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·ENZYMATIC CONCEPT OF
ANAPHYLAXIS AND ALLERGY
and
the Role of Eosinophils in Anaphylactic
Reactions related to Hormonal Alterations
BY
Z. Z. GODLOWSKI
D D (Cracow), Ph D(Edın ). BIRCP (Edin.)
Research Fellow of the Carnegie Trust, Edinburgh,
Physician to Ballochmyle Hospital, Ayrshire, Acting
Professor of Physio-patholngy and Lecturer in Clinical
Medicine at the late Polish School of Medicine,
Edinburgh University
FOREWORD BY
A. MURRAY DREWNAN
MD, FRCPE, FRSE,
Professor of Pathology, University of Edinburgh
E. & $. LIVINGSTONE LTD.
EDINBURGH AXD LONDON
1953

·PREFACE essentially involved in this branch of biology, becauso the author is not qualified for such a discussion, Only the minimum of established facts and working theories has been borrored from these basic sciences, a minimum necessary for the elucidation and understanding of those manifestations of allergy which the clinician faces in everyday practice. This work, therefore, intends to help those medical practitioners who are interested in the pathogenesis and basic atiology of clinical allergy For purely technical reasons the whole essay is divided into three main chapters, followed by "General Conclusions" In each chapter stress has been lad on one particular object, to make the presentation of this object moro comprehensivo certain fact, and theories are repeated in each chapter Tho terminology used for the designation of the intracellular proteolytic enzymes is based in principle on that accepted by M. Bergmann (1942), with some modifications For example, pepsinase, repsin-like proteinase, Cathepain I, and proteaso are synonyms, and they denominate an enzyme whose proteolytic activity 19 similar to but not identical with gastric pepsin. Trypsinase, trypsin-like proteinase, and Cathepsin II are synonyms, and they denominate an enzynie whose proteolytic activity is similar to but not identical with that of intestinal trypsin. The intracellular proteinases, however, differ in many respects from the corresponding enzymes of alimentary canal. I wish to take this opportunity of expressing my thanks to the Carnegie Trust for the Universities of Scotland for meeting the expenses of production and printing of the colour photo. micrograph plates as well as covering part of the cost of the printing of this book. Ny thanks are also due to Professor A. 21. Drennan for his help and criticisin and for putting at my dispo al all the rescarch facilities of his Department, to Drs F. A Richards, J S. Robson, M. MacDonald-Robson, and A. McLean for their assistance with my English and reading proofs (they have not, however, corrected the whole work); to Dr T. C Dodds for taking colour photomicrographs, and to Mr W. Robb for his supervision of the animals and for his assistance in animal experiments, Colour photomicrographis and tables are used by kind permission of the Editor of the Journal of Endocrinology EDINBL RGB. Z. Z. GODLOWVSKI. May 1953

·ENZYMATIC CONCEPT OF
ANAPHYLAXIS AND ALLERGY
and
the Role of Eosinophils in Anaphylactic
Reactions related to Hormonal Alterations
BY
Z. Z. GODLOWSKI
M D (Cracow), PhD (Edın), MRCP (Edin)
