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Protease Inhibitor Reduces Airway Response and Inflammation In Mouse Model Of Cockroach Allergy
AB224 Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
The Effects Of Pollen Season On Reactions To Subcutaneous Immunotherapy Dr. Devi Jhaveri, DO1, Dr. Julie Abraham, MD2, Megan Betteley, BS3, Dr. Mary Ann O’Riordan, PhD2, Dr. Theodore H. Sher, MD, FAAAAI4, Dr. Robert W. Hostoffer, DO4, Dr. Haig Tcheurekdjian, MD, FAAAAI4; 1 University Hospitals, South Euclid, OH, 2Rainbow Babies and Children’s Hospital, Cleveland, OH, 3Case Western Reserve University School of Medicine, Cleveland, OH, 4Allergy/Immunology Associates, Inc., South Euclid, OH. RATIONALE: It is common practice to hold build-up dosing of subcutaneous allergen immunotherapy (SCIT) during pollen season. We hypothesized that build-up dosing during pollen season, as compared to held dosing, would not increase the rate of adverse reactions to SCIT. METHODS: A prospective, blinded, randomized study was conducted _5 years old on build-up over four months in northeast Ohio. Subjects > phase of SCIT for tree and/or grass pollens were randomized to one of two groups: build-up SCIT dosing (Group 1) or held SCIT dosing (Group 2) through the pollen seasons. Each SCIT visit was analyzed for immediate and delayed reactions via patient questionnaire. RESULTS: 47 patients were randomized; 36 had 108 visits during tree season and 37 had 100 visits during grass season. Group 1 and 2 had no differences in age, sex, or asthma history. Tree season immediate reaction rate was 48% (all local) vs. 50% (5% of which were systemic) for group 1 and 2, respectively (p50.81). Tree season delayed reaction rate was 10% (all local) vs. 8% (3% of which were systemic) for group 1 and 2, respectively (p50.76). Grass season immediate reaction rate was 34% vs. 39% for group 1 and 2, respectively (p50.59). All were local. Grass season delayed reaction rate was 7% vs. 5% for group 1 and 2 respectively (p51). All were local. CONCLUSIONS: There is no significant difference in the rate of reactions in build up versus held dosing of SCIT in tree/grass pollen season.
774
PhD1; 1Division of Allergy & Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Sher-I-Kashmir Institute of Medical Sciences, Jammu and Kashmir, India. RATIONALE: Aryl hydrocarbon Receptor (AhR) is a multifunctional regulator that senses and responds to environmental stimuli and plays a critical role in linking environmental exposure to the development of allergic diseases. We sought to investigate whether AhR plays a role in mediating cockroach allergen induced immune responses. METHODS: AhR expression in fibroblasts of human airway from asthmatic and healthy individuals was examined by immune-staining. FITC-labeled Bla g 2 uptake by fibroblasts was also tested. Differential levels of Transforming growth factor b1 (TGFb1) and IL6 were measured when fibroblasts were treated with cockroach extract (CRE) in the presence or absence of AhR agonist, TCDD and antagonist CH122319, or using fibroblasts with AhR knock-down. a-SMA, MMP2, and ADAM33 expression were also examined in AhR knock-down fibroblasts. Similar observation will be made in lung fibroblasts from wild type (WT) and knock-out (AhR-/-) mice. RESULTS: Airway tissues from patients with asthma have more fibroblasts in the basement membrane as compared to those from healthy controls. AhR expression was higher in airway fibroblasts from asthmatic subjects. TGFb1 production was significantly increased in CRE-treated fibroblasts, and further enhanced by TCDD, but inhibited by CH122319 and AhR knock-down. Furthermore, a-SMA, MMP2, and ADAM33 expression was reduced in AhR knock-down fibroblasts. Validation for these findings is ongoing in the lung fibroblasts that we have recently cultured from WT and AhR-/ mice. CONCLUSIONS: These results provide evidence supporting a role of AhR in mediating cockroach allergen induced allergic immune responses, cell differentiation, and possibly airway remodeling in asthma.
