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Mouse Bone Marrow Derived Mesenchymal Stem Cells Supress Airway Inflammation In Both Chronic and Acute Murine Asthma Model
Abstracts AB141
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
Mouse Bone Marrow Derived Mesenchymal Stem Cells Supress Airway Inflammation In Both Chronic and Acute Murine Asthma Model Dr. Tunc Akkoc, PhD1, Mr. Ismail Ogulur, MSc1, Ms. Gulben Gurhan1, Ms. Ayca Aksoy, MSc2, Dr. Gokhan Duruksu, PhD2, Dr. Deniz Filinte, MD3, Dr. Erdem Kombak3, Prof. Isil B. Barlan, MD1, Prof. Erdal Karaoz, PhD2; 1Marmara University Division of Pediatric Allergy-Immunology, 2Kocaeli University Center for Stem Cell and Gene Therapies Research and Practise, 3Marmara University Department of Pathology. RATIONALE: The aim of the present study was to investigate the efficacy of mouse compact bone (mCB) derived MSCs on lung histopathology and lymphocyte proliferation. METHODS: mCB-MCS were isolated from BALB/c mice, characterized and marked by GFP. To generate murine models of chronic and acute asthma, mice were i.p. sensitized with OVA and exposed to aerolized OVA. mCB-MSCs (2.5x105 cells) were administrated i.v. after last nebulization. Mice were sacrified, and splenocytes and lung lymphocytes were isolated and marked with CFSE. Cells stimulated with OVA (40mg/ml) were cultured under suitable conditions for 5 days. Flow cytometric analysis and histopathological examination of lungs were evaluated. In histopathological analysis, the measurements were performed from minimum 5 points of each airway and mean values were calculated. Goblet cells stained with PAS enumarated in 1500 cells. RESULTS: In sections stained with H&E, the distal [without MCS chronic:29,9mm acute:32,03mm; with MSC chronic:13,3mm acute:12,25mm] and proximal [without MCS chronic:42,6mm acute:28,97mm; with MSC chronic:17,4mm acute:18,9mm] airway epithelial thicknesses were observed to decrease in both mouse models. Likewise, in sections stained with PAS, a significant reduction in number of hyperplasic goblet cells in the proximal [without MSC chronic:140 acute:1200; with MSC chronic:4 acute:211] and distal [without MSC chronic:55 acute:118; with MSC chronic:0 acute:0] airways was observed. Morever, in the CFSE staining experiment, CB-MSCs inhibited lymphocyte proliferation in both asthma model. CONCLUSIONS: The results reported here provide that mCB-MSCs may provide powerful alternative therapeutic for the treatment of chronic and acute asthma. This study was supported by TUBITAK-SBAG (110S368).
495
Profiling Eicosanoids In Breath Condensates Of Asthmatic and Healthy Children Dr. Li-chen Chen1,2, Prof. Jing-Long Huang1,2, Mrs. AI-Hsuan Wu1, Prof. Ming-ling Kuo3, Prof. Shau-ku Huang4,5; 1Chang-Gung Memorial Hospital, Taoyuan, Taiwan, 2Chang-Gung University, Taoyuan, Taiwan, 3 Department of Microbiology and Immunology, Institute of Basic Medical Science, Taoyuan, Taiwan, 4Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan, 5 Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Eicosanoids are lipid mediators implicated in the regulation of allergic inflammation responses and have been considered as potential biomarkers for asthmatic children. The objective of this study was to investigate profiles of 10 selected eicosanoid metabolites in exhaled breath condensates (EBCs) of children with asthma in comparison to those of healthy children. METHODS: EBCs were from 175 children (aged 962.3 years) with stable atopic asthma (58 using inhaled steroids) and 125 healthy controls (10.861.2 years). Either high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS) or enzymatic immunoassays (EIA) were used to measure 10 different metabolites. In addition, exhaled nitric oxide levels (FeNO) and bronchial hyperresponsiveness were assessed through a methacholine challenge test (PC20) in all subjects. RESULTS: Among the ten eicosanoids, the levels of LTB4 (5.71 pg/ml vs 0.44 pg/ml; P<0.001), LTE4 (9.13 pg/ml vs 5.38 pg/ml; P<0.001), and PGE2 (13.29 pg/ml vs 6.77pg/ml; P<0.018) were significantly higher in asthmatics than in healthy children, while 11-dehydro TXB2 was significantly less abundant (3.55 pg/ml vs 1.0 pg/ml; P50.045) in asthmatics. The levels of eicosanoids demonstrated no appreciable relationship to
asthma severity. From the fasting lipid profiles, we found a slightly higher level of cholesterol, with all other elements being within the normal range. These parameters discriminated asthmatics from healthy children better than FEV1, FeNO or PC20. CONCLUSIONS: In the current study, a composite of LTB4, LTE4 and PGE2 levels in EBCs was distinguishable between asthmatic and healthy subjects, suggesting the potential utility of assessing EBC’s eicosanoids as inflammatory markers for childhood asthma.