Research Fellow of the Carnegie Trust, Edinburgh,
Physician to Ballochmyle Hospital, Ayrshire, Acting
Professor of Physio-pathology and Lecturer in Clinical
Bledicine at the late Polish Schnol of Medicine,
Edinburgh University
FORETFORD BY
A. MURRAY DRENNAN
MD, FRCPE, FRSE
Profesant of Pathology. Uolversity of Edinburgh
E. & S. LIVINGSTONE LTD.
EDINBURGH AND LONDON
1953

·vi PREFACE essentially involved in this branch of biology, because the author is not qualified for such a discussion, Only the minimum of established facts and working theories has been borrowed from these basic sciences, a minimuni necessary for the elucidation and understanding of those manifestations of allergy which the clinician faces in everyday practice. This work, therefore, intends to help those medical practitioners who are interested in the pathogenesis and hasíc xtiology of clinical allergy. For purely technical reasons the whole essay is derided into three main chapters, follaned by "General Conclusions." In each chapter stress has been laid on one particular object, to make the presentation of tliis object more comprehensive certain facts and theories are repeated in each chapter. The terminology used for the designation of the intracellular proteolytic enzymes is based in principle on that accopted by MI Bergmann (1949), with some modifications. For example, pepsinase, pepsin-like proteinase, Cathepsin I, and proteaso are synonyms, and they denominate an enzyme whoso proteolytic activity is similar to but not lentical with gastric pepsin Trypsinnse, trypsin-like proteinase, and Cathepsın II are synonyms, and they denominate an enzyme whose proteolytic activity is similar to but not identical with that of intestinal trypsın. The intracellular proteinases, however, differ in many respects from the corresponding enzymes of alimentary canal I wish to take this opportunity of expressing my thanks to the Carnegie Trust for the Universities of Scotland for meeting the expenses of production and printing of the colour photomicrograph plates as well as covering part of the cost of the printing of this book My thanks are also due to Professor A. M. Drennan for his help and criticism and for putting at my disposal all the rescarch facilities of his Department, to Dra F. A. Richards, J S. Robson, M. JacDonald-Robson, and A. McLean for their assistance with my English and reading proofs (they bare not, however, corrected the wholo work), to Mr T. C Dodds for taking colour photomicrographs; and to Mr W. Robb for his supervision of the animals and for lus assistance in animal experiments Colour photomicrographs and tables are used by kind permission of the Editor of the Journal of Endocrinology, LDINBURGA, 2. Z GODLOVSKI. lay 1953 ·CONTENTS CHAPTER I ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY PAGE INTRODUCTION 1 2 4 9 ANTIGEN. Definition and Inechanism of action-Fate of antigen in the body- Haptens ANTIBODY Definition and mechanism of action-Site of antibody productson- Local immunity-Relation of lymphatic tisave to the release of antibodies Specificity of proteolytic antibodies SENSITISATION Mechanisms of sensitisation-Sensitization by the mechanism of slow spread of antigen--Sensitisation by the mechanism of rapid spread of antigen-Lymphoid tasue in rapid spread of antigen---Anti-anaphylaxis -- Passive sensitisation or passive anaphylazıs-Anamnestie reaction- Histo-chemical and sub-microscopical structure of the cell —Antigenic conditioning of proteins--Terminology and classification of proteinases Specificity of mtracellular proteinases--Reorganisation or adaptation of intracellular proteinases-Toxicity of enzymes-Protein catabolism seen in the sub-microscopical structure of the cell ANAPHYLACTIC SHOCK AND THE ANAPHYLACTIC REACTION Peptone shock--Anaphylactic shock-Chronic anaphylax33-Heparin in anaphylactic reaction Histamine in the anaphylactic reaction- Proteolytic enzymes and proteolytic catabolites in the anaphylactic reaction - Syndromes simular to the snaphylactic reactson - The enzymatic concept versus the concept of antigen-antibody reactions in anaphylaxis and allergy 30 CHAPTER 11 HORMONAL INVOLVEMENT IN ANAPHYLAXIS 17 47 INTRODUCTION. PITUITARY-ADREXAL RESPONSE. Mechanism of pituitary-adrenal response--Relation of pituitary-adrenal response to anaphylaxis INTERRELATION BETWEEX LIPOIDS AND CORTICOSTEROIDS Influence of exogenous corticosteroids on blood upoids-Mechanism of hpoid inactiration by corticosteroids Effects of lecithin on biological action of cortiosteroids-Physico-chemical, physical and chemical factors nactivating lipoids-Tentative explanation of the action of C1.01ygenated corticosteroida 10 anapbylazis 51 Yu ·viii COYTEXTS CHAPTER II THE ROLE OF EOSINOPHILS IY AYAPHYLAXIS PAGE 61 INTRODOCTION Hematopoietic and pon-hæmatopoietic formation of cosmophila. MECHANISM OF EOSINOPHIL FORMATION Factors conditioning Openopli formation Classification of eosinophila - Effects of antigenic and de-antigenised dieta on circolatmg and tissue eosinophus Effects of the diet with de-antigenised proteine on intestinal eosinophilia-Inilgence of large dosage of heparin on circulating eosinopbila Ongin of eosinophilisation of tbe cryptal epithelial cells Enzymatic adaptation as worhing theory of eosinophilation-Function of lymphoid barrier in the lamina propria mucose of intestine-Merbanism of the arrest of posmophils in the intestinal lymphocytic barner-Role of hepari in mobilisation of eosinophus-Place of origin of cosinophils. 