Genetic Environmental Factors in Clinical Allergy [1 ed.] 9780816655410, 9780816617364

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Genetic and Environmental Factors in Clinical Allergy

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Genetic and

Environmental Factors in Clinical Allergy

David G. Marsh and Malcolm N. Blumenthal, Editors

University of Minnesota Press, Minneapolis

Copyright © 1990 by the Regents of the University of Minnesota All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher. Published by the University of Minnesota Press 2037 University Avenue Southeast, Minneapolis MN 55414. Printed in the United States of America. Library of Congress Cataloging-in-Publication Data Genetic and environmental factors in clinical allergy / David G. Marsh and Malcolm N. Blumenthal, editors. p. cm. ISBN 0-8166-1736-8. — ISBN 0-8166-1737-6 (pbk.) 1. Allergy—Genetic aspects. 2. Allergy—Environmental aspects. 3. Immunogenetics. I. Marsh, David G. II. Blumenthal, Malcolm N. [DNLM: 1. Allergens—immunology. 2. Hypersensitivity— genetics. 3. Hypersensitivity—immunology. QW 900 G3276] RC585.G46 1990 616.97'042-dc20 DNLM/DLC for Library of Congress 89-20544 CIP The University of Minnesota is an equal-opportunity educator and employer.

Contents Preface Chapter 1. Chapter 2.

Historical Introduction. David G. Marsh Population Selection and Methods of Evaluating Immunologic Responses to Allergens. Malcolm N. Blumenthal

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10 Allergens and Allergen Nomenclature David G. Marsh 20 The Relevance of Allergen Standardization and Purity to Epidemiologic and Genetic Studies of Immune Responses to Allergens. Marianne Roebber 32 Chapter 5. Epidemiology of Atopic Allergy. Linda R. Freidhoff 53 Supplement. Prevalence of Percutaneous Immediate Hypersensitivity and Histamine Reactivity in the U.S. Population: Data from the Second National Health and Nutrition Examination Survey. Peter J. Gergen and Paul C. Turkeltaub 73 Chapter 6. Environmental and Developmental Factors in Allergic Disease in Infancy and Early Childhood. Bengt Bjorksten and John W. Gerrard 84 Chapter 7. Immunogenetic and Immunochemical Factors Determining Immune Responsiveness to Allergens: Studies in Unrelated Subjects. David G. Marsh 97 Chapter 8. Genetic Control of IgE Antibody Responses in Humans: The Amb V (Ra5) Model. Kelsye M. Coulter, Guerin Dorval and Lawrence Goodfriend 124 Chapter 9. Immunogenetics of Specific Immune Responses to Allergens in Twins and Families. Malcolm N. Blumenthal and Sergio Bonini 132 Chapter 10. Genetic Aspects of Bronchial Hyperreactivity. Russell J. Hopp, Nicki M. Nair, Againdra K. Bewtra and Robert G. Townley 143

Chapter 3. Chapter 4.

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Chapter 11.

Chapter 12. Glossary Contributors Index

CONTENTS

The Role of Genetic and Environmental Factors in the Control of Basophil and Mast Cell Releasability. Gianni Marone, Vincenzo Casolaro, Domenico Celestino and Sergio Bonini Family Analysis and Genetic Counseling for Allergic Diseases. Deborah A. Meyers

153 161 177 181 185

Preface This monograph presents the first comprehensive review of the role of genetic and environmental factors in the clinical expression of atopic allergy, which includes diseases such as hay fever, asthma, atopic dermatitis (eczema) and certain types of food intolerance. Atopic allergy is very prevalent; it afflicts about 20% of Caucasoid populations and usually higher proportions of non-Caucasoid populations; moreover, it places a considerable socioeconomic burden on those afflicted. In 1979, a Task Force on Asthma and the Other Allergic Diseases organized by the U. S. Department of Health and Human Services* gave a conservative estimate that 35 million U.S. residents then suffered from asthma and other serious allergies. The report also estimated that U.S. allergy sufferers "spend well in excess of $1 billion each year for physicians' services, drugs, and hospital and nursing care," with annual indirect costs, such as lost wages, being in excess of $800 million. Asthma deaths in the U.S. were estimated to be between 2,000 and 4,000 per year. Further deaths are known to result from severe allergic reactions to insect stings, foods, and certain drugs. An understanding of the genetic and environmental factors that determine atopic allergy is vital to the diagnosis, prevention, and treatment of this multifaceted disease. This monograph is, therefore, of particular importance to clinicians and researchers in allergy and clinical immunology. However, the monograph has been organized with a wider audience in mind, since recent research findings are also highly relevant to understanding how the human immune system works and the nature of human susceptibility to immunologic diseases. Indeed, many experts now regard atopic allergy as the most appropriate model for approaching such problems. The monograph covers a broad range of theoretical, experimental, and clinical topics written by experts in the genetic, epidemiologic, and developmental as* Young P. Asthma and Allergies, an Optimistic Future. Based on the Report of the Task Force on Asthma and the Other Allergic Diseases, National Institute of Allergy and Infectious Diseases. NIH Publication No 80-388. U.S. Dept Health and Human Services 1980;14-26.

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PREFACE

pects of atopic allergy in its several different manifestations. Following a brief historical introduction to the subject (Marsh), the importance of the criteria used to select the subjects for study, and choice of methodology used to evaluate the subjects' atopic symptomatology and specific immunologic responses, are discussed (Blumenthal). The subsequent chapter by Marsh provides a tabulation of the physicochemical properties of most of the well-characterized purified allergens and describes the new IUIS Allergen Nomenclature, which is used throughout the book. Next, the selection of well-standardized, high quality allergenic extracts needed for the quantitative immunologic assays of specific allergies is reviewed (Roebber). The highly purified individual allergenic components required for measuring specific IgE and IgG antibody and cellular immune responses are also covered in the same chapter. Current knowledge of the epidemiology of atopic allergy is then thoroughly reviewed (Freidhoff), followed by a supplement covering the most extensive national survey of allergic sensitivity ever performed in the U.S. population (Gergen and Turkeltaub). The subsequent review of environmental and developmental determinants of atopic disease by Bjb'rksten and Gerrard, which focuses especially on early childhood, will be of particular interest to the pediatrician. The next five chapters deal with the genetic determination of specific immune responses to highly purified allergens and the clinical expression of allergic disease. Studies of specific antibody responses to highly purified allergens in groups of unrelated people are reviewed by Marsh and by Coulter et al. These studies focus on some remarkable associations between particular HLA types and specific IgE and IgG antibody responses to allergens, which illustrate the utility of the allergy model for basic immunogenetic studies of human immune responsiveness. Twin and family studies are covered in the next three chapters. The first, by Blumenthal and Bonini, reports on family and twin studies of the genetics of IgE production; even in twins raised apart, both total and specific IgE antibody levels are seen to be determined primarily by genetic factors. Hopp et al. then discuss the genetic determination of bronchial hyperreactivity and its importance in asthma and Marone et al. provide evidence that "releasability" factors important in allergy are inherited. In the final chapter, Meyers discusses the genetic analysis of basal total serum IgE levels in families; high IgE levels provide perhaps the best predictive marker for the overall presence of atopy. This chapter will be of interest to clinicians in counseling their patients and the patients' families, since the analysis of IgE levels and of skin-test sensitivity to a panel of environmental allergens can be used to predict atopic disease in families. D. G. M. andM. N. B.