496
Increased IP-10 Expressions In Nasal Fibroblasts From Patients With Refractory Chronic Rhinosinusitis and Asthma Dr. Mamoru Yoshikawa, MD1, Dr. Kota Wada, MD1, Dr. Tsuyoshi Yoshimura, MD2, Dr. Daiya Asaka, MD2, Prof. Hiroshi Moriyama, MD2; 1Department of Otorhinolaryngology, Toho University School of Medicine, Tokyo, Japan, 2Department of Otorhinolaryngology, Jikei University School of Medicine, Tokyo, Japan. RATIONALE: Chronic rhinosinusitis (CRS) is characterized by local inflammation of the sinonasal tissues. CRS patients with nasal polyps and asthma often develop acute exacerbation of sinonasal symptoms after upper respiratory tract infections. However, the influence of concomitant asthma on the nasal immune response to viral infection remains unclear. METHODS: Specimens of nasal polyp and mucosal tissues were obtained from 3 groups of CRS patients (n514 per group): 1) patients without asthma (CRS group), 2) patients with aspirin-tolerant asthma (ATA group), and 3) patients with aspirin-intolerant asthma (AIA group). Nasal fibroblasts isolated from the specimens were stimulated with poly I:C. IP-10 and I-TAC expressions were analyzed by the quantitative real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay. RESULTS: Nasal fibroblasts from the ATA and AIA groups showed significantly enhanced expression of IP-10 mRNA and protein after poly I:C stimulation compared with cells from the CRS group and the control group (normal nasal mucosa). However, the expression of I-TAC protein was not detected. CONCLUSIONS: Our findings suggest that CRS associated with asthma may become intractable through the over-production of IP-10 in response to viral infection.
497
Vitamin D Regulating TGF-b Induced Epithelial-Mesenchymal Transition Ms. Kimberly Fischer1, Devendra K. Agrawal2; 1Creighton University, Omaha, 2Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, NE. RATIONALE: Subepithelial fibrosis is a hallmark characteristic of airway remodeling in asthma. An important regulator of fibrosis is transforming growth factor b (TGF-b). TGF-b can induce airway remodeling in epithelial cells through induction of epithelial-mesenchymal transition (EMT). This represents a novel therapeutic target in asthma. Vitamin D has immunomodulatory functions that could inhibit subepithelial fibrosis in asthma. METHODS: Human bronchial epithelial cells (BEAS-2B) were stimulated with the active form of Vitamin D, calcitriol (100 nM). After 24 hours, TGF-b1 (10 ng/ml) or TGF-b2 (10 ng/ml) was added to the cells for an additional 48 hours. Following stimulation, mRNA and protein was isolated and mRNA transcripts for E-cadherin, Snail, MMP2, MMP9 were analyzed by qPCR while E-cadherin and Snail were examined by Western blot. An invasion assay and scratch wound assay were performed to identify the migratory properties of the cells following stimulation. RESULTS: TGF-b1 and TGF-b2 decreased E-cadherin expression and increased the expression of Snail, MMP2, and MMP9 mRNA transcript levels. TGF-b also increased cell invasion. The effect of TGF-b on these markers and motility was impeded by the presence of calcitriol as ascertained at the mRNA, protein levels, and invasion assays. CONCLUSIONS: These data suggest that both TGF-b1 and TGF-b2 can regulate EMT expression markers, which can be impeded by stimulation with calcitriol in human airway epithelial cells. Therefore, calcitriol could be a potential therapeutic agent in the prevention and management of subepithelial fibrosis.
