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The Effect of Etanercept on the Human Cutaneous Allergic Response
S154 Abstracts
Blood Collection, Processing Methods and Maternal Characteristics and the Potential Impact on Study Results in a Longitudinal Birth Cohort Study K. A. Roberg1, K. T. Sullivan-Dillie1, K. S. Anklam1, L. A. Rosenthal1, D. F. DaSilva1, C. J. Tisler1, T. E. Pappas1, R. E. Gangnon2, J. E. Gern1, R. F. Lemanske, Jr.1; 1Allergy Asthma Research, UW Medical School, Madison, WI, 2Biostatistics and Medical Informatics and Population Health Sciences, UW Madison, Madison, WI. RATIONALE: Blood processing factors and maternal and infant characteristics may influence the cell yield or pattern of cytokine secretion by umbilical cord blood cells. METHODS: Cord blood samples were analyzed on 287 subjects as part of the Childhood Origins of ASThma project (COAST), a prospective study of children at high risk for developing asthma/allergies. Mononuclear cells from samples were separated by centrifugation, counted and stimulated with the mitogen PHA and a suspension of killed staphylococcus, and IFN-, IL-10 and IL-13 were quantitated by ELISA. Cell yield and cytokine production were then related to processing factors including method of collection, time between sample collection and preparation, changes in responses over time; as well as maternal and infant characteristics or exposures. RESULTS: Clots in the cord blood samples were associated with a reduction in median cell yield (21% reduction, p < 0.0001), and changes in cytokine secretion, including an 8% reduction in PHA-induced IL-10 (p = 0.004). In addition, season of collection affected total cell yield (p=0.004), with highest median yields in fall and lowest in summer, (difference of 22%). PHA-induced IL-13 responses were also sensitive to season of collection (p=0.002); and were higher in the spring (23% increase) and summer (27% increase) relative to fall or winter. No other variables had significant (p<0.01) effects in multivariate regression models. CONCLUSIONS: Careful documentation of processing and environmental variables is important when collecting cord blood samples. This data does suggest that maternal seasonal exposure affects cord blood response in the baby. Funding: National Institutes of Health
598
SUNDAY
The Effect of Etanercept on the Human Cutaneous Allergic Response E. R. Conner1, M. Brummet1, C. Bickel1, L. Bankova1, L. McGirt1, T. Meusel1, S. Stevens2, B. S. Bochner1, L. A. Beck1; 1Johns Hopkins University School of Medicine, Baltimore, MD, 2Amgen, Thousand Oaks, CA. RATIONALE: TNF-alpha, derived from mast cells and other sources, induces a variety of inflammatory events. We therefore examined the effect of TNF-alpha inhibition in a human cutaneous allergen challenge model. METHODS: Intradermal skin testing with standardized dust mite allergen was performed on sensitive subjects with perennial allergic rhinitis. The acute phase response (APR) was measured at 15 minutes. Wheal size and skin biopsies were taken at 2 hours and again at 16 hours (the late phase response (LPR)). Subjects then received open-label etanercept, a soluble TNF-alpha receptor inhibitor, 50 mg subcutaneously every three days for a total of 3 doses. Intradermal skin testing and biopsies were repeated 24 hours after the last etanercept dose. RESULTS: So far, 6 subjects have completed the study. Following administration of etanercept, APR size was reduced in 4 of 6 subjects by a mean of 22%. Two of the subjects did not have a LPR. In 3 of the 4 subjects who developed an LPR, etanercept reduced the LPR by a mean of 59%. To date, levels of tissue eosinophils, and endothelial staining for E-selectin and VCAM-1, counted blindly, do not appear to correlate with reductions in either the APR or LPR. No adverse events were noted in response to anti-TNF therapy. CONCLUSIONS: Anti-TNF therapy appears to inhibit both the APR and LPR macroscopic responses in this intradermal allergen challenge model. This inhibition may be independent of endothelial activation and eosinophil recruitment. Funding: NIH