77 79 81 THE FATE OF EOSINOPHILS IN EOSINOPEXIA HORMOYALLY INDUCED INTRODCCTION METHOD Eosinophil counting--Assessment of tisspe eosinophilia-Effects of eosinopec hormones on white blood cells Intestinal perfusion- Hypophysectomy-Clotting time-Adrenalme infusion and insulin hypoglycæma. RESULTS Clinical experiments-Tissue posinophūia animals-Argentafline cells in alimentary tract --Intestinal coenopbla related to eosinopenic hormones Intestinal perfusion-The direct action of eosinopenic bormones on white blood cells. DISCUSSION Physiological furtuations mendatang xxsinophile-Hormonal eosinopenia and t1990e eosinnphile- Direct action of bobinopen hormones on white blood cells Merhanism of polymorphonuclear leucocytoes following wjection of corticosteroids. FUNCTION OF EOSINOPHILS Summary of easinophil formation--Relation of eosinophils to lymphoid tissue Eosinophils as source of antigen-Eosinophils a3 source of anapbylactic reactio–Pathogenetic significance of eosinophils 94 1) 101 CHAPTER TY GENERAL CONCLUSIONS 111 REFERENCES .' 116 INDEX ·CHAPTER I ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY INTRODUCTION B. EFORE entering upon the discussion of the problems of anaphylaxis and allergy, their terminology needs to be clearly defined Anaphylaxis and allergy, as will be shown below, are pathologically and ætiologically identical conditions. The only difference between them lies in the route and in the rate of penetration of antigens into the affected organism. Such dual terminology may cause confusion, hence, as a matter of convenience, the term anaphylaxis has been used for the phenomena of both experimental and clinical reactions due to sensitisation The generally accepted hypothesis of anaphylaxs is based on the antigen-antibody reaction The exact nature, place, and mechanism are, however, a subject of unsettled dispute It will be shown below that manifestations of anaphylaxis are not directly caused by the antigen-antibody interaction, very often no anaphylactic symptoms accompany such interaction. In the suggested enzymatic concept of anaphylaxis the anaphylactic phenomena should be regarded rather as resulting from the failure of the antigen-antibody reaction. The nature and mode of action between antigen and antibody, as well as the mechanism of sensitisation and anaphylactic shock or its equvalents, are most simply explained by the alterations in intracellular and extracellular proteolysis The easiest way of presenting the enzymatic concept of anaphylaxis is to give a brief description of the nature and the mode of action of antigen and antibody, and of the mechanism of sensitisation and anaphylactic shock The basic principles of this concept are not new, they were suggested by Biedl and Kraus in 1909 and were supported by other workers during the following fifteen years. Contemporary knowledge of cellular biochemistry and structure was inadequately advanced to permit of sufficient supporting evidence, and this hypothesis has not been generally accepted 1 ·2 EXZYBIATIC CONCEPT OF AXAPHYLAXIS AND ALLERGY ANTIGEN Definition and Mechanism of Action. According to Topley and Wilson (1936) the definition of antigen is as follows An antigen is any substance which, when introduced parenterally into the animal tissues, stimulates the production of antibody, and which, when mixed with that antibody, reacts with it in some observable way. This definition lays particular stress on antibody In the enzymatic approach to anaphylaxis the stress is laid upon the close interdependence of antigen and sensitisation which is the basic feature of anaphylaxis Sensitisation is a condition in which toxic enzymes appear in the cells involved as a result of antigenic action. Antigen may therefore be described as any substance which, when introduced into the animal organism, whether orally or parenterally, is able to induce the formation of (1) a toxic enzyme system in the cells liable to be sensitised, and (2) specific antibodies in the tissues concerned. These two fundamental features of antigen are not necessarily coupled with each other, ie, the production of specific antibodies may take place in an organism in which sensitisation has not occurred, and vice versa. This is a brief outline of the hypothesis which is discussed in this work. Antigen may enter the organism by several portals of entry, eg, skin, lungs, alimentary canal, etc., or it may originate in the organism's