. The variations were examined by single-strand conformational polymorphism analysis followed by sequencing. Two polymorphisms were identified, +853 GIA and -418 G/C nucleotide substitutions. There was a significant deviation in the genotypic frequencies at +853 and the allele frequencies for G were significantly higher in the COPD patient group than in the control group. For locus -418, the allele frequencies for C in the COPD patient group also tended to be higher than those in the control group. The +853 G/A nucleotide substitution was a silent variant. The -418 G/C substitution was located in the consensus sequence for the Sp1 binding site. These polymorphisms may be associated with the development of chronic obstructive pulmonary disease, decreasing the transcription and stability of the messenger ribonucleic acid, and available as genetic markers of susceptibility to the disease.

Dislocation of E-cadherin in the airway epithelium during an antigen-induced asthmatic response.

The airway epithelium plays a critical role in asthma. E-cadherin, located on the basolateral side of the epithelial cells, forms adherent junctions. To investigate the role of E-cadherin on the regulation of permeability of molecules and fluid in asthmatic responses, we observed the dynamics of E-cadherin after an immunochallenge against guinea pigs. Immunohistochemical studies revealed that E-cadherin was expressed on the lateral sides of epithelial cells before the immunochallenge and after immediate airway responses (IAR). However, E-cadherin immunoreactivities decreased from the basolateral region in late airway responses (LAR) 6 h after the challenge. Simultaneously, soluble E-cadherin immunoreactivities were detected in lavage fluid only in LAR, suggesting that E-cadherin is partly cleaved and released into the lumen in LAR. Airway permeability, which was examined by penetration of horseradish peroxidase from the airway side into the epithelium, increased in both IAR and LAR. These results suggest that E-cadherin detachment from the lateral side of the epithelial cells increased airway permeability in LAR but not IAR. We conclude that an antigen challenge causes an opening of adherent junctions as well as increases airway permeability in LAR. This mechanism would participate in airflow limitation during attacks and the increase of airway permeability and hyperresponsiveness in asthmatics.

[Vascular anastomoses in free jejunal reconstruction to the neck vessels: an experience of consecutive 20 cases without using surgical microscope].

Twenty consecutive cases of pharyngoesophageal cancer who underwent free jejunal reconstruction were reported. The common carotid or external carotid artery was used for a feeder of the free graft. The internal jugular vein were served as a drainage vein. All anastomoses were performed in an end-to-side fashion without using surgical microscopes. Mean carotid artery clamping time was 16 minutes and no neurological complications were noticed postoperatively. Graft failure was occurred in 1 patient. The presenting technique, showing 95% success rate, is recommended as a simple option for vascular anastomosis in free jejunal reconstructive surgery.

Initial steroid hormone dose in the treatment of idiopathic sudden deafness.

OBJECTIVE: The purpose of this study was to clarify whether higher doses of steroids improve the prognosis of idiopathic sensorineural hearing loss (ISHL) and the suitable dose of steroid hormone. STUDY DESIGN: The study was a retrospective statistical analysis. SETTING: This study was performed at the Department of Otolaryngology, Head Neck Surgery, Kumamoto University School of Medicine. PATIENTS: Two hundred fifty patients with ISHL were analyzed in this study. They were divided into two groups: those receiving less than a specified daily dose of steroid and those receiving a daily dose greater than or equal to the specified dose. INTERVENTIONS: The patients received systemic steroid therapy combined with adenosine triphosphate, vitamins, diuretics, vasodilators, hyperbaric oxygen therapy, stellate ganglion block, or volume expander. MAIN OUTCOME MEASURES: The correlation between the initial dose of steroid hormone and the improvement rate was analyzed. RESULT: Spearman's correlation coefficients and partial correlation coefficients between the initial dose and the prognosis were all significantly negative. On the other hand, the correlations between the initial dose and the prognosis were positive in the group receiving <30 mg/day, whereas they were negative in the group receiving > or =30 mg/day, although these correlations were not significant. CONCLUSION: The general use of steroid hormone to treat ISHL is not recommended. Furthermore, if steroid hormone is used for treatment, the use of <30 mg/day of prednisolone is preferable.

Early molecular and cellular events of oxidant-induced pulmonary fibrosis in rats.

