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235 Cloning and sequencing of a Walnut (Juglans regia) food allergen and its sequence similarity with other seed storage proteins
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART3
241
233
Maternal Food Antigen Avoidance During Lactation and Allergy During the First |O years of age.Gunnar Hattevig, M.D.Ph D., Nele Sigurs, M.D.Ph D., and Bengt Kjellman, M.D.Ph D. Dept of Ped, SkOvde and BorAs, SWEDEN. Sixtyfive infants with family history of atopy and with mothers on a diet free from eggs, cow's mitk and fish during the f i r s t threemonths of lactation (D-group) and 50 infants in a matched group withouth diet restrictions (ND-group) have been followed six times up to the age of four years (Pediatrics 1992;89:735-9). At the age of ten years all 115 children reported for the 7th follow-up, including interview, examination, and skin-prick-test (STP) to 15 food-, and inhalant allergens. The cummutative incidences and the current prevalences of atopic symptoms/sign, and the prevalence of positive STP did not differ significantly between the groups. The cummulative incidences of atopic symptoms/signs (%) were (D-/ND-group);total:54/72, atopic dermatitis (AD): 43/60, asthma (A): 12/26, allergic rhinoconjunct i v i t i s (ARC): 33/38. The current prevalences (%) were; total: 43/56, AD: 28/28, A: 14/22, ARC: 31/38. Positive STP (%) were; total: 37/49, food: 12/22, pollen: 26/37, animal danders 29/35. Conclusion: The significant lower incidence of AD in the D-group observed up to four years did not remain at 10 years.
235
C l o n i n g a n d S e q u e n c i n g o f a W a l n u t (Juglans regia) F o o d A l l e r g e n a n d Its S e q u e n c e Similarity w i t h O t h e r S e e d Storage Proteins. SS Teuber, ]VII), AM Dandekar, PhD, WR Peterson, BS, SL Uratsu, BS, Davis, California, USA. Walnuts rank second in per capita consumption of tree nuts in the USA and can be associated with systemic, IgE-mediated reactions in some individuals. A eDNA expression library in 3.Uni-ZAP. prepared from walnut embryo, was screened using a patient's sera which reacted with multiple protein bands on immunoblotting. Reactive clones were purified and subcloned by excision into pBluescript in E. cell A eDNA encoding a 598 AA allergen (about 66kDa), was previously isolated and found to be a member of the vicilin family of seed storage proteins. A recombinant fusion protein from a second clone, of approximately 19 kDa, was induced and bound sera IgE from 7 of 12 patients tested. The eDNA insert size was 642 bp, with an intact poly-A tail but missing the start codon. It encodes a protein 143 AA in length. Searches of protein databases revealed that this clone exhibits 46.1% identity with the brazil nut (Bertholletia excelsa) methionine rich 2S albumin seed storage protein precursor. Interestingly, the gene encoding this protein has been successfully used as a transgene to enhance the nutritional quality of legumes, which are deficient in methionine. This finding has possible profound implications for the allergenicity of genetically engineered food products since patients allergic to walnut could potentially crossreact with brazil nut protein in the genetically engineered plant product. However, the clinical significance of this homology is not currently known.
234
F o o d a l l e r g e n s a r e s t a b l e to d i g e s t i o n in a s i m p l e model of the gastrointestinal tract. JD Astwood PhD and RL Fuchs PhD St. Louis, M e Protein allergens occur in a wide range of plant and animal foodstuffs. While there are no reliable prospective methods for assessing the allergenicity of a protein, it is reasonable to propose that food allergens share physicochemicai properties which distinguish them from non-allergens. One candidate property is stability to gastrointestinal digestion. We have systematically evaluated the stability of 17 major food allergens in simple models of gastric and intestinal fluids. The data show that without exception, the important food allergens of egg, milk, soybean, peanut, and Brassicacae are stable to digestion in the gastric model. For example, [3-conglycinin (7S globulin fraction of soybean) was stable for 60 minutes, in contrast, it was shown that other food proteins such as Ribulose bis-phosphate carboxylase/oxygenase (the most abundant protein on earth) were rapidly and completely digested in the gastric model within 15 seconds. The model of gastric digestion was further characterized by considering potential confounding factors such as effects of a whole food matrix on digestion times. The whole food matrix had no effect on the stability of allergens or lability of non-allergen food proteins in the gastric model. It was concluded that stability to digestion is a significant parameter distinguishing food allergens from nonallergens. Proteolytic stability favors the likelihood that a protein will reach the intestinal mucosa, the presumed access point of sensitization. Therefore, digestive stability is one criteria that should be evaluated to assess the safety of new proteins introduced into food crops by genetic engineering.
