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Lipoxin A4 (LXA4) and Leukotriene C4 (LTC4) Levels in Nasal Lavages after Intranasal Provocation with Aspirin in Aspirin Sensitive and Aspirin Tolerant Patients
S144 Abstracts
Potential Role of the Glucocorticoid Receptor (GCR) Beta in Glucocorticoid (GC) Dependent Histone H4 Acetylation L. B. Li, D. Y. M. Leung, E. Goleva; National Jewish Medical and Research Center, Denver, CO. RATIONALE: Upon steroid binding GCRalpha translocates to the nuclei of target cells, induces histone H4 acetylation to active gene transcription and engages histone deacetylases (HDACs) to transrepress. The majority of PBMC from steroid resistant (SR) asthmatics have impaired nuclear localization of GCRalpha despite treatment with GCs. The role of GCRbeta (dominant negative isoform of the steroid receptor) in this process has not been addressed. METHODS: Histone H4 lysines (K) 5, 8, 16 acetylation was evaluated by immunofluorescent staining in PBMC from 3 normal, 3 SS, 3 SR asthmatics and in wild type or retrovirally transduced with bicistronic GFPGCRbeta construct murine lymphoid DO11.10 cell line. GCRalpha cellular was assessed by immunofluorescent staining and Western blotting in DO11.10 cells. Expression of HDACs mRNA was evaluated by real time PCR. RESULTS: Dexamethasone (DEX) treatment for 6h significantly induced H4 K5, K16 acetylation in normal PBMC (p=0.0038 as compared to baseline). This was also observed in PBMC of SS patients (p=0.026 as compared to baseline), but not in SR patients. Impaired GCRalpha nuclear translocation in response to DEX was observed in a GCRbeta expressing DO11.10 cell line, but not in a non-transduced cell line. DEX treatment enhanced H4 K16 acetylation in the wild type DO11.10 cell line (p=0.027), importantly, no change was observed in the GCRbeta transgenic cell line. The pattern of HDAC mRNA expression was similar between two cell lines. CONCLUSIONS: GCRbeta inhibits GCRalpha nuclear translocation in response to GCs and this results in inhibition of steroid induced histone H4 acetylation.
559
SUNDAY
Lipoxin A4 (LXA4) and Leukotriene C4 (LTC4) Levels in Nasal Lavages after Intranasal Provocation with Aspirin in Aspirin Sensitive and Aspirin Tolerant Patients M. Kupczyk, I. Kupry´s-Lipi´nska, M. Boche´nska-Marciniak, A. Antczak, P. Górski, P. Kuna; Department of Pneumonology and Allergy, Medical University of Lodz, Lodz, POLAND. RATIONALE: The aim of our study was to determine lipoxin A4 and leukotriene C4 (LTC4) levels in nasal lavages after intranasal provocation with aspirin in aspirin intolerant (AIA) in comparison to aspirin tolerant (ATA) subjects. METHODS: In 12 AIA and 8 ATA asthmatics intranasal challenge were performed with placebo or 16 mg of lysine-aspirin (Lys-ASA) on a consecutive day. Nasal lavages were collected 2 hours after challenge and lipoxin A4 and leukotrien C4 levels were determined using ELISA method. RESULTS: In AIA group the mean level of LXA4 was 43±21,5 pg/ml after placebo with decrease after Lys-ASA challenge (29±17 pg/ml, p=0,015). We found increase in LXA4 level in ATA group after ASA provocation as compared to placebo (33±16 pg/ml and 52±31 pg/ml after placebo and ASA respectively, p=0,046). The comparison between AIA and ATA group revealed statistically significant difference in LXA4 levels after Lys-ASA challenge (p=0,044) but not after placebo (p=0,266). In AIA group the mean level of LTC4 was 56,5±12 pg/ml before challenge and statistically significant increase after Lys-ASA challenge to 109,5±50 pg/ml, p<0.05). In ATA group we did not observed any statistically significant differences in LTC4 level before to after ASA challenge, levels were respectively 32,8±10 pg/ml and 38,0±7 pg/ml. Although the difference between AIA and ATA groups did not reach the statistical significance before provocation there is a mark difference between groups after aspirin challenge (p<0,05). CONCLUSIONS: The results of our study show that aspirin sensitive subjects are not able to generate LXA4 after ASA challenge in contrast to ATA group. Funding: Medical University of Lodz