775
777
MONDAY
Protease Inhibitor Reduces Airway Response and Inflammation In Mouse Model Of Cockroach Allergy Mr. Sanjay Saw, MSc, Dr. Naveen Arora, PhD; CSIR Institute of Genomics and Integrative Biology, New Delhi, India. RATIONALE: Serine protease(s) are potent inducer of airway resistance and inflammation and the allergic response may be modified by proteases inhibitor. In the present study, effect of serine protease inhibitor in cockroach allergen induced airway resistance and inflammation was determined in mouse model. METHODS: Mice were immunized with cockroach extract (CE) or Per a 10 and treated with 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) 1 h before challenge. AHR was measured, one day after the last challenge and mice were euthanized to get BALF, blood and lung to evaluate of inflammation and oxidative stress. RESULTS: AEBSF treatment to mice reduced more than 90% AHR in CE and in Per a 10 challenged mice (p<0.05). EPO, IgE, IL-4, IL-5 and IL-13 levels were significantly (p<0.05) reduced in AEBSF treated mice as compared to untreated group. The treatment lowered the total cell, eosinophil and neutrophil count in BALF to 90, 80 and 83 % in CE and 100, 62 and 90 % in Per a 10 challenged mice (p<0.05), respectively. Lung histology revealed that AEBSF treated mice had reduced inflammation and low mucus production in airways. Furthermore, the treatment had significantly reduced 8-isoprostane and ROS level to 88 and 86 in CE and 82 and 83 % in per a 10 challenged mice in comparison to untreated group (p<0.05). CONCLUSIONS: AEBSF reduces allergen induced airway resistance and inflammation indicating that it might be used in ‘‘add-on’’ therapy for respiratory diseases. The therapeutic effect of inhibitor is independent of protease activity of allergen.
776
Aryl Hydrocarbon Receptor (AhR) Modulates Cockroach Allergen Induced TGF Beta 1 Secretion In Fibroblasts Sarah Mirza1,2, Yufeng Zhou, MD, PhD1, Priya Tripathi, PhD1, Liang Yuan1, Beverly Plunkett, MS1, Allen Myers, Ph.D1, Peisong Gao, MD,
Effect Of Peanut Allergens On Intestinal Barrier Permeability and Tight Junction Localisation In Caco-2 Cell Cultures Prof. Cenk Suphioglu, PhD1, Ms. Dwan Price, BSc Hons1, Prof. Leigh Ackland, PhD2, A. Wesley Burks, MD, FAAAAI3, Dr. Matthew Knight, PhD4; 1Deakin University, Australia, 2Deakin Universisty, Australia, 3 University of North Carolina, Chapel Hill, NC, 4Department of Primary Industries, Australia. RATIONALE: To investigate allergen-epithelial interactions of peanut allergens with the human intestinal epithelium. METHODS: We investigated the intestinal epithelial transport of peanut extract using the human Caco-2 cell culture model, exposed to varying concentrations of crude peanut extract. Western and immunofluorescence analysis were used to identify the cellular and molecular changes of peanut extract on the intestinal epithelium. RESULTS: Following exposure of Caco-2 cells to 1 or 3mg/mL peanut extract, strong binding of the peanut allergens Ara h 1 and Ara h 2 to the apical cellular membrane was observed. These allergens were also observed to cross the Caco-2 monolayers. Treatment with peanut extract resulted in a decrease in transepithelial electrical resistance, indicating compromised barrier integrity, but not to the extent to allow increased flux of Lucifer Yellow. Total cellular protein levels of the tight junction proteins occludin, junctional adhesion molecule-A (JAM-A), claudin-1 and Zonula occludin-1 (ZO-1) remained unchanged. However, colocalisation of the transmembrane tight junction proteins occludin, JAM-A and claudin-1, with the intracellular adhesion protein ZO-1, were significantly altered. CONCLUSIONS: The disruption of Caco-2 barrier integrity through disrupted tight junctions may enable movement of peanut proteins across the intestinal epithelium without M cell involvement. This may account for peanut’s increased allergenicity, compared to other food allergens, and provide an explanation for the potency of peanut allergens in eliciting an immune response. Indeed, this may be similar to that of immune adjuvants that alter barrier integrity to obtain artificial immune stimulation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2014