SUNDAY
494
J ALLERGY CLIN IMMUNOL VOLUME 133, NUMBER 2
Mouse Bone Marrow Derived Mesenchymal Stem Cells Supress Airway Inflammation In Both Chronic and Acute Murine Asthma Model Dr. Tunc Akkoc, PhD1, Mr. Ismail Ogulur, MSc1, Ms. Gulben Gurhan1, Ms. Ayca Aksoy, MSc2, Dr. Gokhan Duruksu, PhD2, Dr. Deniz Filinte, MD3, Dr. Erdem Kombak3, Prof. Isil B. Barlan, MD1, Prof. Erdal Karaoz, PhD2; 1Marmara University Division of Pediatric Allergy-Immunology, 2Kocaeli University Center for Stem Cell and Gene Therapies Research and Practise, 3Marmara University Department of Pathology. RATIONALE: The aim of the present study was to investigate the efficacy of mouse compact bone (mCB) derived MSCs on lung histopathology and lymphocyte proliferation. METHODS: mCB-MCS were isolated from BALB/c mice, characterized and marked by GFP. To generate murine models of chronic and acute asthma, mice were i.p. sensitized with OVA and exposed to aerolized OVA. mCB-MSCs (2.5x105 cells) were administrated i.v. after last nebulization. Mice were sacrified, and splenocytes and lung lymphocytes were isolated and marked with CFSE. Cells stimulated with OVA (40mg/ml) were cultured under suitable conditions for 5 days. Flow cytometric analysis and histopathological examination of lungs were evaluated. In histopathological analysis, the measurements were performed from minimum 5 points of each airway and mean values were calculated. Goblet cells stained with PAS enumarated in 1500 cells. RESULTS: In sections stained with H&E, the distal [without MCS chronic:29,9mm acute:32,03mm; with MSC chronic:13,3mm acute:12,25mm] and proximal [without MCS chronic:42,6mm acute:28,97mm; with MSC chronic:17,4mm acute:18,9mm] airway epithelial thicknesses were observed to decrease in both mouse models. Likewise, in sections stained with PAS, a significant reduction in number of hyperplasic goblet cells in the proximal [without MSC chronic:140 acute:1200; with MSC chronic:4 acute:211] and distal [without MSC chronic:55 acute:118; with MSC chronic:0 acute:0] airways was observed. Morever, in the CFSE staining experiment, CB-MSCs inhibited lymphocyte proliferation in both asthma model. CONCLUSIONS: The results reported here provide that mCB-MSCs may provide powerful alternative therapeutic for the treatment of chronic and acute asthma. This study was supported by TUBITAK-SBAG (110S368).
495
Profiling Eicosanoids In Breath Condensates Of Asthmatic and Healthy Children Dr. Li-chen Chen1,2, Prof. Jing-Long Huang1,2, Mrs. AI-Hsuan Wu1, Prof. Ming-ling Kuo3, Prof. Shau-ku Huang4,5; 1Chang-Gung Memorial Hospital, Taoyuan, Taiwan, 2Chang-Gung University, Taoyuan, Taiwan, 3 Department of Microbiology and Immunology, Institute of Basic Medical Science, Taoyuan, Taiwan, 4Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan, 5 Johns Hopkins Asthma and Allergy Center, Baltimore, MD. RATIONALE: Eicosanoids are lipid mediators implicated in the regulation of allergic inflammation responses and have been considered as potential biomarkers for asthmatic children. The objective of this study was to investigate profiles of 10 selected eicosanoid metabolites in exhaled breath condensates (EBCs) of children with asthma in comparison to those of healthy children. METHODS: EBCs were from 175 children (aged 962.3 years) with stable atopic asthma (58 using inhaled steroids) and 125 healthy controls (10.861.2 years). Either high performance liquid chromatography coupled with tandem mass spectrometry (LC/MS) or enzymatic immunoassays (EIA) were used to measure 10 different metabolites. In addition, exhaled nitric oxide levels (FeNO) and bronchial hyperresponsiveness were assessed through a methacholine challenge test (PC20) in all subjects. RESULTS: Among the ten eicosanoids, the levels of LTB4 (5.71 pg/ml vs 0.44 pg/ml; P<0.001), LTE4 (9.13 pg/ml vs 5.38 pg/ml; P<0.001), and PGE2 (13.29 pg/ml vs 6.77pg/ml; P<0.018) were significantly higher in asthmatics than in healthy children, while 11-dehydro TXB2 was significantly less abundant (3.55 pg/ml vs 1.0 pg/ml; P50.045) in asthmatics. The levels of eicosanoids demonstrated no appreciable relationship to
asthma severity. From the fasting lipid profiles, we found a slightly higher level of cholesterol, with all other elements being within the normal range. These parameters discriminated asthmatics from healthy children better than FEV1, FeNO or PC20. CONCLUSIONS: In the current study, a composite of LTB4, LTE4 and PGE2 levels in EBCs was distinguishable between asthmatic and healthy subjects, suggesting the potential utility of assessing EBC’s eicosanoids as inflammatory markers for childhood asthma.