560
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Bronchoalveolar Lavage Leukotriene C4/D4/E4 Levels in Acute Asthma and Acute Bronchiolitis C. K. Kim1, J. Y. Koh1, M. Hoekendijk1, Y. Y. Koh2; 1Pediatric Asthma /Allergy Center, Inje University, Sanggyepaik Hospital, Seoul, REPUBLIC OF KOREA, 2Department of Pediatrics, Seoul National University Hospital, Seoul, REPUBLIC OF KOREA. RATIONALE: The pathogenesis for the relationship between acute bronchiolitis and asthma has not yet been completely elucidated. Cys-LTs are pivotal mediators in the pathophysiology of asthma, specifically, bronchoconstriction, airway inflammation and oedema. The aim of this study was to determine the levels of cys-LTs(LTC4/D4/E4 ) in bronchoalveolar lavage (BAL) fluid from patients with acute RSV bronchiolitis and acute asthma. METHODS: This study investigate the levels of cys-LTs in BAL between children with acute asthma with no identifiable RSV infection (n=18) and infants with acute bronchiolitis caused by respiratory syncytial virus (RSV) (n=20). Comparisons of the cys-LTs were made with normal controls (n=14). LTC4/D4/E4 levels were measured in concentrated BAL fluids by ELISA. RESULTS: The LTC4/D4/E4 level in both the asthma group (P<0.001) and the bronchiolitis group (P<0.05) were significantly higher than in the control group. When infants in the bronchiolitis group were divided into eosinophil (+) and eosinophil (-) subgroups, the eosinophil (+) subgroup had significantly higher LTC4/D4/E4 levels (P<0.001) compared to the control group, whereas the eosinophil(-) subgroup showed no difference in LTC4/D4/E4 levels when compared. The numbers of BAL eosinophils correlated significantly with the level of BAL LTC4/D4/E4 in the asthma group (r=0.70, P=0.001) and in the whole bronchiolitis group (r=0.53, P=0.012), respectively. CONCLUSIONS: These findings suggest that only a subgroup of RSV bronchiolitis results in an cys-LTs elevation like asthma, and this could provide a valuable framework to explain the link between RSV bronchiolitis and asthma.
561
Unc119, a Novel Src Kinase Activator, Enhances Myofibroblast Transformation of Human Lung Fibroblasts R. S. Vepachedu, N. A. Singhania, M. M. Gorska, R. Alam; Medicine, National Jewish Medical and Research Center, Denver, CO. RATIONALE: Unc119 was identified as an activator of Src Family kinases (SFK). SFK regulates -smooth muscle actin (SMA) induction and myofibroblasts transformation of fibroblasts. We examined whether Unc119 influences myofibroblast transformation of fibroblasts. METHODS: Mouse fibroblasts and Human lung fibroblasts with stable over expression Unc119 protein were obtained by retroviral infection. Unc119 deficiency was achieved by SiRNA against Unc119. The activation of SFK was checked by autophosphorylation of the proteins. The effect of Unc119 over expression on -SMA formation under TGF- 1 activation was studied on western blots and by staining -SMA and observing under immunoflorescence microscope. RESULTS: Unc119 co-localized with Fyn in fibroblasts. Overexpression caused Fyn activation in vitro and in vivo, and decreased cell migration in wound healing assay. SiRNA-mediated knockdown of Unc119 caused inhibition of Fyn activation in vivo and fibroblast proliferation. Overexpression and knockdown of Unc119 was associated with increased and decreased, respectively, expression of a-SMA in fibroblasts. CONCLUSIONS: Unc119 activates Fyn in fibroblasts and regulates its activation and proliferation. Further, Unc119 is important for myofibroblasts transformation of lung fibroblasts as determined by a-SMA expression. Unc119 and SFK may play an important role in airway remodeling. Funding: National Jewish Medical and Research Center
