- Email: [email protected]
A Novel Assay to Detect Specific IgE Antibodies to Helicobacter pylori in Serum
AB114 Abstracts
449
SUNDAY
Allergen-Derived Proteases and PAR-2 Participate in Allergic Sensitization K. Page, J. Ledford, P. Zhou, M. Wills-Karp; Cincinnati Children’s Medical Center, Cincinnati, OH. RATIONALE: While most aeroallergens have intrinsic protease activity, the role proteases play in modulating allergic airway disease remains unclear. Using a physiologically relevant method of sensitization, we recently showed that German cockroach (GC) feces (frass) regulated airway hyperresponsiveness (AHR) and mucin production in a protease-dependent manner. Since GC proteases can activate protease activated receptor (PAR)-2, the goal of these studies was to clarify the role of proteases and PAR-2 in the initiation of allergic airway disease. METHODS: GC frass was rendered protease-free by pretreatment with aprotinin. We compared sensitization to GC frass or protease-depleted GC frass in wild type and PAR-2-deficient mice by sensitization via intraperitoneal injection of antigen bound to alum or intratracheal inhalation of antigen. Parameters of allergic airway inflammation (AHR, serum IgE, Th2 cytokine production, cellular infiltration and mucin production) were measured. RESULTS: Sensitization to GC frass resulted in allergic airway inflammation and AHR in wild type mice using either sensitization strategy. Sensitization by inhalation of GC frass in PAR-2-deficient mice resulted in a significant decrease in all aspects of allergic airway disease. Importantly, when mice were sensitized by intraperitoneal injection, proteases and PAR-2 had no effect on allergic airway inflammation or AHR. CONCLUSIONS: Our data suggests the importance of active serine proteases in GC frass and PAR-2 during allergic sensitization when the allergen is administered by inhalation. These data suggest the importance of these intrinsic proteases in the initiation of allergic airway disease and implicate an important role for resident airway cells in mediating sensitization to an allergen.
450
Development of Severe Atopic Disease in Children after Cardiac Transplant S. Chhabra, E. A. Frazier, T. T. Perry, A. M. Scurlock, S. M. Jones; University of Arkansas for Medical Sciences/Arkansas Children’s Hospital, Little Rock, AR. RATIONALE: Recent literature indicates increased incidence of atopic disease among children following solid organ transplantation. We have identified a case series of post-cardiac transplant pediatric patients with severe atopic disease. METHODS: Case series report of 6 cardiac transplant patients followed for severe atopic disease in the Allergy/Immunology Clinic at Arkansas Children’s Hospital. RESULTS: Six post cardiac transplant patients (50% male) with mean age of 5.5 years (range 3.6-12.3 years) were identified. Mean age at transplant was 5.1 months (range 1.2-15.2 months). All patients received initial immunosuppression with prednisolone for 6-8 months and at least two other immunosuppressive agents including: tacrolimus (2/6), azathioprine (2/6), mycophenolate (4/6), and cyclosporine (3/6). Five of six patients had 3 atopic disorders with atopic dermatitis being the most prevalent (5/6 with 2 refractory cases). Other disorders included multiple food allergies (4/6), moderate-severe persistent asthma (3/6), allergic rhinitis (2/6), and drug allergy (1/6). Median peak IgE was 258 kU/L (range 4.7-2296 kU/ L) and median absolute eosinophil count one year after transplant was 745 kU/L (range 162-1400). Family history was positive for atopic disease in 5/6 patients. Four of six patients have recurrent infections, three requiring immunoglobulin replacement. CONCLUSIONS: Further work is needed to define factors that influence development of post-transplant atopic disease in pediatric patients including the role of duration and type of immunosuppressive therapy, environmental exposures, dietary exposures, and recipient/donor atopic history.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
451
A Novel Assay to Detect Specific IgE Antibodies to Helicobacter pylori in Serum H. Gutekunst1, H. James1, P. Hoffman2, L. Workman1, A. Custovic3, P. Cooper4, T. Platts-MIlls1; 1University of Virginia - Division of Allergy and Immunology, Charlottesville, VA, 2University of Virginia, Division of Infectious Disease, Charlottesville, VA, 3North West Lung Centre, Wythenshawe Hospital, University of Manchester, Manchester, UNITED KINGDOM, 4Instituto de Microbiologia, Universidad San Francisco de Quito, Quito, ECUADOR. RATIONALE: Infectious diseases can influence the development of allergic disorders. While Helicobacter pylori (H. pylori) infection is generally regarded as inducing a Th1-type immune response, its role in the development of gut mucosal inflammation remains unclear. As such, we developed a novel assay to detect specific IgE (sIgE) to three different strains of H. pylori in serum. METHODS: Sera from patients (n5289) at four clinical sites: Virginia, Ecuador, Ghana, and Ohio were assayed for levels of total and specific IgE. Using a modification of the ImmunoCAP protocol, proteins from three different strains of H. pylori (HP26695, G27, SS1) were biotinylated and bound to streptavidin to generate specific allergen CAPs used to measure sIgE to H. pylori. RESULTS: Among the four clinical sites, we detected sIgE to H. pylori in 25 patients. These positive results had a range of 0.36 - 7.43 IU/mL and similar titers were detected by CAPs from each of the three specific strains. The sample with the highest sIgE to H. pylori was in a patient with known eosinophilic esophagitis. The level of total IgE was higher in the sera positive for sIgE to H. pylori compared to sera that tested negative (p < 0.01). CONCLUSIONS: To our knowledge, this study represents the first validation of an assay to detect sIgE to H. pylori. The range of IgE detected begins to establish the validity of this assay and further investigations are currently underway to elucidate the role sIgE to H. pylori may have in diseases such as asthma and eosinophilic esophagitis.