496
Increased IP-10 Expressions In Nasal Fibroblasts From Patients With Refractory Chronic Rhinosinusitis and Asthma Dr. Mamoru Yoshikawa, MD1, Dr. Kota Wada, MD1, Dr. Tsuyoshi Yoshimura, MD2, Dr. Daiya Asaka, MD2, Prof. Hiroshi Moriyama, MD2; 1Department of Otorhinolaryngology, Toho University School of Medicine, Tokyo, Japan, 2Department of Otorhinolaryngology, Jikei University School of Medicine, Tokyo, Japan. RATIONALE: Chronic rhinosinusitis (CRS) is characterized by local inflammation of the sinonasal tissues. CRS patients with nasal polyps and asthma often develop acute exacerbation of sinonasal symptoms after upper respiratory tract infections. However, the influence of concomitant asthma on the nasal immune response to viral infection remains unclear. METHODS: Specimens of nasal polyp and mucosal tissues were obtained from 3 groups of CRS patients (n514 per group): 1) patients without asthma (CRS group), 2) patients with aspirin-tolerant asthma (ATA group), and 3) patients with aspirin-intolerant asthma (AIA group). Nasal fibroblasts isolated from the specimens were stimulated with poly I:C. IP-10 and I-TAC expressions were analyzed by the quantitative real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay. RESULTS: Nasal fibroblasts from the ATA and AIA groups showed significantly enhanced expression of IP-10 mRNA and protein after poly I:C stimulation compared with cells from the CRS group and the control group (normal nasal mucosa). However, the expression of I-TAC protein was not detected. CONCLUSIONS: Our findings suggest that CRS associated with asthma may become intractable through the over-production of IP-10 in response to viral infection.
497
Vitamin D Regulating TGF-b Induced Epithelial-Mesenchymal Transition Ms. Kimberly Fischer1, Devendra K. Agrawal2; 1Creighton University, Omaha, 2Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, NE. RATIONALE: Subepithelial fibrosis is a hallmark characteristic of airway remodeling in asthma. An important regulator of fibrosis is transforming growth factor b (TGF-b). TGF-b can induce airway remodeling in epithelial cells through induction of epithelial-mesenchymal transition (EMT). This represents a novel therapeutic target in asthma. Vitamin D has immunomodulatory functions that could inhibit subepithelial fibrosis in asthma. METHODS: Human bronchial epithelial cells (BEAS-2B) were stimulated with the active form of Vitamin D, calcitriol (100 nM). After 24 hours, TGF-b1 (10 ng/ml) or TGF-b2 (10 ng/ml) was added to the cells for an additional 48 hours. Following stimulation, mRNA and protein was isolated and mRNA transcripts for E-cadherin, Snail, MMP2, MMP9 were analyzed by qPCR while E-cadherin and Snail were examined by Western blot. An invasion assay and scratch wound assay were performed to identify the migratory properties of the cells following stimulation. RESULTS: TGF-b1 and TGF-b2 decreased E-cadherin expression and increased the expression of Snail, MMP2, and MMP9 mRNA transcript levels. TGF-b also increased cell invasion. The effect of TGF-b on these markers and motility was impeded by the presence of calcitriol as ascertained at the mRNA, protein levels, and invasion assays. CONCLUSIONS: These data suggest that both TGF-b1 and TGF-b2 can regulate EMT expression markers, which can be impeded by stimulation with calcitriol in human airway epithelial cells. Therefore, calcitriol could be a potential therapeutic agent in the prevention and management of subepithelial fibrosis.
SUNDAY
494