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Latex sensitization prior to secondary allergen sensitization enhances systemic Th2 immune responses without augmenting airway inflammation
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
Abstracts
871 Validation of a Flow Cytometric Assay Detecting Basnphil Activation (BAT) for the Diagnosis of ]gE-Mediated
In Vitro
Natural Rubber Latex (NRL) Allergy DG Ebo, B Lechkar, AJ Schuerwegh, CH Bridts, LS De Clerck, WJ Stevens University of Antwerp, Antwerp, Belgium BACKGROUND: After in vitro allergen-specific stimulation, basophils become activated and demonstrate an enhanced expression of the membrane marker CD63. This alteration can easily be analyzed by flow cytometry (FCM) using monoclonal antibodies like Alexa 488-conjugated antibodies against IgE and PE-conjugated anti-CD63. OBJECTIVE: To investigate if this technique (BAT) could be applied in the diagnosis of NRL allergy, and to evaluate if the BAT could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and latex skin test. M E T H O D S : We studied 12 healthy controls without a history of NRL allergy and a negative latex IgE and skin test (group 1), 24 individuals without a history of NRL allergy with a negative latex IgE and skin test but with other inhalant allergies (group 2), and 29 NRL allergic patients with a history of NRL allergy with a positive latex IgE and prick test (group 3). Results are expressed as percentages of CD63 up-regulation (i.e., percentage latexinduced CD63 expression minus spontaneous CD63 expression. Sensitivity and specificity of the BAT was calculated between NRL allergic patients and a pooled group that was composed out of all non-NRL hypersensitive individuals. After validation, the diagnostic performances of the BAT were further evaluated in 13 individuals with a history of NRL allergy but with negative specific IgE and/or skin test (group 4). Twenty-four individuals with positive latex IgE without history of NRL allergy and negative latex skin tests, were also analyzed (group 5). RESULTS: According, to the ROC-generated threshold value of 17% between NRL allergic patients and the pooled group of non-NRL allergic individuals, the sensitivity and specificity of the BAT was 93.1% and 91.7 %, respectively. The numbers of positive BATs in the different groups are summarized in the table. CONCLUSION: The BAT seems a highly sensitive and specific tool for diagnosing NRL allergy. The technique might also help to determine the clinical relevance of positive lgE quantification in the absence of overt NRL allergy.
Group
1
2
3
4
5
Number of cases N=12 N=24 N=29 N=13 N=24 CD63 expression median 0% 0% 48% 40% 4% (range) (0 - 6%) (0 - 49%) (12 - 92%) (24 - 94%) (0 - 81%) Number of positive BATs* 0 3 27 13 4 * Number of cases with CD63 up-regulation > 17%.
Properties of Cornstarch Glove Powder 8 "7~lmmunoadjuvant Demonstrated by Bronchial Challenge in Guinea Pigs I
L-
J Barbara*, M-C Santais§, D Levy*, F Ruff§, F Leynadier* *Hospital Tenon, Paris, France §University Paris V, Paris, France INTRODUCTION: Cornstarch powder present in medical gloves plays an important role in latex-induced hypersensitivity as allergen carrier, either by the inhalation route or by direct contact with mucous membranes. Our objective was to test the hypothesis that inhaled allergen-bearing cornstarch glove powder acts as an immunoadjuvant in latex-induced allergy. M A T E R I A L S AND METHODS: Adult male guinea pigs (n = 10 in each group) were exposed daily in a closed chamber to non-ammoniated natural rubber latex (NRL) either as an aerosolized latex solution (LS) or as nebulized latex-contaminated cornstarch glove powder (LCCGP). Both groups were exposed to 10 mg NRL protein daily for two weeks. Control groups were exposed either to cornstarch without latex (CS) or to normal saline solution (NSS). Sensitization to NRL was evaluated by airway responsiveness to bronchial challenge, measured as airway resistance, in the second week after the final immunization dose. For this procedure, animals were anesthetized, tracheotomized, then challenged by an intratra-
$285
cheal injection of 1 mg NRL protein in 20 ul of saline. Airway resistance, expressed as cm H 2 0 x rain, was measured continuously over the next 15 min. The level of latex-specific IgG and IgG1 in serum obtained immediately before and after the challenge was determined by ELISA. RESULTS: Guinea pigs sensitized with LCCGP showed significantly greater bronchoconstriction after latex challenge compared to animals sensitized with LS (74.4 + 10.8 vs. 43.7 _+ 11.9 cm H 2 0 x min, p<0.02) and both groups had significantly greater bronchoconstriction (p<0.02) compared to control animals (CS = 22.7 _+4.5 cm H 2 0 x rain, NSS 20 =17.2 + 3.4 cm H 2 0 x min). Neither anti-latex IgG nor anti-latex IgG1 was detected in any of the sera. CONCLUSION: These results show that repeated exposure to NRL either as LS or as LCCGP induced specific airway hyper-responsiveness to latex allergen challenge but sensitization with LCCGP produced greater hyper-responsiveness than sensitization with LS. Thus, cornstarch glove powder appears to act as an adjuvant in NRL sensitization. The fact that IgG and IgG1 were not detected in the sera of these animals might be the result of an immune response limited to the airways.