The Effects Of Pollen Season On Reactions To Subcutaneous Immunotherapy Dr. Devi Jhaveri, DO1, Dr. Julie Abraham, MD2, Megan Betteley, BS3, Dr. Mary Ann O’Riordan, PhD2, Dr. Theodore H. Sher, MD, FAAAAI4, Dr. Robert W. Hostoffer, DO4, Dr. Haig Tcheurekdjian, MD, FAAAAI4; 1 University Hospitals, South Euclid, OH, 2Rainbow Babies and Children’s Hospital, Cleveland, OH, 3Case Western Reserve University School of Medicine, Cleveland, OH, 4Allergy/Immunology Associates, Inc., South Euclid, OH. RATIONALE: It is common practice to hold build-up dosing of subcutaneous allergen immunotherapy (SCIT) during pollen season. We hypothesized that build-up dosing during pollen season, as compared to held dosing, would not increase the rate of adverse reactions to SCIT. METHODS: A prospective, blinded, randomized study was conducted _5 years old on build-up over four months in northeast Ohio. Subjects > phase of SCIT for tree and/or grass pollens were randomized to one of two groups: build-up SCIT dosing (Group 1) or held SCIT dosing (Group 2) through the pollen seasons. Each SCIT visit was analyzed for immediate and delayed reactions via patient questionnaire. RESULTS: 47 patients were randomized; 36 had 108 visits during tree season and 37 had 100 visits during grass season. Group 1 and 2 had no differences in age, sex, or asthma history. Tree season immediate reaction rate was 48% (all local) vs. 50% (5% of which were systemic) for group 1 and 2, respectively (p50.81). Tree season delayed reaction rate was 10% (all local) vs. 8% (3% of which were systemic) for group 1 and 2, respectively (p50.76). Grass season immediate reaction rate was 34% vs. 39% for group 1 and 2, respectively (p50.59). All were local. Grass season delayed reaction rate was 7% vs. 5% for group 1 and 2 respectively (p51). All were local. CONCLUSIONS: There is no significant difference in the rate of reactions in build up versus held dosing of SCIT in tree/grass pollen season.
774
PhD1; 1Division of Allergy & Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Sher-I-Kashmir Institute of Medical Sciences, Jammu and Kashmir, India. RATIONALE: Aryl hydrocarbon Receptor (AhR) is a multifunctional regulator that senses and responds to environmental stimuli and plays a critical role in linking environmental exposure to the development of allergic diseases. We sought to investigate whether AhR plays a role in mediating cockroach allergen induced immune responses. METHODS: AhR expression in fibroblasts of human airway from asthmatic and healthy individuals was examined by immune-staining. FITC-labeled Bla g 2 uptake by fibroblasts was also tested. Differential levels of Transforming growth factor b1 (TGFb1) and IL6 were measured when fibroblasts were treated with cockroach extract (CRE) in the presence or absence of AhR agonist, TCDD and antagonist CH122319, or using fibroblasts with AhR knock-down. a-SMA, MMP2, and ADAM33 expression were also examined in AhR knock-down fibroblasts. Similar observation will be made in lung fibroblasts from wild type (WT) and knock-out (AhR-/-) mice. RESULTS: Airway tissues from patients with asthma have more fibroblasts in the basement membrane as compared to those from healthy controls. AhR expression was higher in airway fibroblasts from asthmatic subjects. TGFb1 production was significantly increased in CRE-treated fibroblasts, and further enhanced by TCDD, but inhibited by CH122319 and AhR knock-down. Furthermore, a-SMA, MMP2, and ADAM33 expression was reduced in AhR knock-down fibroblasts. Validation for these findings is ongoing in the lung fibroblasts that we have recently cultured from WT and AhR-/ mice. CONCLUSIONS: These results provide evidence supporting a role of AhR in mediating cockroach allergen induced allergic immune responses, cell differentiation, and possibly airway remodeling in asthma.
