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Genetic Variability Of Tbxa2r Promoter Is Associated With Asa Intolerant Acute Urticaria
Abstracts AB189
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
Allergen Recognition by Human Dendritic Cells: The Critical Role of DC-SIGN A. M. Ghaem Maghami, M. Emara, F. Shakib; The University of Nottingham, Nottingham, UNITED KINGDOM. RATIONALE: The initial recognition of allergens by DCs is of key importance in determining downstream events including T cell subset polarization. The mannose receptor (MR), expressed by DCs, has been shown to be partially responsible for the uptake of the house dust mite (HDM) allergen Der p 1. However, MR contributes only about 40-60% of the total Der p 1 uptake, suggesting the existence of other receptors. Thus, the aim of this study was to identify other putative allergen recognition receptors expressed on human DCs. METHODS: A re-tagging technique was used to identify new receptors for Der p 1 on DCs. Various techniques including flow cytometry, ELISA and gene silencing were used to study the role of the newly identified receptor in the uptake of different allergens by DCs. RESULTS: DC-SIGN was identified as a novel receptor for Der p 1. Blocking and gene silencing experiments showed that DC-SIGN is partly responsible for the uptake of the HDM allergen Der p 1 (n 5 9) (p 5 0.004) and the dog allergen Can f 1 (n 5 5) (p < 0.05). Using different glycoforms of Der p 1, we also showed that Der p 1 uptake by DCs is proportional to its level of glycosylation (n 5 5) (p < 0.05). CONCLUSIONS: The c-type lectin DC-SIGN acts as a receptor for the uptake of Der p 1 and Can f 1 by DCs, and the glycosylation state of the allergen is likely to play a key role in this process.
741
Genetic Variability Of Tbxa2r Promoter Is Associated With Asa Intolerant Acute Urticaria N. Shrestha Palikhe, S. H. Kim, J. H. Kim, G. S. Choi, Y. M. Ye, H. S. Park; Ajou University, Suwon, REPUBLIC OF KOREA. Genetic variability of TBXA2R promoter is associated with ASA intolerant acute urticaria N. Shrestha Palikhe, S.H. Kim, J.H. Kim, G.S. Choi, Y.M. Ye, H.S.Park Department of Allergy and Rheumatology, School of Medicine, Ajou University RATIONALE: The pathogenic difference between two groups of ASA intolerant urticaria ASA intolerant acute urticaria (AIAU) and ASA intolerant chronic urticaria (AICU) is still not clarified. Thromboxane A2, a key mediator of arachidonate pathway, is decreased by ASA exposure. We investigated the genetic association of TBXA2R promoter polymorphism which was compared between AIAU and AICU patients. METHODS: Two promoter polymorphisms of the TBXA2R gene, -4684T>C and 795T>C were genotyped using primer extension methods from 167 AIAU, 149 AICU and 265 as normal controls (NCs). In-vitro functional study was performed by dual luciferase system. Electrophoretic mobility shift assay (EMSA) was carried out in human mast cell (HMC-1). Thromboxane A2 production was measured by sera of AIAU, AICU and NC. RESULTS: AIAU patients carrying TT genotype had significantly higher frequency than NC (p 5 0.015), while no difference was noted between AICU and NC. The luciferase activity was lowered significantly in the construct containing TBXA2R -4684T compared to the -4684C (P < 0.001) in HMC-1, A549 and U937 cell lines. EMSA showed that -4684T produced specific band with higher affinity than -4684C in HMC-1 cells. Thromboxane A2 production was significantly lowered in AIAU patients compared to NC (p 5 0.016) and AICU (p 5 0.049). CONCLUSION: The TBXA2R -4684T>C polymorphism which could induce lower expression and production of thromboxane A2 may lead to develop the phenotype of AIAU.
742
Screening for Autoimmune Mechanisms in Chronic Idiopathic Urticaria (CIU) Utilizing an Immortalized Human Mast Cell Line (LUVA) A. M. Tinana1, J. Mozena2, J. W. Steinke2, L. Borish2; 1University of Texas Medical Branch, Galveston, TX, 2University of Virginia, Charlottesville, VA.