599
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
In Vivo Efficacy in Mouse Asthma Model of Basic Fibroblast Growth Factor I. Lee1, M. Son1, J. Jeong1, B. Kim1, S. Kang1, Y. Kim2; 1DONG-A PHARM. CO., LTD, Yongin, REPUBLIC OF KOREA, 2College of medicine, Seoul National University, Seoul, REPUBLIC OF KOREA. RATIONALE: It has been believed that eosinoiphilic inflammation induced by Th2 cytokines is a main feature of asthma and FGF2 is implicated in asthma. However more than half of adult asthma patients were non-eosinophilic. The immunological role of FGF2 remains unclear. We have investigated the anti-asthmatic and anti-inflammatory effects of FGF2 in non-eosinophilic asthma animal model. METHODS: BALB/c mice induced asthma-like symptoms using 75ug OVA and 10ug LPS were given 500ug/kg FGF2 intra-nasally three times with the OVA sensitization. The efficacy of FGF2 was judged by measurement of the number of leukocytes in bronchoalveolar lavage (BAL) fluids and inflammatory cytokines (IP-10, vEGF and TGF-1). RESULTS: BALB/c mice induced asthma with OVA/LPS showed the increase of leukocytes, especially neutrophil, and inflammatory cytokines. Intra-nasal administration of 500ug/kg FGF2 suppressed the indicators of asthma which included the number of the leukocytes of the BAL fluid and the related cytokines. FGF2 significantly suppressed the leukocytes, especially neutrophils. The number of total leukocytes of the control group was 1.51 ± 0.15 x 106 whereas that of 500ug/kg FGF2 was 7.11 ± 1.78 x 105 (p<0.05). The production of inflammatory cytokines decreased in BAL fluid from FGF2-treated mice. The levels of IP-10, vEGF and TGF-1 were 498.83 ± 283.90, 8.34 ± 2.05 and 108.29 ± 55.61 pg/ml, respectively, for the control group but 9.58 ± 6.86, 2.77 ± 0.63 and 27.36 ± 7.72 pg/ml, respectively, for the FGF2 administered group. CONCLUSIONS: The results indicate that FGF2 can be expected to be a important role for the treatment of asthma. Funding: Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea
600
Development of Autoreactive Antibodies in Allergic Mice after Chronic Respiratory Allergen Exposure H. Garn1, I. Mittermann2, R. Valenta2, H. Renz1; 1Department of Clinical Chemistry and Molecular Diagnostics, Medical Faculty, Philipps University of Marburg, Marburg, GERMANY, 2Division of Immunopathology, Department of Pathophysiology, Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA. BACKGROUND: It has been demonstrated that patients suffering from severe and chronic manifestations of allergy such as atopic dermatitis and chronic asthma exhibit signs of humoral and cellular autoreactivity of a mixed Th2/Th1 phenotype. We asked whether allergic inflammation induced by chronic allergen exposure may result in the development of autoreactivity. METHODS: Allergic sensitization was induced in Balb/c mice with Al(OH)3-adsorbed ovalbumin (OVA) followed by repeated respiratory OVA aerosol challenge. Serum samples were taken at different time points from these animals and, for control purposes, from PBS-treated mice. The development of autoantibodies was monitored by indirect immunofluorescence using HEp-2 cells and rat tissue specimens, ELISA and Western blotting. RESULTS: Sera from sensitized and acutely challenged animals showed already IgG reactivity to HEp-2 cells and rat tissue specimens. The total number of animals with autoreactivity and the reactivity score increased after chronic allergen challenge. In animals exposed to allergen for 8 weeks IgG antibodies to various antigens in the murine EL-4 lymphocyte cell line were detected by ELISA and by Western blotting. No autoantibody reactivities were observed in animals treated with PBS alone. CONCLUSIONS: Our observations demonstrate that allergic sensitization to a foreign antigen followed by airway challenge induces the development of autoreactive antibodies. The role of these autoantibodies can now to be investigated in our animal model. Funding: Deutsche Forschungsgemeinschaft SFB/TR22