own proteins In the latter case the protein is altered in some ray, whether chemically or physico-chemically, and the organism thereafter treats it as a foreign protein. For example, during and after a surgical operation material from damaged cells enters the circulation and may exert its antigenic activity upon various organs The chemical constitution of antigens varies considerably. The substance which actually exerts antigenic activity on the affected cells is always a protein, either wholly or in part. The substance which originally entered the organism may not be a protein and may not possess, per se, antigenic power. Such a substance after entering the organism interacts with the organism's own proteins. The resulting compound acquires antigenic activity (Haptens). Antigen, per se, is not toxic: when it enters an organism ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 3 not previously sensitised it produces no anaphylactic reaction. Antigen must be distinguished from a group of substances which are not antigens but which, when injected into a nonsensitised organism, produce symptoms identical with those of anaphylaxis Examples of this pseudo-antigenic activity are the reaction following the intravenous injection of chloroform (p. 43), peptone shock (p. 30), and the reaction following intravenous injection of human gamma globulin into nonsensitised dogs or men (p. 42). The difference between antigenic and pseudo-antigenic actions 18 of fundamental importance, because it explains the mechanism of anaphylactic reaction The sensitising dose of antigen entering the cell for the first time causes toxic protease to appear in the cell. A second ("shock ") dose of the antigen will then initiate toxic proteolysis which liberates intracellularly toxic breakdown products. By contrast, in pseudo-antigenic action, the pathological proteolysis is begun without previous appearance of toxic enzymes (for details see p. 40) Fate of Antigen in the Body. The antigenic protein, in its initial invasion in experimental anaphylaxis, as well as in clinical allergy, may be distributed all over the organism It was demonstrated by the radioactive tracer technique that practically all organs show the presence of radioactive antigen (Crampton et al, 1950; Haurowitz et al, 1951), and that tracer antigen 19 demonstrable in the cytoplasm as well as in the nucleus of the cells (Coons et al, 1951) Another group of experiments with radioactive antigen showed that soon after its injection into specifically sensitised rabbits radioactive antigen appears first in the microsomes and later in the mitochondria (site of numerous enzymes (Peters, 1952 b)) of the invaded cells (Haurowitz et al., 1951). Since the latter are regarded as the place where the proteins are synthesised (Claude, 1943), the above workers have suggested that antigenic protein may be incorporated into the organism's own proteins The final fate of the antigen in the organism is not definitely known. Experiments with antigenic proteins marked with azo-dyes showed that their molecules are first phagocytosed by polymorph leucocytes, and later that these molecules appear in the phagocytes of the reticuloendothelial system (Heidelberger et al., 1933; Sabın, 1939, ·4 ENZYMATIO CONCEPT OF ANAPHYLAXIS AND ALLERGY and others). Dixon et al. (1951), using a double marking method of proteins with azo-dyes and with radioactive 1131, proved that dyed proteins are somewhat different from the non-dyed proteins in so far as their transportation into the interior of the organism is concerned. They also demonstrated that antigenic protein gradually disappears from various organs pari passu with the rise of the titre of the specific antibodies in the blood (for explanation see p 15) The longest period in which antigen in its native form was demonstrated in the organism was 120 days after its injection (Ifelaster et al., 1951). The ultimate fate of the antigen or its derivatives (produced by union of antigen with antibody) is presumably proteolysis. According to the theory suggested in this essay, antigenic proteins may, in swtable conditions, be incorporated into the structure of the host-cell. In such a form they may remain intracellular as long as the invaded cell lives They may or may not alter the whole character of these cells. Further more, this physiopathological state may be transmitted to the noxt generation according to the Mendelian law, Haptens. Primarıly non-antigenic substances, such as benzoic acid derivatives, may acquire antigenic character if, after entering the organisin, they couple themselves with the organism's own protein molecules. these newly formed antigenic compounds are designated as haptens (Landsteiner, 1920) or derivative antigens (Chase, 1952) These attached substances are then called determinant groups because they confer specificity upon the proteins with which they are linked (Marrack, 1950). Haptens can also be produced artificially in