452
Identification of the Compounds in Ku Shen (Sophora flavescentis), an active constituent of ASHMI (Anti-asthma Herbal Medicine Intervention) that Inhibit IgE synthesis A. Castillo, I. Lopez Exposito, L. Brown, W. Slotkin, M. Mishoe, B. Liang, N. Yang, X. Li; Mount Sinai School of Medicine, New York, NY. RATIONALE: Previous experiments demonstrated that the ASHMI constituent Ku Shen inhibited IgE production by a human myeloma B cell line (IC505 45.68 mg/mL). This herb was fractionated into 8 different fractions (KSF1-KSF8) with respect to polarity and acidic vs basic properties. KSF1 (a flavanoid rich fraction) showed the strongest inhibition of IgE secretion (IC50510.41 mg/mL). The aim of this study was to identify the compounds in KSF1 responsible for this suppression. METHODS: KSF1was sub-fractionated by preparative reverse-phase high performance liquid chromatography (RP-HPLC) into four fractions (KSF1a, b, c, and d). These fractions were tested for reduction of IgE production by a human myeloma B-cell line cultured at 2x105 cells/mLfor 6 days. IgE levels in supernatants were quantified by Immunocap. RESULTS: Among KSF1 sub-fractions, KSF1a had no effect on IgE production. Sub-fractions b, c and d inhibited IgE secretion in a dose dependent manner (KSF1c IC50 value 1.33 mg/mL, KSF1b IC50 value 2.32 mg/mL and KSF1d IC50 value 8.16 mg/mL). No cytotoxicity was detected using MTT assay. CONCLUSION: KSF1c fraction contains the compounds most responsible for the anti-IgE effects of Ku Shen. This fraction will be further investigated to elucidate the chemical structures of the compounds most responsible for the anti-IgE activity of Ku Shen.
449
SUNDAY
Allergen-Derived Proteases and PAR-2 Participate in Allergic Sensitization K. Page, J. Ledford, P. Zhou, M. Wills-Karp; Cincinnati Children’s Medical Center, Cincinnati, OH. RATIONALE: While most aeroallergens have intrinsic protease activity, the role proteases play in modulating allergic airway disease remains unclear. Using a physiologically relevant method of sensitization, we recently showed that German cockroach (GC) feces (frass) regulated airway hyperresponsiveness (AHR) and mucin production in a protease-dependent manner. Since GC proteases can activate protease activated receptor (PAR)-2, the goal of these studies was to clarify the role of proteases and PAR-2 in the initiation of allergic airway disease. METHODS: GC frass was rendered protease-free by pretreatment with aprotinin. We compared sensitization to GC frass or protease-depleted GC frass in wild type and PAR-2-deficient mice by sensitization via intraperitoneal injection of antigen bound to alum or intratracheal inhalation of antigen. Parameters of allergic airway inflammation (AHR, serum IgE, Th2 cytokine production, cellular infiltration and mucin production) were measured. RESULTS: Sensitization to GC frass resulted in allergic airway inflammation and AHR in wild type mice using either sensitization strategy. Sensitization by inhalation of GC frass in PAR-2-deficient mice resulted in a significant decrease in all aspects of allergic airway disease. Importantly, when mice were sensitized by intraperitoneal injection, proteases and PAR-2 had no effect on allergic airway inflammation or AHR. CONCLUSIONS: Our data suggests the importance of active serine proteases in GC frass and PAR-2 during allergic sensitization when the allergen is administered by inhalation. These data suggest the importance of these intrinsic proteases in the initiation of allergic airway disease and implicate an important role for resident airway cells in mediating sensitization to an allergen.