601
Blood Collection, Processing Methods and Maternal Characteristics and the Potential Impact on Study Results in a Longitudinal Birth Cohort Study K. A. Roberg1, K. T. Sullivan-Dillie1, K. S. Anklam1, L. A. Rosenthal1, D. F. DaSilva1, C. J. Tisler1, T. E. Pappas1, R. E. Gangnon2, J. E. Gern1, R. F. Lemanske, Jr.1; 1Allergy Asthma Research, UW Medical School, Madison, WI, 2Biostatistics and Medical Informatics and Population Health Sciences, UW Madison, Madison, WI. RATIONALE: Blood processing factors and maternal and infant characteristics may influence the cell yield or pattern of cytokine secretion by umbilical cord blood cells. METHODS: Cord blood samples were analyzed on 287 subjects as part of the Childhood Origins of ASThma project (COAST), a prospective study of children at high risk for developing asthma/allergies. Mononuclear cells from samples were separated by centrifugation, counted and stimulated with the mitogen PHA and a suspension of killed staphylococcus, and IFN-, IL-10 and IL-13 were quantitated by ELISA. Cell yield and cytokine production were then related to processing factors including method of collection, time between sample collection and preparation, changes in responses over time; as well as maternal and infant characteristics or exposures. RESULTS: Clots in the cord blood samples were associated with a reduction in median cell yield (21% reduction, p < 0.0001), and changes in cytokine secretion, including an 8% reduction in PHA-induced IL-10 (p = 0.004). In addition, season of collection affected total cell yield (p=0.004), with highest median yields in fall and lowest in summer, (difference of 22%). PHA-induced IL-13 responses were also sensitive to season of collection (p=0.002); and were higher in the spring (23% increase) and summer (27% increase) relative to fall or winter. No other variables had significant (p<0.01) effects in multivariate regression models. CONCLUSIONS: Careful documentation of processing and environmental variables is important when collecting cord blood samples. This data does suggest that maternal seasonal exposure affects cord blood response in the baby. Funding: National Institutes of Health
598
SUNDAY
The Effect of Etanercept on the Human Cutaneous Allergic Response E. R. Conner1, M. Brummet1, C. Bickel1, L. Bankova1, L. McGirt1, T. Meusel1, S. Stevens2, B. S. Bochner1, L. A. Beck1; 1Johns Hopkins University School of Medicine, Baltimore, MD, 2Amgen, Thousand Oaks, CA. RATIONALE: TNF-alpha, derived from mast cells and other sources, induces a variety of inflammatory events. We therefore examined the effect of TNF-alpha inhibition in a human cutaneous allergen challenge model. METHODS: Intradermal skin testing with standardized dust mite allergen was performed on sensitive subjects with perennial allergic rhinitis. The acute phase response (APR) was measured at 15 minutes. Wheal size and skin biopsies were taken at 2 hours and again at 16 hours (the late phase response (LPR)). Subjects then received open-label etanercept, a soluble TNF-alpha receptor inhibitor, 50 mg subcutaneously every three days for a total of 3 doses. Intradermal skin testing and biopsies were repeated 24 hours after the last etanercept dose. RESULTS: So far, 6 subjects have completed the study. Following administration of etanercept, APR size was reduced in 4 of 6 subjects by a mean of 22%. Two of the subjects did not have a LPR. In 3 of the 4 subjects who developed an LPR, etanercept reduced the LPR by a mean of 59%. To date, levels of tissue eosinophils, and endothelial staining for E-selectin and VCAM-1, counted blindly, do not appear to correlate with reductions in either the APR or LPR. No adverse events were noted in response to anti-TNF therapy. CONCLUSIONS: Anti-TNF therapy appears to inhibit both the APR and LPR macroscopic responses in this intradermal allergen challenge model. This inhibition may be independent of endothelial activation and eosinophil recruitment. Funding: NIH
