- Email: [email protected]
Allergen specific induction of proinflammatory cytokines and chemokines in peripheral blood mononuclear cells of latex allergic patients
S110 Abstracts
Allergen Specific Induction of Proinflammatory Cytokines and Chemokines in Peripheral Blood Mononuclear Cells of Latex Allergic Patients M. Lehto1, K. Palosuo2, A. Kotovuori3, E. Varjonen2, S. Lehtimäki1, M. Majuri1, T. Reunala4, N. Kalkkinen3, T. Palosuo5, H. Alenius1; 1Finnish Institute of Occupational Health, Helsinki, FINLAND, 2Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, FINLAND, 3Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND, 4Tampere University and University Hospital, Tampere, FINLAND, 5National Public Health Institute, Helsinki, FINLAND. RATIONALE: IgE responses and skin reactivity against the two major natural rubber latex (NRL) allergens, Hev b6.01 and Hev b5, have been extensively studied previously. In the present study we examined cellular responses to Hev b6.01 and Hev b5 in NRL-allergic patients and controls. METHODS: Hev b6.01 and Hev b5 were purified under non-denaturating conditions using chromatographic methods. Allergen specific IgE antibodies were measured by ELISA. Peripheral blood mononuclear cells (PBMC) were isolated and their proliferation was examined. Allergen specific cytokine and chemokine mRNA production of PBMC were measured by real-time PCR. RESULTS: More than 70% of the patients had positive skin prick test reactions and IgE antibodies against Hev b6.01, but less than 40% of the patients responded to Hev b5. Proliferation of PBMC against Hev b6.01, but not against Hev b5, was significantly enhanced in NRL-allergic patients compared to controls. There were no significant differences in the levels of Th1 or Th2 cytokines between patients and controls after allergen stimulation. However, stimulation with Hev b6.01 and Hev b5 elicited significantly stronger induction of TNF-, IL-12p40 and MIP-1 mRNA in NRL-allergic patients compared to controls. CONCLUSION: Allergen specific induction of proinflammatory cytokines and chemokines in circulating mononuclear cells may play an important role in the pathogenesis of latex allergy. Funding: Finnish Institute of Occupational Health
439
SUNDAY
Prevalence of IgE Antibodies to Extensively Purified Hev b2, Hev b5, Hev b6.01, and Hev b13 in Latex Allergic Patients in Three Geographic Areas T. Palosuo1, M. Lehto2, A. Kotovuori3, N. Kalkkinen3, C. Blanco4, R. G. Hamilton5, H. Alenius2, T. Reunala6, K. Turjanmaa6; 1National Public Health Institute, Helsinki, FINLAND, 2Finnish Institute of Occupational Health, Helsinki, FINLAND, 3Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND, 4Dr. Negrin University Hospital., Las Palmas de Gran Canaria, SPAIN, 5Johns Hopkins University School of Medicine, Baltimore, MD, 6Tampere University Hospital, Tampere, FINLAND. RATIONALE: Hev b13 and Hev b2 reportedly represent major latex allergens. However, native allergens are difficult to purify to homogeneity and other allergens or their fragments may copurify with them. Only a minority of latex allergic patients in Finland had IgE to these extensively purified allergens. Our study assesses the prevalence of sensitization to these allergens in other populations from diverse geographic areas. METHODS: Highly purified Hev b2 and Hev b13 were isolated from latex B-serum using multiple chromatographic steps under non-denaturing conditions. Similarly purified native Hev b5 and Hev b6.01 were included for comparison. IgE reactivity against the four allergens was assayed by ELISA in sera from 200 latex allergic patients and 175 control subjects from Spain, USA and Finland. RESULTS: IgE antibody levels exceeding the 98th percentile of local controls to Hev b2 were found in 4%, 15% and 31% of Spanish, US and Finnish latex allergic patients, respectively. IgE antibodies specific for Hev b13 were seen in 17%, 27% and 26% of Spanish, US and Finnish patients. In contrast, Hev b5-specific IgE was seen in 46% Spanish, 60% US and 26% Finnish patients and Hev b6.01-specific IgE was detected in the serum of 70%, 66% and 62% of Spanish, US and Finnish patients, respectively. CONCLUSION: Prevalence of IgE to Hev b2 and Hev b13 in latex allergic patients differs among various geographic areas but it is in general low (<32%) when assayed using extensively purified allergens. The reasons for the different sensitization rates require further study. Funding: National Public Health Institute