Genetic and Environmental Factors in Clinical Allergy

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Historical Introduction David G. Marsh Heredity has long been suspected to play an important role in the development of asthma, hay fever, and other conditions generally referred to as "atopic allergic diseases," which are now known to be mediated by IgE (1). Since much of the research carried out prior to 1960 has been extensively reviewed elsewhere (2-6), only the major findings will be discussed here. The early findings will be reviewed in the context of today's knowledge about the genetics of the disease and as a prelude to discussions of recent data on the genetics and epidemiology of allergic disease in subsequent chapters. Family Studies High prevalence rates for hay fever and asthma in particular families were observed long before the immunologic, pharmacologic, and physiologic bases of these diseases were investigated. As early as 1650, Sennertus (cited in ref. 7) reported the occurrence of asthma in three successive generations of his wife's family; in 1868, Salter (8) reported that 39% of 217 asthmatics had positive family histories of asthma. It is interesting that Salter (9) had, four years earlier, alluded to the multifactorial determination of asthma when he wrote, "several brothers and sisters in a family may be asthmatic without the parents having been so. This would seem to suggest . . . that certain combinations produce certain results, and lead to the creation of certain peculiarities, and that the combined qualities of the progeny are not the mere results of the combined qualities of the parents." In 1872, Wyman (10) found that "some families suffer more than others from 3

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Autumnal Catarrh [ragweed pollen hay fever] . . . [and] that not infrequently, while some members of a family have this disease, others have June Cold [grass pollen hay fever], an observation that was readily apparent in his own family. With today's hindsight, one might interpret Wyman's observations as indicative of at least two genetic controls of atopic allergy: a) transmission of a gene determining the general predisposition toward allergic disease, perhaps controlling the overall level of responsiveness of the IgE-producing system (Chapter 12), and b) transmission of genes controlling the predisposition toward antigen-specific responsiveness, i.e., specific immune response (Ir) genes (Chapter 7). Most of the early researchers failed to recognize the genetic significance of the heterogeneity of allergic disease, both in terms of the different types of symptomatology and in the allergen-specificity of allergic responsiveness, which was seen in Wyman's and many subsequent findings. Rather than considering atopic allergy as a complex multifactorial trait (influenced by many genetic and environmental factors), "one-gene hypotheses" were generally invoked to explain the inheritance of allergy. There are historical reasons for this bias. First, singlegene models were then favored as explanations for a number of complex human diseases. Second, it seemed quite appropriate to explain atopic allergy, a "strange (Gr aToma), other (Gr aXXos)" disease, by the presence of a single, dominant gene causing a generalized "supersensitivity" to allergens—which were then regarded as unique entities, set apart from antigens (11, 12). The most impressive early study was carried out in 1916 by Cooke and VanderVeer (13). They analyzed the inheritance of allergy in the families of 504 "allergic" patients (including a number having diseases of questionable atopic origin) in a report which covered over 100 pages of The Journal of Immunology. Cooke and VanderVeer found that 48.4% of their patients had a positive allergic family history, versus only 14.5% of the families of a control group of 76 nonallergic individuals. They made two interesting findings: a) people with a bilateral family history tend to develop allergies before puberty, those with a unilateral history, after puberty, and those with a negative history generally do not become allergic; b) children are not born allergic, but inherit the "tendency to develop allergy" equally from both parents. Cooke and VanderVeer concluded that the primary mode of transmission of the predisposition toward all types of allergies is genetic, not placental, and postulated autosomal dominant inheritance. Most of the early authors (13-20) favored dominant inheritance, except for Adkinson (21), who considered recessive inheritance more probable. In 1936, Wiener et al. (7) analyzed their own and previously published data and postulated the existence of an incompletely recessive allergy-determining gene. They suggested that individuals homozygous for the allergy allele (hh) develop atopy early in life, whereas a variable proportion (about one-sixth to one-fifth) of the heterozygotes (Hh) develop allergy, and then only after puberty. Wiener et al. and,

Ha earlier, Balyeat (16) pointed out "that the stronger the hereditary influence, the greater the tendency to multiple sensitivity" —i.e., hh individuals are allergic to more allergens than atopic Hh individuals. These postulates have their modern equivalents in one set of hypotheses relating to the genetic control of IgE production and its influence on multiple versus restricted IgE antibody responses. According to these views, IgE production is largely determined by a major gene, where high IgE is inherited as a recessive trait (22-24; Chapter 12); high IgE producers (rr) tend to be sensitive to multiple allergens, and low IgE producers (RR and Rr) are either nonallergic (in most cases) or are sensitive to very few allergens (25; Chapter 7). Several investigators (2, 3, 26, 27) subsequently failed to confirm Wiener's postulated relationship between age of onset and familial incidence of allergy. Also, there was evidence for genetic heterogeneity both in the allergenic specificity of atopic disease noted earlier (10) and in symptomatology (atopic rhinitis, asthma, and eczema, which may exist either alone or in any combination; 2, 13, 21, 28). To try to explain these observations, Tips (29) proposed a three-gene hypothesis. He suggested that three independent loci determine the aforementioned three principal types of atopic disease, with the expression of each disease being inherited as a recessive trait. No support for this view has been published subsequently, although many consider that independent gene(s) may control the airways hyperreactivity seen particularly in asthma (Chapter 10).

Twin Studies Twin studies provide a useful approach in evaluating the relative importance of hereditary versus environmental factors in the expression of disease (5; Chapter 9). Unfortunately, in most of the early studies the criteria for zygosity were not well defined. The first detailed study of allergy using twins was performed in 1936 by Spaich and Ostertag (30), who studied 71 twin pairs aged 3 to 79 years: 38 monozygotic (MZ), 28 like-sexed dizygotic (DZ), and 5 male-female pairs. They found higher concordance rates for asthma, hay fever, and eczema among monozygotics than among dizygotics. In a much larger study, Edfors-Lubs (31) questioned 7,000 Swedish twin pairs about their allergies. Zygosity diagnosis was also determined by questionnaire, except in a panel of 200 pairs where five serological markers were evaluated, the results of which correlated well with the questionnaire data. She obtained the following concordance rates: for asthma/hay fever/eczema (any combination), 25.3% for MZ pairs and 16.3% for DZ pairs; for asthma, 19% in the MZ twins and 4.8% in the DZ twins. These data suggest that nongenetic factors play an important role in the expression of allergies, a conclusion which is in accord with three smaller studies (32-34).