vitro, and their specificity may be tested serologically by using specific antisera (Landsteiner et al., 1927, 1929, 1932, 1934, 1939, Pauling et al., 1942, Pauling et al., 1914). ANTIBODY Definition and Mechanism of Action, Antibodies are proteins of globulin type, some of which are gamma globulin (Kass, 1945; and others) They appear in the body as a result of antigenic action and aro marked ·ENZYBIATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY 5 chemically in such a way as to enable them to interact with the specific antigen, rendering it inactive (Blarrack, 1950). If the criteria for the classification of antibodies are their effects on the antigenic protein, there are at least five main types of antibody to be distinguished 1. If the new protein compound, formed from the protein of antibody and protein of antigen, disintegrates completely to the level of non-toxic catabolites, such antibody may be called lysın 2 If the newly formed protein molecules become insoluble in the existing chemical environment and are precipitated, such antibody is called precipitin (Eagle, 1935) 3 If the viscosity of the newly formed protein molecules, localsed on the surface of bacteria or cells, is such as to cause aggregation of these bacteria or cells, this antibody is called agglutinin 4. If an antibody attaches itself to the antigen molecules on the surface of bacteria or cells, forming a layer which alters the surface-acting forces of the bacteria or cells, thus permitting phagocytosis by phagocytes, this antibody is called opsonin (Mudd et al, 1934) 5 Complement fixation and tozın neutralisation also take place as a result of attachment of antibody to the specific antigen (Rich, 1914) The first type of reaction may be regarded as enzymatic, and the antibody concerned as proteolytic enzyme This action may take place either inside the cells or in the body fluids, and may be directed either (a) towards one particular type of antigenic protein (by formation of a "bridge” or by spatial adaptability of the interacting molecules (p 24)), or (6) towards any protein which contacts them in their active form. does not mean that all proteolytic enzymes existing in the organism are proteolytic antibodies (for details see p. 8.) The tryptic action of antibodies and enzymes is the main topic in the further discussion on sensitisation and anaphylaxis. The second, third, and fourth types of antidod's may be regardes as acting through physico-chemical channels; the changes in solubility and viscosity result from union of antigenic protein with the protein of antibody. The mode of action of the fifth This ·6 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY type of antibody is as yet not clearly understood; its action, unlike that of the first type, is usually antigen-specific. The task of the last four types of antibody is to neutralise the antigen by one of the above-mentioned forms of antigenantibody reaction By so doing, the antigen-antibody reaction prevents the spread of antigen and so protects the organism from sensitisation by the initial dose or from anaphylactic reaction if the organism is already sensitised. Site of Antibody Production. There are various reports claiming that all cells of a normal organism are able to produce antibodies (Cannon et al., 1932; Walsh et al., 1934, Westwater, 1910, Oakley et al. 1919). However, their evidence is not conclusive (critically reviewed by Cannon et al., 1934) The main source of the production of antibodies is lymphoid tissue (Drinker et al., 1941, Ehrich et al, 1942. Dougherty et al, 191412, and others). Whether the whole lymphatic system or only part of it is engaged in the production of antibodies depends on several factors. If the regional lymphoid tissue is sufficiently active in neutralisation of the invading antigen (by antigen-antibody reaction) so that antigen cannot pass the regional lymphatic barrier, the actual task of antibody production rests with the regional lymph glands If, however, antigen penetrates through the lymphoid barrier or is brought into the interior of the organism by hæmatogenous spread, the whole lymphatic system may be engaged in manufacturing antibodies (Howell, 1928; McMaster et al., 1935: Sabun, 1939). The lymphatic tissue invaded by antigen may hypertrophy as a result of its increased activity. Such hypertrophy presents as a local or general enlargement of the lymph nodes Thus parenteral administration of foreign protein in ammals or men may produce enlargement of the spleen and lymph nodes (Epstein, 1929, Wiseman, 1931 ; Rich et al., 1939). Local Immunity. The interior of the organism may be invaded by the antigen if it travels via blood capillaries, or if antigen-antibody reaction fails to neutralise the penetrating antigen in the regional lymphoid barrier or in the body Auds. In both cases various organs are invaded by the antigen and theoretically all these ·ENZYMATIC CONCEPT OF AXAPHYLAXIS AND ALLERGY 7 invaded