440
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
N-Terminal Hevein-Like Domains (4.3 kDa) but Not 31 kDa Endochitinases Are Responsible for IgE-Mediated In Vitro and In Vivo Reactions in Latex-Fruit Syndrome P. Karisola1, A. Kotovuori2, S. Poikonen3, E. Niskanen1, N. Kalkkinen2, K. Turjanmaa3, T. Palosuo4, T. Reunala3, H. Alenius5, M. Kulomaa1; 1Biological and Environmental Sciences, University of Jyvaskyla, Jyvaskyla, FINLAND, 2Institution of Biotechnology, University of Helsinki, Helsinki, FINLAND, 3Department of Dermatology, Tampere University Hospital, Tampere, FINLAND, 4National Public Health Institute, Helsinki, FINLAND, 5Finnish Institute of Occupational Health, Helsinki, FINLAND. RATIONALE: Natural rubber latex (NRL) allergic patients often show immediate reactions to fresh fruits including avocado and banana. Based on immunoblot studies, 31 kDa endochitinases containing an N-terminal hevein-like domain (HLD) are recognized by IgE of the NRL allergic patients. To study the endochitinase-induced reactions in vivo, we tested the clinical significance of 31 kDa fruit endochitinases and corresponding HLDs in latex-fruit syndrome. METHODS: Endochitinases (31 kDa) and corresponding HLDs (4.3 kDa) were purified from avocado, banana, and rubber latex. We examined these proteins in inhibition-ELISA, and in skin prick test (SPT) with 15 NRLallergic patients. To define the molecular basis of clinical reactions, we compared the experimentally resolved or modeled structures of these proteins. RESULTS: In ELISA-tests, HLDs from avocado and banana inhibited binding of IgE to prohevein (Hev b 6.01) in 59 % and 38 % of patients, respectively. Corresponding percentages for 31 kDa endochitinases from avocado and banana were 17 % and 20 %, respectively. Isolated HLDs of avocado and banana caused SPT reaction to 73 % of patients but only 7 % of patients reacted to corresponding 31 kDa endochitinases. CONCLUSIONS: IgE-mediated clinical reactions in latex-fruit syndrome seem to be evoked by 4.3 kDa HLD molecules alone but not when linked to 31 kDa endochitinases. Therefore, a reliable diagnosis of latexfruit syndrome needs careful selection of relevant allergens in their proper molecular forms. Funding: Academy of Finland, National Health Institute of Finland
441
Microarray-Based Improvement of Diagnosis for Latex Allergy S. Wagner1, C. Harwanegg2, B. Wagner1, C. Hafner3, A. Mari4, C. Ebner1, R. Hiller2, O. Scheiner1, H. Breiteneder1; 1Dept. of Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA, 2VBC - Genomics Bioscience Research, Vienna, AUSTRIA, 3Dept. of Dermatology, Medical University of Vienna, Vienna, AUSTRIA, 4Allergy Unit, National Health Service, Rome, ITALY. RATIONALE: Currently used reagents for latex allergy diagnosis are still subjected to criticism. Improved and reliable diagnostic reagents are required to identify sensitized individuals and to monitor the possible emergence of a new latex allergy epidemic. METHODS: A microarray-based latex allergy diagnostic test was developed with newly generated latex protein extracts, a panel of recombinant latex allergens, and horseradish peroxidase (HRP). Sera of 74 patients with a compelling history of latex allergy and a positive latex skin test (group 1), 44 individuals without a history of latex hypersensitivity but with other inhalant allergies (group 2), 29 individuals with a compelling history but negative skin and/or serological test (group 3), and 62 individuals without symptoms but positive skin test and/or positive CAP (group 4) were analyzed. RESULTS: The sensitivity of the microarray-based test was 97.3% for group 1, 85.2% for group 3, and 98.4% for group 4. Interestingly, only 83.8% of the allergic subjects (group 1) had a positive CAP result. The specificity of the test was 65, 9% (group 2). In group 1 most reactions were observed to Hev b 6 (60.8%) and Hev b 5 (31.1%), whereas in the group of sensitized individuals (group 4), most reactions were detectable to Hev b 8 (40.4%) and HRP (38.7%). CONCLUSIONS: Our microarray-based test showed greater diagnostic sensitivity than the CAP assay. The analysis of the patient’s IgE binding pattern allows discrimination of the sensitization to allergens of different clinical relevance and the prediction of possible cross-reactivities. Supported by the Austrian Science Fund grant SFB-F01802.