To evaluate the early molecular events of oxidant-induced pulmonary fibrosis, rats were continuously exposed to 0.4 ppm ozone and 7 ppm nitrogen dioxide. The early responses to the combined gases could be divided into three phases. Acute pulmonary inflammation indicated by an increase in pulmonary edema as well as an influx of neutrophils into the airspaces first occurred on days 1 to 3 of the exposure. The pulmonary inflammation was reversed by day 8, and no biochemical or morphologic aspects of tissue responses were detected from days 15 to 45, suggesting that rats adapted to the stimuli during that period. Pulmonary fibrosis could be detected by an increase in the biomarker of lung collagen content at day 60 and by histopathologic evaluation by day 90. Enhanced expression of macrophage inflammatory protein-2 was observed only at day 1, whereas the pulmonary expression of transforming growth factor-beta was upregulated on days 60 and 90 of the exposure. Macrophage expressions of interleukin-1beta and interleukin-6 were enhanced during acute pulmonary inflammation; however, macrophage expression of tumor necrosis factor-alpha was elevated at both day 1 and days 60-90. Activation of nuclear factor-kappa B and increased expression of thioredoxin in the lungs was also observed at day 1 and days 60-90. The expression of antioxidant enzymes, such as manganeous superoxide dismutase and glutathione peroxidase, was not altered during exposure. These results indicate that macrophage activation and the expression of macrophage-derived cytokines may play an important role in the early pulmonary responses against the combined gases.

Dynamic helical CT of T1 and T2 glottic carcinomas: predictive value for local control with radiation therapy.

BACKGROUND AND PURPOSE: Tumor volume and cartilage invasion have been suggested as prognostic factors of glottic carcinomas following definitive radiation therapy. Radiologic examinations provide additional information regarding the deep extension of tumor. We determined whether dynamic helical CT can predict local control of early (T1 and T2 stage) glottic carcinomas treated with definitive radiation therapy. METHODS: Sixty-eight patients with early glottic carcinoma evaluated on pretreatment dynamic helical CT were treated with definitive radiation therapy. Tumor detectability, maximum dimension, tumor volume, and involvement of anatomic subsites (anterior commissure, ventricle, subglottic region, and thyroid and arytenoid cartilages) were determined by consensus by three radiologists without previous knowledge of the clinical information. The CT findings were correlated with local control. RESULTS: The two-year local control rate was 76%; 91% for T1 and 60% for T2 lesions. Univariate analysis revealed clinical T stage, tumor detectability, maximum dimension, tumor volume, anterior commissure involvement, ventricle involvement, and thyroid cartilage involvement as significant prognostic factors. Thyroid cartilage involvement was an independent predictor by multivariate analysis. The lesions separate from the thyroid cartilage had a 95% probability of local control, whereas the lesions adjacent to the cartilage had only a 42% control rate. CONCLUSION: Dynamic helical CT provides prognostic information for the results of definitive radiation therapy. Patients with a tumor adjacent to the thyroid cartilage had an increased risk of local failure.

Ebselen decreases ozone-induced pulmonary inflammation in rats.

We studied the effects of ebselen on rat lung inflammatory responses against ozone exposure. Rats were treated with ebselen every 12 h from 1 h before a single 4-h exposure to 2 ppm ozone. Treatment with ebselen (10 mg/kg) significantly decreased pulmonary inflammation as indicated by the albumin concentration and the number of neutrophils in the bronchoalveolar lavage fluid 18 h after the ozone exposure. Although treatment with ebselen did not alter the macrophage expression of inducible nitric oxide synthase after the ozone exposure, it did markedly inhibit the nitration reaction of tyrosine residues, suggesting that ebselen scavenges peroxynitrite during ozone-induced pulmonary inflammation. Treatment with ebselen also enhanced the pulmonary expression of both copper, zinc, and manganous superoxide dismutases at the same time point. These enzymes may also contribute to a decrease in the formation of peroxynitrite by lowering the concentration of superoxide. Thus, ebselen represents a useful compound for protecting against certain acute lung injuries by modulating the oxidant-related inflammatory process.

Early glottic squamous cell carcinoma. Predictive value of MR imaging for the rate of 5-year local control with radiation therapy.