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Summary of the Early Results There are several problems with the early family and twin studies. The reports included different, often ill-defined, and sometimes questionable allergic diseases (e.g., migraine). Most investigators lacked adequate, properly matched control groups and did not investigate the prevalence of allergy among relatives of control groups as thoroughly as among relatives of the allergic propositi. While some of the studies considered the prevalence of allergy only among sibs, parents, or offspring of the propositi, others included more distant relatives. Of course, the likelihood of finding a positive allergic family history increases with an increasing family size, not to mention when aunts and uncles are included. A further major criticism of most of the studies is the widespread use of questionnaires to determine the prevalence of allergic manifestations among family members and twins. In the absence of simple, objective confirmation of atopic sensitivity, such as by skin testing, such data are highly suspect (Chapters 2 and 4). In summary, the early family studies demonstrate a familial aggregation of allergic diseases and suggest a genetic basis, but fail to show a clear-cut mode of transmission. The presence of important genetic and environmental components seems likely from the twin studies. The early data are more consistent with multigenic than with the unigenic inheritance postulated by most authors. Modern Approaches More definitive immunogenetic studies of allergic disease in humans became possible in the early 1970s following two crucial discoveries: Ishizaka and Ishizaka (1) showed that IgE is the immunoglobulin which causes atopic allergy in humans, and McDevitt and Benacerraf and co-workers (35, 36) demonstrated the presence of M//C-linked Ir genes in animals. The discovery of two human myeloma IgEs facilitated the development of radioimmunoassays for both total and specific IgE (1, 37). With the availability of these new technologies, significant relationships were soon discovered between the serum level of total IgE and atopic status (37, 38), as well as between the level of specific serum IgE antibody and the degrees of both skin-test and leukocyte sensitivities to the same allergens (1, 37-39). The mouse experiments of Levine and Vaz (4CM-2) and Watanabe et al. (43) showed that the ability of inbred strains to produce antibodies toward complex protein antigens is controlled by: a) A///C-linked Ir genes determining specific antibody responses of both the IgE and IgG classes toward antigen administered in very low doses, and b) an IgE-regulating gene unlinked to the MHC complex that regulates "non-specific suppressor T-cell function." These mouse studies showed that the familial segregation of specific immune responses could best be studied following immunogenically limiting immunization, with individual dosages of 0.1-1.0 (xg antigen administered with alumina

HISTORICAL INTRODUCTION

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gel adjuvant, two to four times about four weeks apart (total doses ca 10-200 (jig/Kg body weight). By contrast, the seasonal dosages of inhaled pollen allergens for adult humans usually total no more than 0.06-1.0 |xg (about 0.001-0.01 jxg/Kg), inhaled each year over a six- to eight-week pollen season without added adjuvant (44). (These calculations are based on pollen counts in the Baltimore, Maryland, area and normal adult breathing rates. They are certainly overestimates since they assume a 24 hour-a-day outdoor exposure, that all the inhaled pollen grains contain their full complement of allergens, and that complete extraction of the allergens occurs during the brief period the pollen grains are in the nose.) Marsh et al. (44, 45) hypothesized that such ultra-limiting antigenic exposure would facilitate the analysis of the genetics of immune responsiveness in the genetically highly polymorphic human population. This has, indeed, proved to be the case. As previously observed in the mouse, there is now evidence for at least two types of genetic control of IgE responses in humans, namely, MHClinked control of specific immune responses (Chapters 7, 8, and 9) and nonM//C-linked control of the overall production of IgE (Chapter 12). Since a clear causal relationship exists between the induction of specific IgE antibody responses and the subsequent clinical expression of specific allergies, the allergy model is highly relevant in evaluating the genetic factors that control or influence the expression of immunologic diseases in humans. Because most immune responses to inhaled allergens occur as a result of natural exposure to extremely low, immunogenically limiting antigen doses, this exposure may mimic the situation pertaining during the early phase of an infection more closely than do the model studies that have been performed in animals.

References 1. Ishizaka K, Ishizaka T. Human reaginic antibodies and immunoglobulin E. J Allergy 1968; 42: 330-63. 2. Schwartz M. Heredity in bronchial asthma. Acta Allergol 1952; 5 (Suppl 2): 1-288. 3. Ratner B, Silberman DE. Critical analysis of the heredity concept of allergy. J Allergy 1953; 24: 371-8. 4. Vaughan WT, Black JH. Practice of Allergy, 3d ed. St. Louis, cv Mosby Co 1954; 74-. 5. Bias WB. The genetic basis of asthma. In Austen KF, Lichtenstein LM, eds. Asthma: Physiology, Immunopharmacology, and Treatment. New York, Academic Press 1973; 39-53. 6. Black PL, Marsh DG. The genetic basis for atopic allergy in man. In Segal MS, Weiss EB, eds. Bronchial Asthma: Mechanisms and Therapeutics. Boston, Little, Brown and Co 1976; 53-64. 7. Wiener AS, Zieve I, Fries JH. The inheritance of allergic disease. Ann Eugen 1936; 7: 141-62. 8. Salter HH. On Asthma: Its Pathology and Treatment, 2d ed. London, Churchill 1868. 9. Salter HH. On Asthma: Its Pathology and Treatment. Philadelphia, Blanchard and Lea 1864. 10. Wyman M. Autumnal Catarrh (Hay Fever). Cambridge, Mass, Hurd and Houghton 1872; 82.