RATIONALE: We developed an immortalized human mast cell line (LUVA) displaying high concentrations of FceRI. Previous studies have demonstrated basophil histamine release by CIU-derived serum; however, this approach is limited by high variability of basophil releasability. In this study, we wanted to determine if LUVA cells were activated by serum derived from CIU patients and whether this correlated with markers of autoimmunity, specifically the autologous serum skin test (ASST). METHODS: 32 patients with and without CIU were enrolled. LUVA cells were incubated with CIU and control sera. Cysteinyl leukotriene, prostaglandin D2 levels and % histamine release were measured by commercial ELISA. RESULTS: Induction of leukotriene production from LUVA mast cells was higher with serum from patients with CIU than controls (14576916 v. 6356615 pg/ml; p 5 0.004) and this was not influenced by upregulation of the expression of FceRI via incubation with IgE overnight. Leukotriene production was significantly higher in patients with a positive ASST versus those with a negative ASST (140661013 v. 7786615 pg/ml; p 5 0.05). There was no significant difference in PgD2 production or histamine release. CONCLUSIONS: The higher degree of serum-induced leukotriene production correlated to the presence of a positive ASST demonstrating presence of mast cell activating capability suggestive of an autoimmune phenomenon. Further evidence of a specific CIU autoimmune cohort is suggested by the observation that these subjects were also more likely to display anti-thyroid antibodies. Lack of correlation of leukotriene production to histamine release or PgD2 production demonstrates that the CIU mechanism may not involve strict activation of FceRIa.
743
Ecallantide Treatment for Acute Attacks of HAE by Primary Attack Location W. E. Pullman1, M. Riedl2, M. Campion3, P. T. Horn1; 1Dyax Corp., Cambridge, MA, 2UCLA - David Geffen School of Medicine, Los Angeles, CA, 3Consultant, Newton, MA. RATIONALE: Hereditary angioedema (HAE) is a rare, potentially fatal condition characterized by unpredictable, acute swelling attacks. Attack types can be classified as laryngeal, abdominal, and peripheral. Ecallantide is a plasma kallikrein inhibitor developed for the treatment of acute HAE attacks. The efficacy of ecallantide was analyzed by attack type. METHODS: Ecallantide was evaluated in 2 similar, double-blind, placebo-controlled, Phase 3 studies (EDEMA3-DB and EDEMA4). The appropriate IRBs provided approval; all patients provided written informed consent. Eligible patients were randomized to 30 mg subcutaneous ecallantide or placebo. Efficacy endpoints assessed at 4 hours post-dosing included 2 validated, HAE-specific, patient-reported outcome measures: Mean Symptom Complex Severity (MSCS) score and Treatment Outcome Score (TOS). Time to significant improvement was also assessed at 4 hours. The EDEMA3-DB and EDEMA4 databases were integrated for this analysis. RESULTS: A total of 143 patients (N 5 70 ecallantide; N 5 73 placebo) were included. Ecallantide demonstrated improvement over placebo at all attack locations, based on TOS (abdominal: P 5 0.026; laryngeal: P 5 0.041; peripheral: P 5 0.035). Based on change in MSCS score, ecallantide showed improvement over placebo for abdominal attacks (P 5 0.001); and was numerically superior to placebo for laryngeal and peripheral attacks, although statistical significance was not reached (laryngeal: P 5 0.335; peripheral: P 5 0.111). Ecallantide was significantly better than placebo for time to significant improvement for abdominal (P 5 0.011) and laryngeal (P 5 0.033) but not peripheral attacks (P 5 0.584). Significant improvement was reached by 4 hours for 60.0% of ecallantide-treated versus 11.1% of placebo-treated patients with laryngeal attacks. CONCLUSIONS: Ecallantide therapy demonstrates efficacy for HAE attacks at all anatomic locations, including life-threatening laryngeal attacks.