,,,~B~ Latex Sensitization Prior to Secondary Allergen Sensitization
Enhances Systemic Th2 Immune Responses Without Augment8 E ~ I I ing Airway Inflammation Katharina Bluemchen*, Marcus Schwede*, Kerstin Gerhold§, Philippe Stock§, Bodo Niggemann§, Birgit Wagner¥ Heimo Breiteneder~, Eckard Hamelmann§ *University Hospital Charit& Berlin, Germany §Charitt, Humboldt-University, Berlin, Germany YDepartment of Pathophysiology, University of Vienna, Vienna, Austria CUniversity of Vienna, Vienna, Austria Epidemiologic studies suggest that early sensitization in childhood is a risk factor for secondary allergen sensitization. A good example for this phenomenon are patients with spina bifida and latex allergy who have a high prevalence for atopy, i.e. with marked sensitization rates towards other allergens. Still, there is no direct evidence for a link between an early sensitization boosting Th2 immune responses and increased development of allergic airway inflammation (AI) and asthma. Aim of this study was to investigate if a primary latex sensitization induces increased immune responses and/or AI following secondary allergen sensitization and airway challenges in a mouse model of allergic AI. BALB/c mice were intraperitoneally (ip) immunized with either natural rubber latex (NRL), or PBS six times over a period of three weeks. Subsequently, all mice were ip immunized with ovalbumin (OA) or PBS (negative controls) three times during the next 2 weeks. Finally, all mice were challenged with OA via the airways on days 43, 44 and 45. Two days later, NRL and OA specific and total immunoglobulins (Ig), in vitro cytokine production of mononuclear spleen (MNC) and peribronchial lymph node (PBLN) cells cultured with allergen or mitogen, differential cell counts in broncho-alveolar lavage fluids (BAL), eosinophilic inflammation of lung tissues (immunohistochemistry), and development of in vivo airway reactivity (AR) to inhaled methacholine (barometric whole-body plethysmography) were determined. Sensitization with OA induced a significant increase of OA specific and total IgE and IgG1, as well as increased in vitro IL-5 and low IFN-gamrna production, compared to negative controls. Further, following OA airway challenges of OA sensitized mice, marked eosinophilic AI and increased in vivo AR were detected. NRL sensitization prior to OA immunization significantly increased production of OA-specific IgE, total IgE and IgG1 serum levels, as well as. in vitro IL-4 production of MNCs, compared to exclusively OA-sensitized mice. Surprisingly, following OA airway challenges, PBLN cells of NRL/OA-sensitized animals showed even decreased in vitro IL-5 and IL-4 production compared to PBLN cells of OA-sensitized mice. However, development of eosinophilic AI and in vivo AR were equally enhanced in both groups, compared to controis. In summary, latex sensitization prior to OA sensitization leads to a systemic enhancement of Th2 type immune responses without augmenting the development of AI and AR following OA airway challenges. These data indicate that primary sensitization may lead to enhanced Th2 type immune responses following secondary sensitization, but does not necessarily enhance allergen-induced airway disease.