organs may produce antibodies Even if these organs are able to manufacture specific antibodies, the amount of these antibodies contributed to the general pool in the blood can be of little significance The ability of other than lymphoid tissues to produce antibodies may explain their local immunity Such a mechanism of local immunity is not generally accepted (critically reviewed by Cannon et al, 1934) Another more plausible explanation of local immunity suggests a mechanism similar to that operating in local fixation of antibodies in inflamed tissues from the blood (Fox, 1936) The enzymatic approach to local immunity offers a different explanation. The presence of normal proteolytic enzymes in normal cells. possessing the power of rapid proteolysis of any protein, antigenic or not, to the level of non-toxic catabolites, deprives the antigenic molecules of their toxicity before they can perform their sensitising action Thus, according to this theory, local immunity depends upon the ability of immune organs (normal organs) to hydrolyse the antigeruc protein by their non-specific proteinases; organs whose proteolytic enzymes are inefficient are not immune and are liable to be sensitised Relation of Lymphatic Tissue to the Release of Antibodies. A specific antigen travelling via the lymphatics guarded by the lymphoid tissue may be totally neutralised in the lymphoid barrier or in the body fluids by one of the five forms of antigenantibody reaction If so, the whole incident of antigen penetration is closed, except that specific antibodies have appeared in the regional lymphoid tissue This lymphoid tissue in the normal process of ageing disintegrates and releases the antibodies into the circulation. Thus localsed penetration of antigen 19 reflected in the body fluids in the form of free specific antibodies. Some of the Antigen may pass through the defence barrier of lymphatic tissue it may by-pass it by travelling in the blood stream, in which case all organs, the lymphatic system included, will be invaded by antigen in hugbly dilute form. Non-lymphatic organs with deficient proteolytic systems become sensitised. If in spite of the dilution of antigen lymphoid tissue has been sufficiently stimulated to produce specific antibodies, these will be temporarily stored there Thus lymphatic tissuo becomes an important source of antibodies. The injection of CH-oxygenated corticosteroids causes ·8 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY rapid dissolution of the lymphoid tissue (Selye, 1946); any antibodies contained therein will be released in such case into the circulation (Dougherty et al., 1944a, 1944b; White et al , 1946). If, however, the dilution of the antigen is too high or the lymphatic tissue insufficiently reactive, no specific antibodies will be formed, and the injection of lympholytic corticosteroids will not influence the titre of the specific antibodies in the blood. This may explain why the findings of the above-mentioned workers have not been confirmed by others (De Vries, 1950; Dougherty, 1951). Specificity of Proteolytic Antibodies. Intracellular proteolytic enzymes are present in varying quantities in all the tissues of a normal organism, whether or not antigens have en encountered. These proteolytic enzymes are non-specific in hydrolysis, re., any protein which makes contact with them in their active form) is split very rapidly to the level of non-toxic catabolites. They can also catabolise antigenic proteins, yet they must not be regarded as proteolytic antibodies because the hydrolysis of the antigenic protein has no specific character. These proteolytic intracellular enzymes are designated by various terms, such as proteinase, trypsinase, Cathepsin II, or by various other names according to their hydrolytic action on the synthetic substrates. The specific proteolytic antibodies are those proteolytic enzymes which can proteolyse specific antigen only, which were not present in the cell before the entry of antigen, and which by the hydrolysis of antigen abolish its toxic action. The specific proteolytic antibody may react with antigen in at least one of two ways 1. Formation of a bridge between molecule of enzyme and molecule of substrate by a particular polyvalent metal ion. 2. Spatial adaptability of the " binding planes" between two interacting molecules. Details of these two theories are more fully discussed on page 24, Proteolytic antibodies neutralise antigen by hydrolysing its protein to the level of non-toxic catabolites. Specific proteolytic antibodies have to be clearly distinguished from specific ·ENZYBATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 9 proteolytic toxic enzymes. Toxic proteolytic enzymes act only on specific antigens, but their final breakdoun products are toric. In other words, the specific proteolytic antibodies have the task of defending the organism against the noxious actions of antigeri, and the specific proteolytic toxic enzymes