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11. von Pirquet C. Allergic. Munch Med Wochenschr 1906; 53: 1457. Translated by Prausnitz C in Cell PGH, Coombs RRA, eds. Clinical Aspects of Immunology. Philadelphia, F A Davis Co 1963; 805-7. 12. Coca AF, Cooke RA. On the classification of the phenomena of hypersensitiveness. J Immunol 1923; 8: 163-74. 13. Cooke RA, VanderVeer A. Human sensitization. J Immunol 1916; 1: 201-305. 14. Drinkwater H. Mendelian heredity in asthma. Br Med J 1909; 1: 88-. 15. Spain WC, Cooke RA. Studies in specific hypersensitiveness. XI. The familial occurrence of hay fever and bronchial asthma. J Immunol 1924; 9: 521-9. 16. Balyeat RM. The heredity factor in allergic diseases, with special reference to the general health and mental activity of allergic patients. Am J Med Sci 1928; 176: 332-. 17. Bray GW. The hereditary factor in asthma and other allergies. Br Med J 1930; 1: 384-6. 18. Bray GW. The hereditary factor in hypersensitiveness, anaphylaxis, and allergy. J Allergy 1931; 2: 205-11. 19. Richards MH, Balyeat RM. The inheritance of allergy with special reference to migraine. Genetics 1933; 18: 129-34. 20. Bucher CS, Keeler CE. The inheritance of allergy. J Allergy 1934; 5: 611-4. 21. Adkinson J. The behavior of bronchial asthma as an inherited character. Genetics 1920; 5: 363-70. 22. Marsh DG, Bias WB, Ishizaka K. Genetic control of basal serum immunoglobulin E level and its effect on specific reaginic sensitivity. Proc Nat Acad Sci 1974; 71: 3588-92. 23. Gerrard JW, Rao DC, Morton NE. A genetic study of immunoglobulin E. Am J Hum Genet 1978; 30: 46-58. 24. Meyers DA, Beaty TH, Freidhoff LR, Marsh DG. Inheritance of total serum IgE (basal levels) in man. Am J Hum Genet 1987; 41: 51-62. 25. Willcox HA, Marsh DG. Genetic regulation of antibody heterogeneity: its possible significance in human allergy. Immunogenetics 1978; 6: 209-25. 26. Peshkin MM. Asthma in children. IV. Hypersensitiveness and the family history. Am J Dis Child 1928; 36: 89-. 27. Schnyder UW. Neurodermatitis-Asthma-Rhinitis. Eine genetisch-allergologische Studie. Acta Genet 1960; 10 (Suppl). 28. Rackemann FM. A clinical study of one hundred and fifty cases of bronchial asthma. Arch Intern Med 1918; 22: 89-. 29. Tips RL. A study of the inheritance of atopic hypersensitivity in man. Am J Hum Genet 1954; 6: 328^3. 30. Spaich D, Ostertag M. Untersuchungen iiber allergische Erkrankungen bei Zwillingen. Z Menschl Vererb Konstitut Lehre 1936; 19: 731-52. 31. Edfors-Lubs ML. Allergy in 7,000 twin pairs. Acta Allergol 1971; 26: 249-85. 32. Criep LH. Allergy in identical twins. Report of seven pairs of twins. J Allergy 1942; 13: 591-8. 33. Bowen R. Allergy in identical twins. J Allergy 1953; 24: 236-44.

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34. Falliers CJ, Cardoso RR de A, Bane HN, et al. Discordant allergic manifestations in monozygotic twins: genetic identity versus clinical, physiologic, and biochemical differences. J Allergy 1971; 47: 207-19. 35. McDevitt HO, Tynan ML. Genetic control of the antibody response in inbred mice: transfer of response by spleen cells and linkage to the major histocompatibility (H-2) locus. J Exp Med 1968;128: 1-11. 36. Benacerraf B, McDevitt HO. Histocompatibility-linked immune response genes. Science 1972; 175: 273-9. 37. Johansson SCO, Bennich HH, Berg T. The clinical significance of IgE. Progr Clin Immunol 1972; 1: 1-25. 38. Gleich GJ, Averbeck AK, Swedlund HA. Measurement of IgE in normal and allergic serum by radioimmunoassay. J Lab Clin Med 1971; 77: 690-8. 39. Lichtenstein LM, Ishizaka K, Norman PS, et al. IgE measurements in ragweed hay fever. Relationship to clinical severity and the results of immunotherapy. J Clin Invest 1973; 52: 472-82. 40. Vaz NM, Levine BB. Immune responses of inbred mice to repeated low doses of antigen: relationship to histocompatibility (H-2) type. Science 1970; 168: 852-4. 41. Levine BB, Vaz NM. Effects of combinations of inbred strain, antigen, and antigen dose on immune responsiveness and reagin production in the mouse. Intern Arch Allergy Appl Immunol 1970; 39: 156-71. 42. Levine BB, Vaz NM. Two kinds of genetic control of reagin production in the mouse. J Clin Invest 1970; 49: 58a (Abst). 43. Watanabe N, Kojima S, Ovary Z. Suppression of IgE antibody production in SJL mice. I. Nonspecific suppressor T cells. J Exp Med 1976; 143: 833-45. 44. Marsh DG. Allergens and the genetics of allergy. In Sela M, ed. The Antigens, Vol. III. New York, Academic Press 1975; 271-359. 45. Marsh DG, Bias WB, Hsu SH, Goodfriend L. Associations between major histocompatibility (HL-A) antigens and specific reaginic antibody responses in allergic man. In Goodfriend L, Sehon AH, Orange RP, eds. Mechanisms in Allergy: Reagin-Mediated Hypersensitivity. New York, Marcel Dekker 1973; 113-29.

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Population Selection and Methods of Evaluating Immunologic Responses to Allergens Malcolm N. Blumenthal Atopic allergy provides an excellent model for evaluating the genetics of the human immune system in relationship to disease processes. Atopic allergic diseases are common conditions in which we can identify both the antigen and the immune systems involved. Much useful information on the immunogenetics of IgE mediated diseases has been obtained using population, twin, and family studies (1,2). Many factors should be considered in the study of the immunogenetics of atopic disease. These include definition of parameters to be measured, study design, selection of subjects, and method of statistical analysis. The definitions of various atopic diseases vary with the investigator. Asthma, rhinitis, and eczema are allergic conditions for which definitions have not beer fully agreed upon. As a result, separate studies of allergic diseases may contain different types of populations depending upon how the diseases are defined. In an attempt to resolve this problem, some "standard" definitions are presented in Chapter 5. Both in vivo and in vitro procedures have both been used to measure immune responses in allergic conditions. The parameters of an immune response need tc be well delineated. The establishment of the relationships between different methods measuring the same parameter, the same method measuring the same parameter, or different methods measuring different parameters is important.

In Vivo Methods Several in vivo methods exist for studying the humoral antibody responses as well 10

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as responses of the T lymphocyte system (3,4). Skin tests are the primary in vivo method used to evaluate IgE mediated responses. The immediate wheal and flare skin reaction has been used to evaluate atopic conditions. Cutaneous (puncture or prick) and intradermal skin testing may be performed to detect the presence of cytophylic (reaginic) IgE antibody. This reaction reaches a maximum ten to 30 minutes after injection of the allergen. The allergen interacts with the IgE antibodies attached to cells that contain mediators of inflammation or their precursors (e.g., histamine, eosinophilic factor, platelet activating factor, and leukotrienes C4, D4, and E4, collectively known as SRS-A). As a result, mediating substances are released which cause vasodilation, erythema, localized edema, increased permeability, and puritis. Clinically, these may constitute the "immediate" wheal and flare and subsequent "late-phase" reactions. Many factors influence the skin test and its interpretation (4-9; chapter 5).