562
Potential Role of the Glucocorticoid Receptor (GCR) Beta in Glucocorticoid (GC) Dependent Histone H4 Acetylation L. B. Li, D. Y. M. Leung, E. Goleva; National Jewish Medical and Research Center, Denver, CO. RATIONALE: Upon steroid binding GCRalpha translocates to the nuclei of target cells, induces histone H4 acetylation to active gene transcription and engages histone deacetylases (HDACs) to transrepress. The majority of PBMC from steroid resistant (SR) asthmatics have impaired nuclear localization of GCRalpha despite treatment with GCs. The role of GCRbeta (dominant negative isoform of the steroid receptor) in this process has not been addressed. METHODS: Histone H4 lysines (K) 5, 8, 16 acetylation was evaluated by immunofluorescent staining in PBMC from 3 normal, 3 SS, 3 SR asthmatics and in wild type or retrovirally transduced with bicistronic GFPGCRbeta construct murine lymphoid DO11.10 cell line. GCRalpha cellular was assessed by immunofluorescent staining and Western blotting in DO11.10 cells. Expression of HDACs mRNA was evaluated by real time PCR. RESULTS: Dexamethasone (DEX) treatment for 6h significantly induced H4 K5, K16 acetylation in normal PBMC (p=0.0038 as compared to baseline). This was also observed in PBMC of SS patients (p=0.026 as compared to baseline), but not in SR patients. Impaired GCRalpha nuclear translocation in response to DEX was observed in a GCRbeta expressing DO11.10 cell line, but not in a non-transduced cell line. DEX treatment enhanced H4 K16 acetylation in the wild type DO11.10 cell line (p=0.027), importantly, no change was observed in the GCRbeta transgenic cell line. The pattern of HDAC mRNA expression was similar between two cell lines. CONCLUSIONS: GCRbeta inhibits GCRalpha nuclear translocation in response to GCs and this results in inhibition of steroid induced histone H4 acetylation.
559
SUNDAY
Lipoxin A4 (LXA4) and Leukotriene C4 (LTC4) Levels in Nasal Lavages after Intranasal Provocation with Aspirin in Aspirin Sensitive and Aspirin Tolerant Patients M. Kupczyk, I. Kupry´s-Lipi´nska, M. Boche´nska-Marciniak, A. Antczak, P. Górski, P. Kuna; Department of Pneumonology and Allergy, Medical University of Lodz, Lodz, POLAND. RATIONALE: The aim of our study was to determine lipoxin A4 and leukotriene C4 (LTC4) levels in nasal lavages after intranasal provocation with aspirin in aspirin intolerant (AIA) in comparison to aspirin tolerant (ATA) subjects. METHODS: In 12 AIA and 8 ATA asthmatics intranasal challenge were performed with placebo or 16 mg of lysine-aspirin (Lys-ASA) on a consecutive day. Nasal lavages were collected 2 hours after challenge and lipoxin A4 and leukotrien C4 levels were determined using ELISA method. RESULTS: In AIA group the mean level of LXA4 was 43±21,5 pg/ml after placebo with decrease after Lys-ASA challenge (29±17 pg/ml, p=0,015). We found increase in LXA4 level in ATA group after ASA provocation as compared to placebo (33±16 pg/ml and 52±31 pg/ml after placebo and ASA respectively, p=0,046). The comparison between AIA and ATA group revealed statistically significant difference in LXA4 levels after Lys-ASA challenge (p=0,044) but not after placebo (p=0,266). In AIA group the mean level of LTC4 was 56,5±12 pg/ml before challenge and statistically significant increase after Lys-ASA challenge to 109,5±50 pg/ml, p<0.05). In ATA group we did not observed any statistically significant differences in LTC4 level before to after ASA challenge, levels were respectively 32,8±10 pg/ml and 38,0±7 pg/ml. Although the difference between AIA and ATA groups did not reach the statistical significance before provocation there is a mark difference between groups after aspirin challenge (p<0,05). CONCLUSIONS: The results of our study show that aspirin sensitive subjects are not able to generate LXA4 after ASA challenge in contrast to ATA group. Funding: Medical University of Lodz