599
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
In Vivo Efficacy in Mouse Asthma Model of Basic Fibroblast Growth Factor I. Lee1, M. Son1, J. Jeong1, B. Kim1, S. Kang1, Y. Kim2; 1DONG-A PHARM. CO., LTD, Yongin, REPUBLIC OF KOREA, 2College of medicine, Seoul National University, Seoul, REPUBLIC OF KOREA. RATIONALE: It has been believed that eosinoiphilic inflammation induced by Th2 cytokines is a main feature of asthma and FGF2 is implicated in asthma. However more than half of adult asthma patients were non-eosinophilic. The immunological role of FGF2 remains unclear. We have investigated the anti-asthmatic and anti-inflammatory effects of FGF2 in non-eosinophilic asthma animal model. METHODS: BALB/c mice induced asthma-like symptoms using 75ug OVA and 10ug LPS were given 500ug/kg FGF2 intra-nasally three times with the OVA sensitization. The efficacy of FGF2 was judged by measurement of the number of leukocytes in bronchoalveolar lavage (BAL) fluids and inflammatory cytokines (IP-10, vEGF and TGF-1). RESULTS: BALB/c mice induced asthma with OVA/LPS showed the increase of leukocytes, especially neutrophil, and inflammatory cytokines. Intra-nasal administration of 500ug/kg FGF2 suppressed the indicators of asthma which included the number of the leukocytes of the BAL fluid and the related cytokines. FGF2 significantly suppressed the leukocytes, especially neutrophils. The number of total leukocytes of the control group was 1.51 ± 0.15 x 106 whereas that of 500ug/kg FGF2 was 7.11 ± 1.78 x 105 (p<0.05). The production of inflammatory cytokines decreased in BAL fluid from FGF2-treated mice. The levels of IP-10, vEGF and TGF-1 were 498.83 ± 283.90, 8.34 ± 2.05 and 108.29 ± 55.61 pg/ml, respectively, for the control group but 9.58 ± 6.86, 2.77 ± 0.63 and 27.36 ± 7.72 pg/ml, respectively, for the FGF2 administered group. CONCLUSIONS: The results indicate that FGF2 can be expected to be a important role for the treatment of asthma. Funding: Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea
600
Development of Autoreactive Antibodies in Allergic Mice after Chronic Respiratory Allergen Exposure H. Garn1, I. Mittermann2, R. Valenta2, H. Renz1; 1Department of Clinical Chemistry and Molecular Diagnostics, Medical Faculty, Philipps University of Marburg, Marburg, GERMANY, 2Division of Immunopathology, Department of Pathophysiology, Center of Physiology and Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA. BACKGROUND: It has been demonstrated that patients suffering from severe and chronic manifestations of allergy such as atopic dermatitis and chronic asthma exhibit signs of humoral and cellular autoreactivity of a mixed Th2/Th1 phenotype. We asked whether allergic inflammation induced by chronic allergen exposure may result in the development of autoreactivity. METHODS: Allergic sensitization was induced in Balb/c mice with Al(OH)3-adsorbed ovalbumin (OVA) followed by repeated respiratory OVA aerosol challenge. Serum samples were taken at different time points from these animals and, for control purposes, from PBS-treated mice. The development of autoantibodies was monitored by indirect immunofluorescence using HEp-2 cells and rat tissue specimens, ELISA and Western blotting. RESULTS: Sera from sensitized and acutely challenged animals showed already IgG reactivity to HEp-2 cells and rat tissue specimens. The total number of animals with autoreactivity and the reactivity score increased after chronic allergen challenge. In animals exposed to allergen for 8 weeks IgG antibodies to various antigens in the murine EL-4 lymphocyte cell line were detected by ELISA and by Western blotting. No autoantibody reactivities were observed in animals treated with PBS alone. CONCLUSIONS: Our observations demonstrate that allergic sensitization to a foreign antigen followed by airway challenge induces the development of autoreactive antibodies. The role of these autoantibodies can now to be investigated in our animal model. Funding: Deutsche Forschungsgemeinschaft SFB/TR22
601