442
Allergen Specific Induction of Proinflammatory Cytokines and Chemokines in Peripheral Blood Mononuclear Cells of Latex Allergic Patients M. Lehto1, K. Palosuo2, A. Kotovuori3, E. Varjonen2, S. Lehtimäki1, M. Majuri1, T. Reunala4, N. Kalkkinen3, T. Palosuo5, H. Alenius1; 1Finnish Institute of Occupational Health, Helsinki, FINLAND, 2Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, FINLAND, 3Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND, 4Tampere University and University Hospital, Tampere, FINLAND, 5National Public Health Institute, Helsinki, FINLAND. RATIONALE: IgE responses and skin reactivity against the two major natural rubber latex (NRL) allergens, Hev b6.01 and Hev b5, have been extensively studied previously. In the present study we examined cellular responses to Hev b6.01 and Hev b5 in NRL-allergic patients and controls. METHODS: Hev b6.01 and Hev b5 were purified under non-denaturating conditions using chromatographic methods. Allergen specific IgE antibodies were measured by ELISA. Peripheral blood mononuclear cells (PBMC) were isolated and their proliferation was examined. Allergen specific cytokine and chemokine mRNA production of PBMC were measured by real-time PCR. RESULTS: More than 70% of the patients had positive skin prick test reactions and IgE antibodies against Hev b6.01, but less than 40% of the patients responded to Hev b5. Proliferation of PBMC against Hev b6.01, but not against Hev b5, was significantly enhanced in NRL-allergic patients compared to controls. There were no significant differences in the levels of Th1 or Th2 cytokines between patients and controls after allergen stimulation. However, stimulation with Hev b6.01 and Hev b5 elicited significantly stronger induction of TNF-, IL-12p40 and MIP-1 mRNA in NRL-allergic patients compared to controls. CONCLUSION: Allergen specific induction of proinflammatory cytokines and chemokines in circulating mononuclear cells may play an important role in the pathogenesis of latex allergy. Funding: Finnish Institute of Occupational Health
439
SUNDAY
Prevalence of IgE Antibodies to Extensively Purified Hev b2, Hev b5, Hev b6.01, and Hev b13 in Latex Allergic Patients in Three Geographic Areas T. Palosuo1, M. Lehto2, A. Kotovuori3, N. Kalkkinen3, C. Blanco4, R. G. Hamilton5, H. Alenius2, T. Reunala6, K. Turjanmaa6; 1National Public Health Institute, Helsinki, FINLAND, 2Finnish Institute of Occupational Health, Helsinki, FINLAND, 3Institute of Biotechnology, University of Helsinki, Helsinki, FINLAND, 4Dr. Negrin University Hospital., Las Palmas de Gran Canaria, SPAIN, 5Johns Hopkins University School of Medicine, Baltimore, MD, 6Tampere University Hospital, Tampere, FINLAND. RATIONALE: Hev b13 and Hev b2 reportedly represent major latex allergens. However, native allergens are difficult to purify to homogeneity and other allergens or their fragments may copurify with them. Only a minority of latex allergic patients in Finland had IgE to these extensively purified allergens. Our study assesses the prevalence of sensitization to these allergens in other populations from diverse geographic areas. METHODS: Highly purified Hev b2 and Hev b13 were isolated from latex B-serum using multiple chromatographic steps under non-denaturing conditions. Similarly purified native Hev b5 and Hev b6.01 were included for comparison. IgE reactivity against the four allergens was assayed by ELISA in sera from 200 latex allergic patients and 175 control subjects from Spain, USA and Finland. RESULTS: IgE antibody levels exceeding the 98th percentile of local controls to Hev b2 were found in 4%, 15% and 31% of Spanish, US and Finnish latex allergic patients, respectively. IgE antibodies specific for Hev b13 were seen in 17%, 27% and 26% of Spanish, US and Finnish patients. In contrast, Hev b5-specific IgE was seen in 46% Spanish, 60% US and 26% Finnish patients and Hev b6.01-specific IgE was detected in the serum of 70%, 66% and 62% of Spanish, US and Finnish patients, respectively. CONCLUSION: Prevalence of IgE to Hev b2 and Hev b13 in latex allergic patients differs among various geographic areas but it is in general low (<32%) when assayed using extensively purified allergens. The reasons for the different sensitization rates require further study. Funding: National Public Health Institute
440