236
A s s e s s m e n t s o f E o s i n o p h i l C a t i o n i c P r o t e i n 0ECP) in Nasal W a s h e s f r o m W h e e z i n g I n f a n t s . JB LaRussa MD, GP Rakes MD. DD Murphy RN, TAE Platts-Mills MD, Ph.D.. PW Heymann MD, Charlottesville VA, USA. Wheezing during infancy is frequently caused by viral infections. Although a large percentage of wheezing infants will no longer wheeze as toddlers, 30-40% will continue to have wheezing episodes. At present it is difficult to judge which infants have or will develop asthma. In a previous study at the University of Virginia, 9/22 (41%) of wheezing children < age 2 had nagal ECP levels > 200 ng/ml, compared to 1/17 (6%) of controls (p< 0.03) (J. Pediatrics, In Press). The wheezing infants, however, were more difficult to distinguish from controls based on measurements of serum ECP, total blood ensinophil counts, total IgE, or IgE antibody by RAST to inhaled allergens. An additional 13 subjects < age 2 have been enrolled. In the wheezing group, 4/8 (50%) had high levels of nasal ECP(ng/ml) 561, 412, 678, and 200, which decreased to 83, 4, 72, and 92, respectively at follow-up one month later when the patients were ussymptomatic. No patient received steroids. One of 5 control infants had elevated nasal ECP (291 ng/ml and then 115 ng/ml at return visit). Similar to the previous study, there were no significant differences in serum ECP, nasal and serum eosinophils, and positive prick skin tests in the wheezing and control groups. Serum levels of CD30, a putative marker for TM2 activity, was >200 U/ml in both acute and follow-up samples from 5/8 wheezing and 2/5 control patients. Geometric mean levels of CD30 in sera from wheezing (n=22) and control (n=17) infants studied previously were similar (165 U/ml, and 140 U/ml, respectively, I>=NS). In conclusion, the reduction in nasal ECP seen at follow-up suggests that nasal ECP levels may be elevated during an acute wheezing episode as a sign of eosinophil activation in the airways.
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART3
241
233
Maternal Food Antigen Avoidance During Lactation and Allergy During the First |O years of age.Gunnar Hattevig, M.D.Ph D., Nele Sigurs, M.D.Ph D., and Bengt Kjellman, M.D.Ph D. Dept of Ped, SkOvde and BorAs, SWEDEN. Sixtyfive infants with family history of atopy and with mothers on a diet free from eggs, cow's mitk and fish during the f i r s t threemonths of lactation (D-group) and 50 infants in a matched group withouth diet restrictions (ND-group) have been followed six times up to the age of four years (Pediatrics 1992;89:735-9). At the age of ten years all 115 children reported for the 7th follow-up, including interview, examination, and skin-prick-test (STP) to 15 food-, and inhalant allergens. The cummutative incidences and the current prevalences of atopic symptoms/sign, and the prevalence of positive STP did not differ significantly between the groups. The cummulative incidences of atopic symptoms/signs (%) were (D-/ND-group);total:54/72, atopic dermatitis (AD): 43/60, asthma (A): 12/26, allergic rhinoconjunct i v i t i s (ARC): 33/38. The current prevalences (%) were; total: 43/56, AD: 28/28, A: 14/22, ARC: 31/38. Positive STP (%) were; total: 37/49, food: 12/22, pollen: 26/37, animal danders 29/35. Conclusion: The significant lower incidence of AD in the D-group observed up to four years did not remain at 10 years.