PURPOSE: To evaluate MR findings in early (T1 and T2 stages) glottic carcinomas and the predictive value of MR imaging for the rate of 5-year local control with radiation therapy. MATERIAL AND METHODS: Eighty-three patients with early glottic carcinomas were prospectively examined with MR at 1.5 T. MR investigation included unenhanced T1-weighted, T2-weighted, dynamic and contrast-enhanced T1-weighted images. Three patients with presumed advanced diseases on MR were initially treated with total laryngectomy and were excluded from the study. The remaining 80 patients were treated with radiation therapy with curative intent. Tumor detectability, size and relationship to the thyroid cartilage were determined on MR images. The MR findings were then correlated with the rate of local control. RESULTS: Forty-eight of 80 lesions (60%) were detected on MR imaging. All detected lesions but 1 demonstrated increased signal on T2-weighted images. The lesions were best delineated on dynamic images (statistically significant). The 5-year local control rate with radiation therapy was 72%. Univariate analysis revealed clinical T stage, MR detectability, tumor size and relationship to the thyroid cartilage as significant predictors. Multivariate analysis revealed that the relationship to the thyroid cartilage was an independent factor. CONCLUSION: MR provides prognostic information about the results of definitive radiation therapy. To evaluate the tumor extension in lesions detected on precontrast MR images, contrast-enhanced dynamic images should be obtained.

Inflammatory cell populations and cytokine mRNA expression in the nasal mucosa in aspirin-sensitive rhinitis.

Aspirin-sensitive rhinitis is characterized by severe perennial nasal congestion and discharge. The study questioned whether this disease, like immunoglobulin E-mediated rhinitis, might be associated with local recruitment and activation of T-lymphocytes, mast cells and eosinophils with parallel increases in "T-helper2-type" cytokines. Nasal biopsies from 10 patients with aspirin-sensitive rhinitis and 12 healthy controls subjects were studied. Nasal mucosal sections were examined by immunohistochemistry in order to determine cell phenotypes and by in situ hybridization to detect cells expressing messenger ribonucleic acid (mRNA) for cytokines. In aspirin-sensitive rhinitis there were increases in total (CD3+) (p=0.05) and activated (CD25+) T-cells (p=0.007), total (major basic protein (MBP) positive) (p=0.004) and activated (monoclonal antibody which recognizes the cleaved form of eosinophil cationic protein (EG2) positive) eosinophils (p=0.003), tryptase+ mast cells (p=0.04) and CD68+ macrophages (p=0.002). Neutrophils and cells expressing human leukocyte antigen-DR were no different. Marked increases were observed in the numbers of interleukin (IL)-5 mRNA+ cells (p=0.004) in aspirin-sensitive patients, whereas lower numbers of IL-4 mRNA+ cells were observed, with a trend for a difference from controls (p=0.07). No differences were observed for either IL-2 or interferon-gamma. In conclusion, in aspirin-sensitive rhinitis there is intense inflammation of the nasal mucosa characterised by T-lymphocytes, eosinophils and mast cells. The predominance of macrophages and disproportionate increase in interleukin-5 compared to interleukin-4 messenger ribonucleic acid expression suggests that factors other than "allergic" mechanisms may be important in this disease.

Novel association of the src family kinases, hck and c-fgr, with CCR3 receptor stimulation: A possible mechanism for eotaxin-induced human eosinophil chemotaxis.

The chemokine eotaxin is a potent and relatively eosinophil-specific chemoattractant implicated in the cell migration to inflammatory sites in allergic diseases. Eotaxin exerts its activity solely through the CCR3 receptor, but the signaling pathways are poorly defined. In this study, we show that eotaxin induces an increase in tyrosine phosphorylation of multiple cellular proteins in normal human eosinophils. Eotaxin-dependent tyrosine phosphorylation was detected 1 min after stimulation and increased for at least 15 min with kinetics similar to those of eotaxin-induced cell shape changes. Herbimycin A, a tyrosine kinase inhibitor, blocked both eotaxin-induced tyrosine phosphorylation and cell shape changes as well as chemotaxis. Immunofluorescence microscopy analyses showed that eotaxin-induced cell shape changes were accompanied by redistribution of tyrosine-phosphorylated proteins and F-actin reorganization that were sensitive to herbimycin A. Coimmunoprecipitation studies revealed that binding of eotaxin to CCR3 greatly enhanced association of the Src family kinases, Hck and c-Fgr, with CCR3 after internalization of CCR3. These results may indicate that recruitment of Hck and c-Fgr to CCR3 in a compartment triggers tyrosine phosphorylation, leading to rapid cell shape changes required for cell migration.