MONDAY
740
J ALLERGY CLIN IMMUNOL VOLUME 125, NUMBER 2
Allergen Recognition by Human Dendritic Cells: The Critical Role of DC-SIGN A. M. Ghaem Maghami, M. Emara, F. Shakib; The University of Nottingham, Nottingham, UNITED KINGDOM. RATIONALE: The initial recognition of allergens by DCs is of key importance in determining downstream events including T cell subset polarization. The mannose receptor (MR), expressed by DCs, has been shown to be partially responsible for the uptake of the house dust mite (HDM) allergen Der p 1. However, MR contributes only about 40-60% of the total Der p 1 uptake, suggesting the existence of other receptors. Thus, the aim of this study was to identify other putative allergen recognition receptors expressed on human DCs. METHODS: A re-tagging technique was used to identify new receptors for Der p 1 on DCs. Various techniques including flow cytometry, ELISA and gene silencing were used to study the role of the newly identified receptor in the uptake of different allergens by DCs. RESULTS: DC-SIGN was identified as a novel receptor for Der p 1. Blocking and gene silencing experiments showed that DC-SIGN is partly responsible for the uptake of the HDM allergen Der p 1 (n 5 9) (p 5 0.004) and the dog allergen Can f 1 (n 5 5) (p < 0.05). Using different glycoforms of Der p 1, we also showed that Der p 1 uptake by DCs is proportional to its level of glycosylation (n 5 5) (p < 0.05). CONCLUSIONS: The c-type lectin DC-SIGN acts as a receptor for the uptake of Der p 1 and Can f 1 by DCs, and the glycosylation state of the allergen is likely to play a key role in this process.
741
Genetic Variability Of Tbxa2r Promoter Is Associated With Asa Intolerant Acute Urticaria N. Shrestha Palikhe, S. H. Kim, J. H. Kim, G. S. Choi, Y. M. Ye, H. S. Park; Ajou University, Suwon, REPUBLIC OF KOREA. Genetic variability of TBXA2R promoter is associated with ASA intolerant acute urticaria N. Shrestha Palikhe, S.H. Kim, J.H. Kim, G.S. Choi, Y.M. Ye, H.S.Park Department of Allergy and Rheumatology, School of Medicine, Ajou University RATIONALE: The pathogenic difference between two groups of ASA intolerant urticaria ASA intolerant acute urticaria (AIAU) and ASA intolerant chronic urticaria (AICU) is still not clarified. Thromboxane A2, a key mediator of arachidonate pathway, is decreased by ASA exposure. We investigated the genetic association of TBXA2R promoter polymorphism which was compared between AIAU and AICU patients. METHODS: Two promoter polymorphisms of the TBXA2R gene, -4684T>C and 795T>C were genotyped using primer extension methods from 167 AIAU, 149 AICU and 265 as normal controls (NCs). In-vitro functional study was performed by dual luciferase system. Electrophoretic mobility shift assay (EMSA) was carried out in human mast cell (HMC-1). Thromboxane A2 production was measured by sera of AIAU, AICU and NC. RESULTS: AIAU patients carrying TT genotype had significantly higher frequency than NC (p 5 0.015), while no difference was noted between AICU and NC. The luciferase activity was lowered significantly in the construct containing TBXA2R -4684T compared to the -4684C (P < 0.001) in HMC-1, A549 and U937 cell lines. EMSA showed that -4684T produced specific band with higher affinity than -4684C in HMC-1 cells. Thromboxane A2 production was significantly lowered in AIAU patients compared to NC (p 5 0.016) and AICU (p 5 0.049). CONCLUSION: The TBXA2R -4684T>C polymorphism which could induce lower expression and production of thromboxane A2 may lead to develop the phenotype of AIAU.
742
Screening for Autoimmune Mechanisms in Chronic Idiopathic Urticaria (CIU) Utilizing an Immortalized Human Mast Cell Line (LUVA) A. M. Tinana1, J. Mozena2, J. W. Steinke2, L. Borish2; 1University of Texas Medical Branch, Galveston, TX, 2University of Virginia, Charlottesville, VA.