Abstracts
871 Validation of a Flow Cytometric Assay Detecting Basnphil Activation (BAT) for the Diagnosis of ]gE-Mediated
In Vitro
Natural Rubber Latex (NRL) Allergy DG Ebo, B Lechkar, AJ Schuerwegh, CH Bridts, LS De Clerck, WJ Stevens University of Antwerp, Antwerp, Belgium BACKGROUND: After in vitro allergen-specific stimulation, basophils become activated and demonstrate an enhanced expression of the membrane marker CD63. This alteration can easily be analyzed by flow cytometry (FCM) using monoclonal antibodies like Alexa 488-conjugated antibodies against IgE and PE-conjugated anti-CD63. OBJECTIVE: To investigate if this technique (BAT) could be applied in the diagnosis of NRL allergy, and to evaluate if the BAT could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and latex skin test. M E T H O D S : We studied 12 healthy controls without a history of NRL allergy and a negative latex IgE and skin test (group 1), 24 individuals without a history of NRL allergy with a negative latex IgE and skin test but with other inhalant allergies (group 2), and 29 NRL allergic patients with a history of NRL allergy with a positive latex IgE and prick test (group 3). Results are expressed as percentages of CD63 up-regulation (i.e., percentage latexinduced CD63 expression minus spontaneous CD63 expression. Sensitivity and specificity of the BAT was calculated between NRL allergic patients and a pooled group that was composed out of all non-NRL hypersensitive individuals. After validation, the diagnostic performances of the BAT were further evaluated in 13 individuals with a history of NRL allergy but with negative specific IgE and/or skin test (group 4). Twenty-four individuals with positive latex IgE without history of NRL allergy and negative latex skin tests, were also analyzed (group 5). RESULTS: According, to the ROC-generated threshold value of 17% between NRL allergic patients and the pooled group of non-NRL allergic individuals, the sensitivity and specificity of the BAT was 93.1% and 91.7 %, respectively. The numbers of positive BATs in the different groups are summarized in the table. CONCLUSION: The BAT seems a highly sensitive and specific tool for diagnosing NRL allergy. The technique might also help to determine the clinical relevance of positive lgE quantification in the absence of overt NRL allergy.
Group
1
2
3
4
5
Number of cases N=12 N=24 N=29 N=13 N=24 CD63 expression median 0% 0% 48% 40% 4% (range) (0 - 6%) (0 - 49%) (12 - 92%) (24 - 94%) (0 - 81%) Number of positive BATs* 0 3 27 13 4 * Number of cases with CD63 up-regulation > 17%.
Properties of Cornstarch Glove Powder 8 "7~lmmunoadjuvant Demonstrated by Bronchial Challenge in Guinea Pigs I
L-
J Barbara*, M-C Santais§, D Levy*, F Ruff§, F Leynadier* *Hospital Tenon, Paris, France §University Paris V, Paris, France INTRODUCTION: Cornstarch powder present in medical gloves plays an important role in latex-induced hypersensitivity as allergen carrier, either by the inhalation route or by direct contact with mucous membranes. Our objective was to test the hypothesis that inhaled allergen-bearing cornstarch glove powder acts as an immunoadjuvant in latex-induced allergy. M A T E R I A L S AND METHODS: Adult male guinea pigs (n = 10 in each group) were exposed daily in a closed chamber to non-ammoniated natural rubber latex (NRL) either as an aerosolized latex solution (LS) or as nebulized latex-contaminated cornstarch glove powder (LCCGP). Both groups were exposed to 10 mg NRL protein daily for two weeks. Control groups were exposed either to cornstarch without latex (CS) or to normal saline solution (NSS). Sensitization to NRL was evaluated by airway responsiveness to bronchial challenge, measured as airway resistance, in the second week after the final immunization dose. For this procedure, animals were anesthetized, tracheotomized, then challenged by an intratra-
$285
cheal injection of 1 mg NRL protein in 20 ul of saline. Airway resistance, expressed as cm H 2 0 x rain, was measured continuously over the next 15 min. The level of latex-specific IgG and IgG1 in serum obtained immediately before and after the challenge was determined by ELISA. RESULTS: Guinea pigs sensitized with LCCGP showed significantly greater bronchoconstriction after latex challenge compared to animals sensitized with LS (74.4 + 10.8 vs. 43.7 _+ 11.9 cm H 2 0 x min, p<0.02) and both groups had significantly greater bronchoconstriction (p<0.02) compared to control animals (CS = 22.7 _+4.5 cm H 2 0 x rain, NSS 20 =17.2 + 3.4 cm H 2 0 x min). Neither anti-latex IgG nor anti-latex IgG1 was detected in any of the sera. CONCLUSION: These results show that repeated exposure to NRL either as LS or as LCCGP induced specific airway hyper-responsiveness to latex allergen challenge but sensitization with LCCGP produced greater hyper-responsiveness than sensitization with LS. Thus, cornstarch glove powder appears to act as an adjuvant in NRL sensitization. The fact that IgG and IgG1 were not detected in the sera of these animals might be the result of an immune response limited to the airways.