450
Development of Severe Atopic Disease in Children after Cardiac Transplant S. Chhabra, E. A. Frazier, T. T. Perry, A. M. Scurlock, S. M. Jones; University of Arkansas for Medical Sciences/Arkansas Children’s Hospital, Little Rock, AR. RATIONALE: Recent literature indicates increased incidence of atopic disease among children following solid organ transplantation. We have identified a case series of post-cardiac transplant pediatric patients with severe atopic disease. METHODS: Case series report of 6 cardiac transplant patients followed for severe atopic disease in the Allergy/Immunology Clinic at Arkansas Children’s Hospital. RESULTS: Six post cardiac transplant patients (50% male) with mean age of 5.5 years (range 3.6-12.3 years) were identified. Mean age at transplant was 5.1 months (range 1.2-15.2 months). All patients received initial immunosuppression with prednisolone for 6-8 months and at least two other immunosuppressive agents including: tacrolimus (2/6), azathioprine (2/6), mycophenolate (4/6), and cyclosporine (3/6). Five of six patients had 3 atopic disorders with atopic dermatitis being the most prevalent (5/6 with 2 refractory cases). Other disorders included multiple food allergies (4/6), moderate-severe persistent asthma (3/6), allergic rhinitis (2/6), and drug allergy (1/6). Median peak IgE was 258 kU/L (range 4.7-2296 kU/ L) and median absolute eosinophil count one year after transplant was 745 kU/L (range 162-1400). Family history was positive for atopic disease in 5/6 patients. Four of six patients have recurrent infections, three requiring immunoglobulin replacement. CONCLUSIONS: Further work is needed to define factors that influence development of post-transplant atopic disease in pediatric patients including the role of duration and type of immunosuppressive therapy, environmental exposures, dietary exposures, and recipient/donor atopic history.
J ALLERGY CLIN IMMUNOL FEBRUARY 2010
451
A Novel Assay to Detect Specific IgE Antibodies to Helicobacter pylori in Serum H. Gutekunst1, H. James1, P. Hoffman2, L. Workman1, A. Custovic3, P. Cooper4, T. Platts-MIlls1; 1University of Virginia - Division of Allergy and Immunology, Charlottesville, VA, 2University of Virginia, Division of Infectious Disease, Charlottesville, VA, 3North West Lung Centre, Wythenshawe Hospital, University of Manchester, Manchester, UNITED KINGDOM, 4Instituto de Microbiologia, Universidad San Francisco de Quito, Quito, ECUADOR. RATIONALE: Infectious diseases can influence the development of allergic disorders. While Helicobacter pylori (H. pylori) infection is generally regarded as inducing a Th1-type immune response, its role in the development of gut mucosal inflammation remains unclear. As such, we developed a novel assay to detect specific IgE (sIgE) to three different strains of H. pylori in serum. METHODS: Sera from patients (n5289) at four clinical sites: Virginia, Ecuador, Ghana, and Ohio were assayed for levels of total and specific IgE. Using a modification of the ImmunoCAP protocol, proteins from three different strains of H. pylori (HP26695, G27, SS1) were biotinylated and bound to streptavidin to generate specific allergen CAPs used to measure sIgE to H. pylori. RESULTS: Among the four clinical sites, we detected sIgE to H. pylori in 25 patients. These positive results had a range of 0.36 - 7.43 IU/mL and similar titers were detected by CAPs from each of the three specific strains. The sample with the highest sIgE to H. pylori was in a patient with known eosinophilic esophagitis. The level of total IgE was higher in the sera positive for sIgE to H. pylori compared to sera that tested negative (p < 0.01). CONCLUSIONS: To our knowledge, this study represents the first validation of an assay to detect sIgE to H. pylori. The range of IgE detected begins to establish the validity of this assay and further investigations are currently underway to elucidate the role sIgE to H. pylori may have in diseases such as asthma and eosinophilic esophagitis.
452
Identification of the Compounds in Ku Shen (Sophora flavescentis), an active constituent of ASHMI (Anti-asthma Herbal Medicine Intervention) that Inhibit IgE synthesis A. Castillo, I. Lopez Exposito, L. Brown, W. Slotkin, M. Mishoe, B. Liang, N. Yang, X. Li; Mount Sinai School of Medicine, New York, NY. RATIONALE: Previous experiments demonstrated that the ASHMI constituent Ku Shen inhibited IgE production by a human myeloma B cell line (IC505 45.68 mg/mL). This herb was fractionated into 8 different fractions (KSF1-KSF8) with respect to polarity and acidic vs basic properties. KSF1 (a flavanoid rich fraction) showed the strongest inhibition of IgE secretion (IC50510.41 mg/mL). The aim of this study was to identify the compounds in KSF1 responsible for this suppression. METHODS: KSF1was sub-fractionated by preparative reverse-phase high performance liquid chromatography (RP-HPLC) into four fractions (KSF1a, b, c, and d). These fractions were tested for reduction of IgE production by a human myeloma B-cell line cultured at 2x105 cells/mLfor 6 days. IgE levels in supernatants were quantified by Immunocap. RESULTS: Among KSF1 sub-fractions, KSF1a had no effect on IgE production. Sub-fractions b, c and d inhibited IgE secretion in a dose dependent manner (KSF1c IC50 value 1.33 mg/mL, KSF1b IC50 value 2.32 mg/mL and KSF1d IC50 value 8.16 mg/mL). No cytotoxicity was detected using MTT assay. CONCLUSION: KSF1c fraction contains the compounds most responsible for the anti-IgE effects of Ku Shen. This fraction will be further investigated to elucidate the chemical structures of the compounds most responsible for the anti-IgE activity of Ku Shen.