The Quality of the Antigen Impurities in the antigen preparation as well as denaturation of the antigen will influence the results. Either complex allergen extracts (e.g., whole extracts of pollens) or highly purified allergens isolated from these extracts are needed for epidemiologic and genetic studies of allergy. The whole extracts are used primarily to assist in diagnosis (e.g., of ragweed pollen allergy) and the highly purified allergens are used for studies of specific immune responses. As discussed in Chapter 4, the quality of these materials is of the utmost importance. Complex allergen extracts as well as highly purified allergens need to be standardized to ensure uniformity. The degree of purity of the allergens needs to be evaluated.

Differences in the Method of Performing and Recording Skin Tests The volume and concentration of antigen injected, equipment used to perform the test (scarifier, small-pox needle, darning needle, size of needle on the syringe), and site of injection (back, forearm, upper arm) are all factors which may influence the final reaction (5, 6). The skin response must be recorded in a standardized fashion. The time period between the injection of the antigen and the recording of the test is critical. The size of the reaction may vary markedly between readings at ten to 30 minutes after injection of the antigen. The methods for recording the results will also vary from study to study (5). These may include measuring of the skin reaction by the size of the wheal, or flare, or both. This can be recorded using the reactions' mean diameters (mm), areas (mm 2 ), or area paper weights. The latter involves tracing the shape of the skin reaction on a piece of paper. The tracing is then cut out and weighed. Other investigations use a "one-to-four plus" grading system depending upon the size and shape of the flare and/or wheal (4). Other methods, e.g., measurement of wheal thickness, wheal volume, and wheal blood flow by ultrasound and laser Doppler technique,

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have been proposed but are not commonly used (7, 8). Reporting of the test results should include the site, technique, and recording method used. Most investigators use the back and volnar surface of the arm for prick testing and the volnar surface of the arm for intradermal testing. It is recommended that skin tests be performed using uniform techniques and when the patient is off medications that may interfere with the skin results. In addition, it should be realized that allergy injection therapy may either increase or decrease the degree of the reaction depending upon its relationship to the time of testing.

Environmental Factors The time of day, month, and year that the tests are performed and the use of medications such as antihistamines and allergy injection therapy may influence the results (5, 9-12). Testing should be performed under consistent environmental conditions.

Human Factors The reproducibility of the test between the same and different technicians may vary. All of these factors must be kept as constant as possible or variations be evaluated and necessary adjustments made (5). The correlation between immediate wheal and flare skin-test reactions and clinical history is high. It is rare to have a negative skin test in a subject with a well-defined history and a known allergen exposure. It is not, however, uncommon to have a positive skin test in a subject with no clinical history. Thus, the skin test is highly sensitive, but is not necessarily correlated with a specific history (3-5, 12). When evaluating a given test, its sensitivity and specificity should be considered. To determine sensitivity, the number of true positive reactions in individuals with sensitivity to that allergen is divided by the total number of individuals tested with allergies to that allergen (true positives plus false negatives) and multiplied by 100. The specificity of a given diagnostic test is obtained by using individuals with a positive test but no clinical sensitivity to the suspected allergen. They are frequently designated as false positives. To calculate specificity, the number of true negatives is divided by the total number of healthy individuals tested without allergies to the particular allergens (true negatives plus false positives) and multiplied by 100.

In Vitro Methods IgE, an immunoglobulin class, appears causally linked to the development of atopic diseases. Measurement of IgE has been extremely helpful in better understanding the immunogenetics of atopic disease as well as useful for its diagnosis and management. Measurement of total serum IgE has been well established us-

EVALUATION OF IMMUNOLOGIC RESPONSE

13

ing radioimmunoassays (competitive radioimmunosorbent test [RIST] and noncompetitive RIST) and enzyme-linked immunosorbent assays (ELISA) (3, 14). Many assay systems are used to quantitate specific antibody levels by in vitro methods. The major ones used to evaluate the immunogenetics of human immune responses to allergens have been the specific IgE, IgG, and IgG4 assay systems (3, 14). The meaning of a proliferative in vitro lymphocyte response to an allergen in atopic conditions is not well established at the present time. The relationships between in vivo and in vitro determinations of the immune response and the clinical picture is not well defined. The radioallergosorbent test (RAST) utilizes an allergen covalently bound to a solid phase polysaccharide, e.g., cellulose or Sepharose (3). Initially, the solid phase allergen is exposed to serum containing antibodies to the allergen. The mixture is incubated and antibodies to the allergen, including IgE antibodies, then bind to the allergen. The mixture is then washed, leaving the specific antibody directed against an allergen which is coupled to the solid phase. Next, antiserum to human IgE (anti-IgE) that has been radio-labeled and purified by affinity chromatography is added. After incubation, the insoluble allergen immunosorbent is washed again. If IgE antibody was present, radioactive anti-IgE serum will bind to the IgE, which is attached to the specific allergen on the particle. The washed complex is counted in a gamma counter; the number of radioactive counts is proportional to the quantity of IgE antibody specific for the particular allergen. The quality of the RAST is dependent upon the allergen preparation, the capacity of different solid phase substances for the IgE antibodies, whether a given allergosorbent activity has enough immunoreactive allergens on its surface, and the quality of the preparation of anti-IgE. In addition, nonspecific binding is directly related to the total IgE level so that in sera containing greater than 5,000 units per milliliter this may become a serious problem (14-16). The test is influenced by high levels of blocking IgG, which competes with IgE antibodies directed toward the same allergen. This results in a false lowering of the binding of the labeled anti-IgE (16). The problem can be lessened by using a high capacity sorbent and high quality of purified allergens under conditions where all the specific antibodies in a sample will be absorbed. An alternative to the radioimmunoassay is the use of an enzyme coupled antibody, which can be used to detect antigen bound to solid phase antibodies. After washing, the antibody-enzyme complex is reacted with substrate and the enzyme activity measured. The amount of color in the final reaction is proportional to the amount of enzyme conjugated anti-IgE bound to the solid phase allergen-IgE complex. Enzyme immunoassay obviates the need for radioisotopes with their associated health hazards and problems of radioactive waste disposal (17). Generally there are significant positive correlations between the specific RAST, the skin test, and the specific atopic history. Not all patients with positive histories and skin tests have a positive RAST. Much less frequent are patients