RATIONALE: We developed an immortalized human mast cell line (LUVA) displaying high concentrations of FceRI. Previous studies have demonstrated basophil histamine release by CIU-derived serum; however, this approach is limited by high variability of basophil releasability. In this study, we wanted to determine if LUVA cells were activated by serum derived from CIU patients and whether this correlated with markers of autoimmunity, specifically the autologous serum skin test (ASST). METHODS: 32 patients with and without CIU were enrolled. LUVA cells were incubated with CIU and control sera. Cysteinyl leukotriene, prostaglandin D2 levels and % histamine release were measured by commercial ELISA. RESULTS: Induction of leukotriene production from LUVA mast cells was higher with serum from patients with CIU than controls (14576916 v. 6356615 pg/ml; p 5 0.004) and this was not influenced by upregulation of the expression of FceRI via incubation with IgE overnight. Leukotriene production was significantly higher in patients with a positive ASST versus those with a negative ASST (140661013 v. 7786615 pg/ml; p 5 0.05). There was no significant difference in PgD2 production or histamine release. CONCLUSIONS: The higher degree of serum-induced leukotriene production correlated to the presence of a positive ASST demonstrating presence of mast cell activating capability suggestive of an autoimmune phenomenon. Further evidence of a specific CIU autoimmune cohort is suggested by the observation that these subjects were also more likely to display anti-thyroid antibodies. Lack of correlation of leukotriene production to histamine release or PgD2 production demonstrates that the CIU mechanism may not involve strict activation of FceRIa.
743
Ecallantide Treatment for Acute Attacks of HAE by Primary Attack Location W. E. Pullman1, M. Riedl2, M. Campion3, P. T. Horn1; 1Dyax Corp., Cambridge, MA, 2UCLA - David Geffen School of Medicine, Los Angeles, CA, 3Consultant, Newton, MA. RATIONALE: Hereditary angioedema (HAE) is a rare, potentially fatal condition characterized by unpredictable, acute swelling attacks. Attack types can be classified as laryngeal, abdominal, and peripheral. Ecallantide is a plasma kallikrein inhibitor developed for the treatment of acute HAE attacks. The efficacy of ecallantide was analyzed by attack type. METHODS: Ecallantide was evaluated in 2 similar, double-blind, placebo-controlled, Phase 3 studies (EDEMA3-DB and EDEMA4). The appropriate IRBs provided approval; all patients provided written informed consent. Eligible patients were randomized to 30 mg subcutaneous ecallantide or placebo. Efficacy endpoints assessed at 4 hours post-dosing included 2 validated, HAE-specific, patient-reported outcome measures: Mean Symptom Complex Severity (MSCS) score and Treatment Outcome Score (TOS). Time to significant improvement was also assessed at 4 hours. The EDEMA3-DB and EDEMA4 databases were integrated for this analysis. RESULTS: A total of 143 patients (N 5 70 ecallantide; N 5 73 placebo) were included. Ecallantide demonstrated improvement over placebo at all attack locations, based on TOS (abdominal: P 5 0.026; laryngeal: P 5 0.041; peripheral: P 5 0.035). Based on change in MSCS score, ecallantide showed improvement over placebo for abdominal attacks (P 5 0.001); and was numerically superior to placebo for laryngeal and peripheral attacks, although statistical significance was not reached (laryngeal: P 5 0.335; peripheral: P 5 0.111). Ecallantide was significantly better than placebo for time to significant improvement for abdominal (P 5 0.011) and laryngeal (P 5 0.033) but not peripheral attacks (P 5 0.584). Significant improvement was reached by 4 hours for 60.0% of ecallantide-treated versus 11.1% of placebo-treated patients with laryngeal attacks. CONCLUSIONS: Ecallantide therapy demonstrates efficacy for HAE attacks at all anatomic locations, including life-threatening laryngeal attacks.
MONDAY
740