235
C l o n i n g a n d S e q u e n c i n g o f a W a l n u t (Juglans regia) F o o d A l l e r g e n a n d Its S e q u e n c e Similarity w i t h O t h e r S e e d Storage Proteins. SS Teuber, ]VII), AM Dandekar, PhD, WR Peterson, BS, SL Uratsu, BS, Davis, California, USA. Walnuts rank second in per capita consumption of tree nuts in the USA and can be associated with systemic, IgE-mediated reactions in some individuals. A eDNA expression library in 3.Uni-ZAP. prepared from walnut embryo, was screened using a patient's sera which reacted with multiple protein bands on immunoblotting. Reactive clones were purified and subcloned by excision into pBluescript in E. cell A eDNA encoding a 598 AA allergen (about 66kDa), was previously isolated and found to be a member of the vicilin family of seed storage proteins. A recombinant fusion protein from a second clone, of approximately 19 kDa, was induced and bound sera IgE from 7 of 12 patients tested. The eDNA insert size was 642 bp, with an intact poly-A tail but missing the start codon. It encodes a protein 143 AA in length. Searches of protein databases revealed that this clone exhibits 46.1% identity with the brazil nut (Bertholletia excelsa) methionine rich 2S albumin seed storage protein precursor. Interestingly, the gene encoding this protein has been successfully used as a transgene to enhance the nutritional quality of legumes, which are deficient in methionine. This finding has possible profound implications for the allergenicity of genetically engineered food products since patients allergic to walnut could potentially crossreact with brazil nut protein in the genetically engineered plant product. However, the clinical significance of this homology is not currently known.
234
F o o d a l l e r g e n s a r e s t a b l e to d i g e s t i o n in a s i m p l e model of the gastrointestinal tract. JD Astwood PhD and RL Fuchs PhD St. Louis, M e Protein allergens occur in a wide range of plant and animal foodstuffs. While there are no reliable prospective methods for assessing the allergenicity of a protein, it is reasonable to propose that food allergens share physicochemicai properties which distinguish them from non-allergens. One candidate property is stability to gastrointestinal digestion. We have systematically evaluated the stability of 17 major food allergens in simple models of gastric and intestinal fluids. The data show that without exception, the important food allergens of egg, milk, soybean, peanut, and Brassicacae are stable to digestion in the gastric model. For example, [3-conglycinin (7S globulin fraction of soybean) was stable for 60 minutes, in contrast, it was shown that other food proteins such as Ribulose bis-phosphate carboxylase/oxygenase (the most abundant protein on earth) were rapidly and completely digested in the gastric model within 15 seconds. The model of gastric digestion was further characterized by considering potential confounding factors such as effects of a whole food matrix on digestion times. The whole food matrix had no effect on the stability of allergens or lability of non-allergen food proteins in the gastric model. It was concluded that stability to digestion is a significant parameter distinguishing food allergens from nonallergens. Proteolytic stability favors the likelihood that a protein will reach the intestinal mucosa, the presumed access point of sensitization. Therefore, digestive stability is one criteria that should be evaluated to assess the safety of new proteins introduced into food crops by genetic engineering.
236
A s s e s s m e n t s o f E o s i n o p h i l C a t i o n i c P r o t e i n 0ECP) in Nasal W a s h e s f r o m W h e e z i n g I n f a n t s . JB LaRussa MD, GP Rakes MD. DD Murphy RN, TAE Platts-Mills MD, Ph.D.. PW Heymann MD, Charlottesville VA, USA. Wheezing during infancy is frequently caused by viral infections. Although a large percentage of wheezing infants will no longer wheeze as toddlers, 30-40% will continue to have wheezing episodes. At present it is difficult to judge which infants have or will develop asthma. In a previous study at the University of Virginia, 9/22 (41%) of wheezing children < age 2 had nagal ECP levels > 200 ng/ml, compared to 1/17 (6%) of controls (p< 0.03) (J. Pediatrics, In Press). The wheezing infants, however, were more difficult to distinguish from controls based on measurements of serum ECP, total blood ensinophil counts, total IgE, or IgE antibody by RAST to inhaled allergens. An additional 13 subjects < age 2 have been enrolled. In the wheezing group, 4/8 (50%) had high levels of nasal ECP(ng/ml) 561, 412, 678, and 200, which decreased to 83, 4, 72, and 92, respectively at follow-up one month later when the patients were ussymptomatic. No patient received steroids. One of 5 control infants had elevated nasal ECP (291 ng/ml and then 115 ng/ml at return visit). Similar to the previous study, there were no significant differences in serum ECP, nasal and serum eosinophils, and positive prick skin tests in the wheezing and control groups. Serum levels of CD30, a putative marker for TM2 activity, was >200 U/ml in both acute and follow-up samples from 5/8 wheezing and 2/5 control patients. Geometric mean levels of CD30 in sera from wheezing (n=22) and control (n=17) infants studied previously were similar (165 U/ml, and 140 U/ml, respectively, I>=NS). In conclusion, the reduction in nasal ECP seen at follow-up suggests that nasal ECP levels may be elevated during an acute wheezing episode as a sign of eosinophil activation in the airways.