,,,~B~ Latex Sensitization Prior to Secondary Allergen Sensitization
Enhances Systemic Th2 Immune Responses Without Augment8 E ~ I I ing Airway Inflammation Katharina Bluemchen*, Marcus Schwede*, Kerstin Gerhold§, Philippe Stock§, Bodo Niggemann§, Birgit Wagner¥ Heimo Breiteneder~, Eckard Hamelmann§ *University Hospital Charit& Berlin, Germany §Charitt, Humboldt-University, Berlin, Germany YDepartment of Pathophysiology, University of Vienna, Vienna, Austria CUniversity of Vienna, Vienna, Austria Epidemiologic studies suggest that early sensitization in childhood is a risk factor for secondary allergen sensitization. A good example for this phenomenon are patients with spina bifida and latex allergy who have a high prevalence for atopy, i.e. with marked sensitization rates towards other allergens. Still, there is no direct evidence for a link between an early sensitization boosting Th2 immune responses and increased development of allergic airway inflammation (AI) and asthma. Aim of this study was to investigate if a primary latex sensitization induces increased immune responses and/or AI following secondary allergen sensitization and airway challenges in a mouse model of allergic AI. BALB/c mice were intraperitoneally (ip) immunized with either natural rubber latex (NRL), or PBS six times over a period of three weeks. Subsequently, all mice were ip immunized with ovalbumin (OA) or PBS (negative controls) three times during the next 2 weeks. Finally, all mice were challenged with OA via the airways on days 43, 44 and 45. Two days later, NRL and OA specific and total immunoglobulins (Ig), in vitro cytokine production of mononuclear spleen (MNC) and peribronchial lymph node (PBLN) cells cultured with allergen or mitogen, differential cell counts in broncho-alveolar lavage fluids (BAL), eosinophilic inflammation of lung tissues (immunohistochemistry), and development of in vivo airway reactivity (AR) to inhaled methacholine (barometric whole-body plethysmography) were determined. Sensitization with OA induced a significant increase of OA specific and total IgE and IgG1, as well as increased in vitro IL-5 and low IFN-gamrna production, compared to negative controls. Further, following OA airway challenges of OA sensitized mice, marked eosinophilic AI and increased in vivo AR were detected. NRL sensitization prior to OA immunization significantly increased production of OA-specific IgE, total IgE and IgG1 serum levels, as well as. in vitro IL-4 production of MNCs, compared to exclusively OA-sensitized mice. Surprisingly, following OA airway challenges, PBLN cells of NRL/OA-sensitized animals showed even decreased in vitro IL-5 and IL-4 production compared to PBLN cells of OA-sensitized mice. However, development of eosinophilic AI and in vivo AR were equally enhanced in both groups, compared to controis. In summary, latex sensitization prior to OA sensitization leads to a systemic enhancement of Th2 type immune responses without augmenting the development of AI and AR following OA airway challenges. These data indicate that primary sensitization may lead to enhanced Th2 type immune responses following secondary sensitization, but does not necessarily enhance allergen-induced airway disease.