560
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
Bronchoalveolar Lavage Leukotriene C4/D4/E4 Levels in Acute Asthma and Acute Bronchiolitis C. K. Kim1, J. Y. Koh1, M. Hoekendijk1, Y. Y. Koh2; 1Pediatric Asthma /Allergy Center, Inje University, Sanggyepaik Hospital, Seoul, REPUBLIC OF KOREA, 2Department of Pediatrics, Seoul National University Hospital, Seoul, REPUBLIC OF KOREA. RATIONALE: The pathogenesis for the relationship between acute bronchiolitis and asthma has not yet been completely elucidated. Cys-LTs are pivotal mediators in the pathophysiology of asthma, specifically, bronchoconstriction, airway inflammation and oedema. The aim of this study was to determine the levels of cys-LTs(LTC4/D4/E4 ) in bronchoalveolar lavage (BAL) fluid from patients with acute RSV bronchiolitis and acute asthma. METHODS: This study investigate the levels of cys-LTs in BAL between children with acute asthma with no identifiable RSV infection (n=18) and infants with acute bronchiolitis caused by respiratory syncytial virus (RSV) (n=20). Comparisons of the cys-LTs were made with normal controls (n=14). LTC4/D4/E4 levels were measured in concentrated BAL fluids by ELISA. RESULTS: The LTC4/D4/E4 level in both the asthma group (P<0.001) and the bronchiolitis group (P<0.05) were significantly higher than in the control group. When infants in the bronchiolitis group were divided into eosinophil (+) and eosinophil (-) subgroups, the eosinophil (+) subgroup had significantly higher LTC4/D4/E4 levels (P<0.001) compared to the control group, whereas the eosinophil(-) subgroup showed no difference in LTC4/D4/E4 levels when compared. The numbers of BAL eosinophils correlated significantly with the level of BAL LTC4/D4/E4 in the asthma group (r=0.70, P=0.001) and in the whole bronchiolitis group (r=0.53, P=0.012), respectively. CONCLUSIONS: These findings suggest that only a subgroup of RSV bronchiolitis results in an cys-LTs elevation like asthma, and this could provide a valuable framework to explain the link between RSV bronchiolitis and asthma.
561
Unc119, a Novel Src Kinase Activator, Enhances Myofibroblast Transformation of Human Lung Fibroblasts R. S. Vepachedu, N. A. Singhania, M. M. Gorska, R. Alam; Medicine, National Jewish Medical and Research Center, Denver, CO. RATIONALE: Unc119 was identified as an activator of Src Family kinases (SFK). SFK regulates -smooth muscle actin (SMA) induction and myofibroblasts transformation of fibroblasts. We examined whether Unc119 influences myofibroblast transformation of fibroblasts. METHODS: Mouse fibroblasts and Human lung fibroblasts with stable over expression Unc119 protein were obtained by retroviral infection. Unc119 deficiency was achieved by SiRNA against Unc119. The activation of SFK was checked by autophosphorylation of the proteins. The effect of Unc119 over expression on -SMA formation under TGF- 1 activation was studied on western blots and by staining -SMA and observing under immunoflorescence microscope. RESULTS: Unc119 co-localized with Fyn in fibroblasts. Overexpression caused Fyn activation in vitro and in vivo, and decreased cell migration in wound healing assay. SiRNA-mediated knockdown of Unc119 caused inhibition of Fyn activation in vivo and fibroblast proliferation. Overexpression and knockdown of Unc119 was associated with increased and decreased, respectively, expression of a-SMA in fibroblasts. CONCLUSIONS: Unc119 activates Fyn in fibroblasts and regulates its activation and proliferation. Further, Unc119 is important for myofibroblasts transformation of lung fibroblasts as determined by a-SMA expression. Unc119 and SFK may play an important role in airway remodeling. Funding: National Jewish Medical and Research Center
562