J ALLERGY CLIN IMMUNOL FEBRUARY 2005
N-Terminal Hevein-Like Domains (4.3 kDa) but Not 31 kDa Endochitinases Are Responsible for IgE-Mediated In Vitro and In Vivo Reactions in Latex-Fruit Syndrome P. Karisola1, A. Kotovuori2, S. Poikonen3, E. Niskanen1, N. Kalkkinen2, K. Turjanmaa3, T. Palosuo4, T. Reunala3, H. Alenius5, M. Kulomaa1; 1Biological and Environmental Sciences, University of Jyvaskyla, Jyvaskyla, FINLAND, 2Institution of Biotechnology, University of Helsinki, Helsinki, FINLAND, 3Department of Dermatology, Tampere University Hospital, Tampere, FINLAND, 4National Public Health Institute, Helsinki, FINLAND, 5Finnish Institute of Occupational Health, Helsinki, FINLAND. RATIONALE: Natural rubber latex (NRL) allergic patients often show immediate reactions to fresh fruits including avocado and banana. Based on immunoblot studies, 31 kDa endochitinases containing an N-terminal hevein-like domain (HLD) are recognized by IgE of the NRL allergic patients. To study the endochitinase-induced reactions in vivo, we tested the clinical significance of 31 kDa fruit endochitinases and corresponding HLDs in latex-fruit syndrome. METHODS: Endochitinases (31 kDa) and corresponding HLDs (4.3 kDa) were purified from avocado, banana, and rubber latex. We examined these proteins in inhibition-ELISA, and in skin prick test (SPT) with 15 NRLallergic patients. To define the molecular basis of clinical reactions, we compared the experimentally resolved or modeled structures of these proteins. RESULTS: In ELISA-tests, HLDs from avocado and banana inhibited binding of IgE to prohevein (Hev b 6.01) in 59 % and 38 % of patients, respectively. Corresponding percentages for 31 kDa endochitinases from avocado and banana were 17 % and 20 %, respectively. Isolated HLDs of avocado and banana caused SPT reaction to 73 % of patients but only 7 % of patients reacted to corresponding 31 kDa endochitinases. CONCLUSIONS: IgE-mediated clinical reactions in latex-fruit syndrome seem to be evoked by 4.3 kDa HLD molecules alone but not when linked to 31 kDa endochitinases. Therefore, a reliable diagnosis of latexfruit syndrome needs careful selection of relevant allergens in their proper molecular forms. Funding: Academy of Finland, National Health Institute of Finland
441
Microarray-Based Improvement of Diagnosis for Latex Allergy S. Wagner1, C. Harwanegg2, B. Wagner1, C. Hafner3, A. Mari4, C. Ebner1, R. Hiller2, O. Scheiner1, H. Breiteneder1; 1Dept. of Pathophysiology, Medical University of Vienna, Vienna, AUSTRIA, 2VBC - Genomics Bioscience Research, Vienna, AUSTRIA, 3Dept. of Dermatology, Medical University of Vienna, Vienna, AUSTRIA, 4Allergy Unit, National Health Service, Rome, ITALY. RATIONALE: Currently used reagents for latex allergy diagnosis are still subjected to criticism. Improved and reliable diagnostic reagents are required to identify sensitized individuals and to monitor the possible emergence of a new latex allergy epidemic. METHODS: A microarray-based latex allergy diagnostic test was developed with newly generated latex protein extracts, a panel of recombinant latex allergens, and horseradish peroxidase (HRP). Sera of 74 patients with a compelling history of latex allergy and a positive latex skin test (group 1), 44 individuals without a history of latex hypersensitivity but with other inhalant allergies (group 2), 29 individuals with a compelling history but negative skin and/or serological test (group 3), and 62 individuals without symptoms but positive skin test and/or positive CAP (group 4) were analyzed. RESULTS: The sensitivity of the microarray-based test was 97.3% for group 1, 85.2% for group 3, and 98.4% for group 4. Interestingly, only 83.8% of the allergic subjects (group 1) had a positive CAP result. The specificity of the test was 65, 9% (group 2). In group 1 most reactions were observed to Hev b 6 (60.8%) and Hev b 5 (31.1%), whereas in the group of sensitized individuals (group 4), most reactions were detectable to Hev b 8 (40.4%) and HRP (38.7%). CONCLUSIONS: Our microarray-based test showed greater diagnostic sensitivity than the CAP assay. The analysis of the patient’s IgE binding pattern allows discrimination of the sensitization to allergens of different clinical relevance and the prediction of possible cross-reactivities. Supported by the Austrian Science Fund grant SFB-F01802.
442