14

MALCOLM N. BLUMENTHAL

with negative histories and skin tests for a particular allergen and a positive RAST(18, 19). The relationships between a particular method such as an immediate skin reaction or a RAST are not well defined. In addition, the reproducibility of the test performed on the same sample or different samples from the same person should be established. Techniques may vary because of internal or external factors, including test allergens and reagents used, reference control samples, and methods of recording results. Additional environmental factors, such as those described during the discussion of skin testing, are difficult to control and standardize. Individuals are exposed to different concentrations of antigens. For example, ragweed pollen exposure may vary within a city because of factors such as wind and use of air conditioning. Furthermore, the time of day, month, or year that the test is performed may be important (5, 6, 9). Measurements of reaginic antibody vary from season to season. A person may show a positive skin reaction with a high RAST titer to ragweed in September, but have a significantly less intense skin response and RAST titer in April or May. Finally, the age and sex of the subject may influence the results (Chapter 5). It appears that atopic diseases such as asthma, hay fever, and eczema are seen more frequently in males, especially before the age of fifteen. Total IgE levels tend to be elevated in males when compared to females. The frequency of allergic asthma, allergic rhinitis, eczema, elevated serum IgE levels, and specific IgE responses decrease with the aging process. Other tests measuring the antibody response (i.e., allergen-specific IgG4 and IgG delineators) as well as the cellular responses (e.g., histamine release assays and lymphocyte transformation) are available. Their proper significance needs to be defined before being used in genetic studies (Chapter 11). The major tests used to evaluate an immune response to allergen in atopic disease have been the prick skin test, intradermal skin test, RAST, and double antibody RIA (Chapter 7). Depending upon the antigen used, correlations between single dose prick and titration intradermal skin tests range from 0.43 to 0.67 using nonparametric correlation and from 0.79 to 0.8 using parametric correlation in studies performed by the author (13). These correlations were higher than those seen between RAST and either type of skin test. Other investigators have shown slightly higher correlations (20). While single-dose prick skin testing, intradermal skin testing, and RAST are correlated, it has not been demonstrated that they can be used in place of one another. The study design, method of selecting subjects, and type of analysis will vary depending upon the information desired. Four major types of studies have been performed for the evaluation of the genetics of allergy: population, twin, sib pair, and family.

EVALUATION OF IMMUNOLOGIC RESPONSE

15

Population Studies Population studies are used to establish frequencies, prevalence rates, and incidence of various types of atopic disease or of specific immune responses to individual allergens. Statistical associations between genetic markers (e.g., HLA antigen) and disease or immune response phenotype can then be studied. The strength of the association is given by the relative risk: the number of patients with the antigen divided by the number of patients without the antigen, and the number of normal subjects without the antigen divided by the number of normal subjects with the antigen. The significance of the relative risk increases as the sample size increases. This type of analysis can give indications for more detailed analysis, e.g., in families. In population studies, comparisons should be made between a group of unrelated allergic patients and a group of unrelated healthy controls. The selection of these individuals is very important because they may represent selected patients or controls from different geographic or ethnic backgrounds, both of which may affect the results of the study (Chapter 12). The unrelated patient group can also be evaluated by comparing responders (e.g., to a particular highly purified allergen) to non-responders. Ideally, a population of randomly selected subjects should be studied to establish prevalence. The problem is more difficult to deal with in atopic diseases since the measure of exposure to aeroallergens, as well as the time of year the patients are studied, are important considerations (9, 18, 19). There are other constraints on population studies as a means to establish the genetics of a disease or immune response (1, 2). First, the finding of no association does not establish that there is no gene for the disease or variable being investigated closely linked to the genetic system under study (i.e., HLA). Second, in population studies, one cannot determine whether an HLA-linked gene is a major gene or one of many genes, whether it is dominant or recessive, or whether it has full or partial penetrance. Last, many associations having p values in the range of 0.01 to 0.05 observed in a single population study are likely to be statistical artifacts. The reason is that when so many antigens and phenotypes are being considered, an observed association due to chance alone is not unlikely. Thus, one needs to perform at least two different population studies or correct for multiple tests. All this points up the fact that population studies by themselves can neither prove nor disprove the presence of a major susceptibility or resistance gene linked to HLA.

Twin Studies Twin study methodology has long been utilized as a test of the effect of genetic and environmental factors on the development of human traits (21-24). Classical

16

MALCOLM N. BLUMENTHAL

twin studies are based on comparisons of the phenotypic similarities observed between monozygotic (MZ) twin pairs and dizygotic (DZ) twin pairs. Since MZ twins are genetically identical (except for rare mutations), the phenotypic differences found between MZ twin pairs must be environmental in origin. DZ twins share an average of only 50% of their genes. If MZ and DZ twin pairs were raised under equally similar intrapair environments, DZ twins can then be used as controls for the effect of being raised as a twin. Comparisons of the variance of MZ and DZ twins for continuously distributed quantitative traits can yield estimates of heritability or the proportion of total trait variance accounted for by genetic factors. Heritability is useful only insofar as its assumptions and restrictions are not violated. These underlying assumptions are frequently difficult if not impossible to test. Concordance rates are another common method of estimating genetic contributions. If a trait has a heritable component, MZ twins should exhibit higher rates of concordance than DZ twins. These methods give only rough estimates of the relative genetic and environmental effects. Moreover, methods such as model testing and multivariable analysis promise to extract more meaningful data from twin samples. Studies of twins raised together and apart conducted by Blumenthai et al. (25) and Bouchard et al. (26) offer a unique approach to the problem. Twins reared apart offer an opportunity to examine genetic effects directly without the confound of similar rearing environments. Other problems of twin studies are that zygosity diagnosis must be accurate. An incorrect determination can have a significant effect on the results. The problem of obtaining an adequate sample size limits twin studies to relatively common traits such as allergy, where the prevalence rate in Caucasoid populations is about 20-25%. Difficulties arise when looking at a less common trait such as the specific IgE response to short ragweed allergen Amb a V (Ra5), where the prevalence rate in Caucasoids is about 2%. It should be remembered that twins are, by virtue of their unique embryological development, not comparable to the general population. Only with the greatest of circumspection may the results of genetic studies in twins be considered to represent the general population or be appropriately melded with data from paired singleton relatives in tests of genetic models. Finally, twin studies cannot give useful information about the mode of inheritance of a trait or disease. Despite uncertainties and criticism, twin studies are still widely used in medical genetics and will continue to be used as more efficient methods of data analysis are introduced. At best, twin studies will give information on the relative importance of any gene or genes versus environment, but they cannot serve to determine details about the genetic mechanisms of a disease (21-25).

EVALUATION OF IMMUNOLOGIC RESPONSE

17

Sib Pair Studies Investigation of the sharing of the HLA haplotype among sibs affected with a disease has been performed (27). Deviations of the sharing of the HLA haplotype from random expectation are taken as evidence of the existence of an HLAlinked "disease" gene. Studies now have been extended so that affected sib data may be used to detect the presence of HLA-linked disease genes, as well as to try to determine the mode of inheritance of the disease genes. This method of analysis has been used by Metro and Thompson (28) and shows some advantages and disadvantages of the formal family and population analyses. A family analysis is often complicated by missing data when a member is unavailable for testing. Although it is easier to obtain sib pairs for study, larger family analyses when assembled are more comprehensive.