775
777
MONDAY
Protease Inhibitor Reduces Airway Response and Inflammation In Mouse Model Of Cockroach Allergy Mr. Sanjay Saw, MSc, Dr. Naveen Arora, PhD; CSIR Institute of Genomics and Integrative Biology, New Delhi, India. RATIONALE: Serine protease(s) are potent inducer of airway resistance and inflammation and the allergic response may be modified by proteases inhibitor. In the present study, effect of serine protease inhibitor in cockroach allergen induced airway resistance and inflammation was determined in mouse model. METHODS: Mice were immunized with cockroach extract (CE) or Per a 10 and treated with 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) 1 h before challenge. AHR was measured, one day after the last challenge and mice were euthanized to get BALF, blood and lung to evaluate of inflammation and oxidative stress. RESULTS: AEBSF treatment to mice reduced more than 90% AHR in CE and in Per a 10 challenged mice (p<0.05). EPO, IgE, IL-4, IL-5 and IL-13 levels were significantly (p<0.05) reduced in AEBSF treated mice as compared to untreated group. The treatment lowered the total cell, eosinophil and neutrophil count in BALF to 90, 80 and 83 % in CE and 100, 62 and 90 % in Per a 10 challenged mice (p<0.05), respectively. Lung histology revealed that AEBSF treated mice had reduced inflammation and low mucus production in airways. Furthermore, the treatment had significantly reduced 8-isoprostane and ROS level to 88 and 86 in CE and 82 and 83 % in per a 10 challenged mice in comparison to untreated group (p<0.05). CONCLUSIONS: AEBSF reduces allergen induced airway resistance and inflammation indicating that it might be used in ‘‘add-on’’ therapy for respiratory diseases. The therapeutic effect of inhibitor is independent of protease activity of allergen.
776
Aryl Hydrocarbon Receptor (AhR) Modulates Cockroach Allergen Induced TGF Beta 1 Secretion In Fibroblasts Sarah Mirza1,2, Yufeng Zhou, MD, PhD1, Priya Tripathi, PhD1, Liang Yuan1, Beverly Plunkett, MS1, Allen Myers, Ph.D1, Peisong Gao, MD,
Effect Of Peanut Allergens On Intestinal Barrier Permeability and Tight Junction Localisation In Caco-2 Cell Cultures Prof. Cenk Suphioglu, PhD1, Ms. Dwan Price, BSc Hons1, Prof. Leigh Ackland, PhD2, A. Wesley Burks, MD, FAAAAI3, Dr. Matthew Knight, PhD4; 1Deakin University, Australia, 2Deakin Universisty, Australia, 3 University of North Carolina, Chapel Hill, NC, 4Department of Primary Industries, Australia. RATIONALE: To investigate allergen-epithelial interactions of peanut allergens with the human intestinal epithelium. METHODS: We investigated the intestinal epithelial transport of peanut extract using the human Caco-2 cell culture model, exposed to varying concentrations of crude peanut extract. Western and immunofluorescence analysis were used to identify the cellular and molecular changes of peanut extract on the intestinal epithelium. RESULTS: Following exposure of Caco-2 cells to 1 or 3mg/mL peanut extract, strong binding of the peanut allergens Ara h 1 and Ara h 2 to the apical cellular membrane was observed. These allergens were also observed to cross the Caco-2 monolayers. Treatment with peanut extract resulted in a decrease in transepithelial electrical resistance, indicating compromised barrier integrity, but not to the extent to allow increased flux of Lucifer Yellow. Total cellular protein levels of the tight junction proteins occludin, junctional adhesion molecule-A (JAM-A), claudin-1 and Zonula occludin-1 (ZO-1) remained unchanged. However, colocalisation of the transmembrane tight junction proteins occludin, JAM-A and claudin-1, with the intracellular adhesion protein ZO-1, were significantly altered. CONCLUSIONS: The disruption of Caco-2 barrier integrity through disrupted tight junctions may enable movement of peanut proteins across the intestinal epithelium without M cell involvement. This may account for peanut’s increased allergenicity, compared to other food allergens, and provide an explanation for the potency of peanut allergens in eliciting an immune response. Indeed, this may be similar to that of immune adjuvants that alter barrier integrity to obtain artificial immune stimulation.