Family Studies Family studies, although difficult to perform, are needed to evaluate the genetics of the immune responses. One of the biases in family studies is the selection of families. Usually families are selected because they contain two or more affected members. While such families are most informative for detecting linkage, they are not appropriate for determining the genetics of immune responses. This is because the genetic analysis of such families will result in biased estimates of penetrance, gene frequencies, and even transmission probability. Furthermore, in family studies there is the problem of evaluating the results of linkage analysis. To date, we do not know to what extent and in what direction unknown disequilibrium will affect the linkage analysis (29—33). A family analysis to demonstrate linkage is conducted by testing a propositus and close relatives for a genetic marker (e.g., HLA antigen, Gm allotype, or blood group antigen) and for evidence of a disease or one of its components. These analyses are often complicated by ascertainment bias as well as the need to compensate for missing data when a member is unavailable for testing. Despite the above mentioned problems, family studies are probably the most informative. The genetic control of atopic disease is complex. There are many genetic as well as nongenetic factors involved. Family, twin, sib pair, and population studies are providing needed information to understand the pathogenesis of atopic conditions.

References 1. Blumenthal MN, Bach FH. Immunogenetics of atopic diseases in allergy. In Middleton E Jr, Reed CE, Ellis EF, eds. Allergy: Principles and Practice, 2d ed. St. Louis, C V Mosby Co 1983; 11-17.

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2. Blumenthal MN, Mendell NR, Yunis EJ. Immunogenetics of atopic diseases. J Allergy Clin Immunol 1980; 65: 403-5. 3. Gleich GJ, Yunginger JW, Stobo J. Laboratory methods for studies of allergy. In Middleton E Jr, Reed CE, Ellis EF, eds. Allergy: Principles and Practice, 2d ed. St. Louis, C V Mosby Co 1983; 271-93. 4. Norman PS. In vivo methods of study of allergy: skin and mucosal tests, techniques, and interpretation. In Middleton E Jr, Reed CE, Ellis EF, eds. Allergy: Principles and Practice, 2d ed. St. Louis, C V Mosby Co 1983; 293. 5. Dreborg S. The skin prick test, methologic studies, and clinical application. (Dissertation). Linkoping, Sweden; Linkoping University 1980; 239: 8-41. 6. Voorhorst R, van Krieken H. Atopic skin tests reevaluated. II. Variability in results of skin testing done in octuplicate. Ann Allergy 1973; 31: 499-508. 7. Serup J. Diameter, thickness, area, and volume of skin prick histamine wheals. Measurement of skin thickness by 15 MHz A-mode ultrasound. Allergy 1984; 39: S359-64. 8. Serup J, Staberg B. Quantification of wheal reactions with laser Doppler flowmetry. Comparative blood flow measurements of the oedematous center and the parilesional flare of skin prick histamine wheals. Allergy 1985; 40: 233-7. 9. Roitman-Johnson B, Sothern RB, Halberg F, Blumenthal MN. Circadian immunologic rhythms and their implications in the diagnosis and treatment of atopic disorders. In Smolensky MH et al., eds. Recent Advances in the Chronobiology of Allergy and Immunology. Oxford & New York, Pergamon Press 1980; 65-72. 10. Smith JM. Skin tests and atopic allergy in children. Clin Allergy 1973; 3: 269-75. 11. Blumenthal MN, Greenberg L, Yunis EJ. Age related immune changes and their enhancement subsequent to routine skin testing. In Recent Advances in Gerontology. International Congress Series Excerpta Medica 1977; 207-9. 12. Turkeltaub P, Rastogi S, Baer H, Anderson N, Norman PS. A standardized quantitative skin test assay of allergen potency and stability. Studies on the allergen-dose response curve and effective wheal, erythema, and patient selection on assay results. J Allergy Clin Immunol 1982; 70: 343-53. 13. Blumenthal MN, Roitman-Johnson B, Walsh G, Mendell NR, Weinberg R. Correlations and relationships between RAST, prick tests, and intradermal tests. In preparation. 14. Platts-Mills TAE. Diagnostic tests. IV. Laboratory techniques in immediate hypersensitivity. In Lessof MH, ed. Philadelphia, J B Lippincott Co 1981: 85-9. 15. Paull B, Jacob G, Yunginger JW, Gleich GJ. Comparison of binding of IgE and IgG antibodies to honeybee venom phosphoase A. J Immunol 1978; 120: 1917-1923. 16. Zimmermann E, Yunginger JW, Gleich GJ. Interference in ragweed pollen and honeybee venom radioallergosorbent tests. J Allergy Clin Immunol 1980; 66: 386-93. 17. Metzger WR, Butler JE, Swanson P, Reinders E, Richerson HB. Amplification of the enzymelinked immunosorbent assay for measuring allergen specific IgE and IgG antibody. Clin Allergy 1981; 11: 523-31. 18. Norman PS, Lichtenstein LM, Ishizaka K. Diagnostic tests in ragweed hay fever. J Allergy Clin Immunol 1973; 52: 210-24. 19. Wuthrich B, Arrendal H. RAST in the diagnosis of hypersensitivity to dog and cat allergens. Clin Allergy 1979; 9: 191-200.

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20. Santilli J, Potsus R, Goodfriend L, Marsh DG. Skin reactivity to purified pollen allergens in highly ragweed sensitive individuals. J Allergy Clin Immunol 1980; 65: 406. 21. Hasema JK, Elston R. The estimation of genetic variance from twin data. Behav Genet 1970; 1: 11-19. 22. Hopp RJ, Bewtra AK, Watt GD, Nair NM, Townley RG. Genetic analysis of allergic disease in twins. J Allergy Clin Immunol 1984; 73: 265. 23. Escobar V, Corey L, Nancy W, Bixler D, Biegel A. The inheritance of immunoglobulin levels. Prog Clin Biol Research 1978; 24C: 171-6. 24. Allansmith M, McClellan B, Butterworth M. The influence of heredity and environment on human immunoglobulin levels. J Immunol 1969; 102: 1504-10. 25. Johnson B, Hanson B, Mendell NR, Blumenthal MN. Genetics of skin test response in twins raised together and apart. J Allergy Clin Immunol 1987; 79: 229. 26. Bouchard T, Heston L, Echert E, Keyes M, Resnick S. The Minnesota study of twins reared apart. Prog Clin Biol Research, Part B. Intelligence, personality, and development. 1980; 69C: 227-33. 27. Cudworth AC, Woodrow J. Evidence for HLA-Iinked genes in "juvenile diabetes mellitus." Br Med J 1975; 3: 133-5. 28. Motro V, Thompson G. The affected sib method. I. Statistical features of the affectic sib-pair method. Genetics 1985; 110: 522-38. 29. Elston RC. Segregation analysis. In Harris H, Hirschhorn K, eds. Advances in human genetics. New York, Plenum Publishing Corp 1981; 11: 63-120. 30. Boyle CR, Elston RC. Multifactorial genetic models for quantitative traits in humans. Biometrics 1979; 35: 55-68. 31. Dawson DV, Elston RC. A bivariate problem in human genetics: ascertainment of families through a correlated trait. Paper presented at the Biometric Society meeting in San Antonio, March 1982. 32. Elston RC, Namboodiri KK, Hames CG. Segregation and linkage analyses of dopamine-betahydroxylase activity. Hum Hered 1974; 29: 284-92. 33. Goldin LR, Elston RC, Graham JB, Miller CH. Genetic analysis of von Willebrand's disease in two large pedigrees: a multivariate approach. Amer J Med Genet 1980; 6: 279-93.

3^

Allergens and Allergen Nomenclature David G. Marsh During the past 80 years, and especially since the early 1960s, there has been considerable effort on the part of many research groups to identify and characterize the specific components which commonly act as allergens in humans. In order to provide a better focus for these efforts, the IUIS Subcommittee for Allergen Nomenclature has introduced a systematic nomenclature (1). Both highly purified allergens and components identified within complex allergen source materials by techniques such as crossed immunoelectrophoresis (CIE) and isoelectricfocusing (IEF) (Chapter 4) are included within this system. The aim is to provide a unified nomenclature which will promote future physicochemical and immunochemical characterization of allergens, thereby facilitating a wide variety of studies that employ allergens, including genetic and epidemiologic studies. Investigators are being encouraged to exchange materials, including monospecific antibodies (polyclonal or monoclonal), to allow allergen identification. Under the new system, highly purified allergens are designated by the first three letters of the genus (italicized), a space, the first letter of the species name (italicized), a space and, finally, a Roman numeral identifying the allergen (see examples in Tables 1 and 2). The major allergen is usually identified by the Roman numeral "I" (e.g., Lolp I for the rye Group I or Rye I allergen). Between related species and genera, structurally homologous (but not necessarily crossreactive) components are assigned the same numbers. Within the same species,

20

ALLERGENS AND ALLERGEN NOMENCLATURE

21

isoallergens* are designated by capital letters in the order of decreasing pi (e.g., Amb a VA and Amb a VB for the components formerly known as Ra5A and Ra5B, which have pi values of 9.6 and 8.5, respectively). An important aspect of the nomenclature system is that the degree of purity of all highly purified allergens should be clearly documented according to several diverse physicochemical and immunochemical properties —for example, molecular size (SDS-PAGE, gel filtration, etc.), molecular charge (IEF, gel electrophoresis, HPLC using an ion-exchanger, etc.), immunochemical techniques (CIE, CRIE, etc.), hydrophobicity (reverse-phase HPLC), and chemical analysis (NH2-terminal amino acid determination and amino acid composition). Each highly purified allergen is then characterized as completely as possible by physical, chemical, and biological criteria, including amino acid sequencing and, especially, sequencing of the respective cDNA or the entire "allergene" (cf., 49, 89). The allergenic importance of the purified allergen is then determined in subjects having IgE antibodies to a well-characterized, standardized crude extract from which the allergen was derived. Where international or national reference allergen extracts are available, such as those produced through the IUIS-WHO Allergen Standardization Program, a quantitative estimate is made of the content of the purified allergen in the reference (cf., Chapter 4). In the case of unpurified or partially purified allergenic extracts, antigens identified by CIE are assigned Arabic numerals, starting with those showing greatest anodic mobility at pH 8.6. IEF bands (Bds) are numbered according to pi, and SDS-PAGE bands are designated according to their relative molecular mass (Mr). Where both the pi and Mr values are known, the corresponding band may be designated using both types of data; e.g., Der/Bd3.75/30K. Any antigenically and allergenically cross-reactive components are identified, and the allergenic activity of defined components (e.g., antigens recognized by CIE) is investigated (e.g., by CRIE). Tables 1 and 2 provide a summary of the known physicochemical properties of most of the highly purified allergens that have so far been isolated, and serve as useful starting points for researchers interested in genetic studies of human immune responsiveness to allergens. It will be seen that allergens constitute a widely divergent group of macromolecules and no distinguishing feature has been found which separates them as a unique subset of antigens (7). Further information about the properties of allergen molecules is given in several older (7, 16, 22) and more recent (50, 98) reviews. However, a much more comprehensive, computerized allergen database ("ALBE") giving all available physical, chemical, and biological properties of well-characterized, highly purified aller* Structurally homologous, immunologically closely related, and often allergenically indistinguishable components (3, 7).

Table 1. Properties of Some Highly Purified Pollen Allergens

Source

Systematic Name

Poaceae (Gramineae; grasses) Lolium perenne Lolpl (perennial rye) Lot p 11 Lol p III Lolp IV LolpX PhlpV Phleum pratense (timothy) Phi p VI Phi p VII Phi p VIII Dae g I Dactylis glomerata (orchard) Poap X Poa pratensis (Kentucky blue/June)

Original Name(s)

B: 27,000 C: 27,000 Group II (Rye II) A: 11,000 Group III (Rye III) B: 11,000 HMBA (Gp IV?) 56,800 12,000 Cytochrome c 15,000 Ag25 15,000 Agl9 34,000 Ag30 -8,000 Ag3 33,000 Dgl

Group I (Rye I)

Cytochrome c

Ambrosiinae (ragweeds) Amb a I AgE Ambrosia artemisiifolia (elatior) Amb a 11 AgK (short ragweed) Amb a III Ra3 Ra3II Amb a IV Ra4 (BPA-R) Amb a V Ra5 Amb a VI Ra6

Ambrosia trifida (giant ragweed)

Amb tV

Rel. Molr. Mass, Mr

Ra5G

B: C: A: A: B: A: B: A: B:

No. Amino Acid Residues 233 233 97C 97C 508 112

300 82

pi

Allergen Nitrogen Carboin Dry EJ^ (%) hydrate (%) Pollen(%)a

5 .9b 15.1 13.3 5 .75b 10.3 5 .1 9.0b 10.6 9 .9 -10.0 4 4 .5 9.4 3 .9 5 .9b

12,000

112

9 .9

37,800 37,800 38,200 12,300C 12,300 22,800 4,990c-d 4,860C ll,500d 11,500" 4,390C

337 343 331 101C 101 189 45C 44C 108 103 40C

5. l b 5 .Ob 5 .9b 8.6b 8 .0 9.6b 8 .5 >11 .0 >11 .0 8 .3b

References

5.4

0.63

2-10

2.0 2.0 17.0 0.0

0.32

2, 3 , 6-9, 11 6-8. , 11 12 3, 14 5, 16 5, 16 5, 16 5, 16 17 13, 18

0.0

0.48

19-22

8.0

(0.12) 0.081

21, 22 23-26

6.5 0.0

(0.1) 0.032

24, 27 28-32

2.