damage the organism by their toxic end products The distinction between specific proteolytic antibody and specific proteolytic toxic enzyme has fundamental significance in the enzymatic concept of anaphylaxis, this theory admits two factors separately operating in the pathogenesis of anaphylaxis 1. Antigen-antbody Reaction - This requires specific antibody, and its task is to prevent anaphylactic Sensitisation in the affected cells Formation of a Proteolytro zuc Enzyme System ---This constitutes the basic phenomenon of anaphylactic sensitisation Both these reactions are fully discussed in the chapter on sensitisation According to the enzymatic approach to anaphylaxis the anaphylactically sensitised cells manufacture a specific toxic protease Once the toxic protease is formed in the cell, it remains in this cell for the rest of its life, this enzymatic pattern may even be stretched beyond the life of that cell by the incorporation of this pathological enzymatic pattern into the biochemical phenotype (Gulck, 1944) In such a way the enzymatic constitution may be transmitted to the next generation of cells and to the next generation of the whole organism. This may explain the hereditary character of anaphylactic phenomena SENSITISATION Mechanisms of Sensitisation. Sensitisation takes place in the cells of so-called shock organs. These are the tissues where most of the anaphylactic reaction takes place. According to the enzymatic concept of anaphylaxis there are two main mechanisms of anaphylactic sensitisation : 1. The invasion by the sensitising dose of antigen takes plaa ·10 ENZYMATIC CONCEPT OP ANAPHYLAXIS AND ALLERGY via the lymphatics from various portals of entry, at a slow rate (e.g., from respiratory tract, alimentary canal, skin, etc.). 2. The sensitising dose invades the organism via blood capillaries and reaches the interior of the organism at & rapid rate, by-passing the lymphatic barrier (eg., by intravenous injection). The first type of sensitisation is regarded by various allergists as occurring in human allergy. The second type of sensitisation is thought to take place in experimental anaphylaxis. Such a classification, however, is not justified, since in human allergy, eg, in serum sickness, an "anaphylactic" mechanism operates; vice versa, in experimental anaphylaxis, the sensitising dose of antigen may be introduced into the animal organism in the same manner as in human allergy Thus the main difference between these two forms of anaphylaxis lies in the route taken hy the antigen and hence the rapidity with which it reaches the shock organs Sensitisation by the Mechanism of Slow Spread of Antigen. In this type of sensitisation antigen travels along the lymphatics. Most or all of its molecules are arrested by antigen-antibody reaction in the lymph nodes (p 8), the cells responsible being lymphocytes, plasma cells, and the cells of the reticulo-endothelial system (Sabin, 1939, Oakley et al., 1949, and others). This artest results in complete, or almost complete, elimination of the penetrating antigen from the circulating lymph In these circumstances sensitisation does not take place at all In the course of a normal life the organism is contmuously invaded by various antigens, but the lymphatic barrier prevents their further penetration. Where the antigen-antibody reaction is inadequate (p. 12), some of the antigenic molecules may escape this defenco mechanism and reach the blood stream via the thoracic duct. These very minute amounts of circulating non-neutralised antigenic molecules penetrate the interior of almost all cells in the organism (Coons et al., 1951). Those cells which originally possess sufficiently active intracellular non-specific proteinases (Cathepsin II) can break down the invading antigenic protein to non-toxic catabolites. These are normal cells, and they may ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY II be described as insensitive to a given antigen or as immunised to it The cells which are liable to be sensitised are those which, at the time of the initial invasion of antigen, do not possess sufficiently active non-specific proteinases capable of proteolysing and thus getting rid of the antigenic protein Such cells are subjected to the toxic action of the unsplit antigenic molecules. The partially incapacitated proteinases of the sensitised cells may be still capable of hydrolysing the structural proteins of their own cytoplasm but they are incapable of proteolysing a foreign protein. To the stimulus exerted by the foreign protein these cells respond with the production of a new proteolytic enzyme manufactured by the mechanism of enzymatic adaptation " (p 25). This new enzyme, however, has two disadvantages . (1) It can proteolyse only to the level of proteose, and (2) it can proteolyse only specific antigenic protem. The time required for the manufacture of this new toxic protease is designated as the period of sensitisation, and its length varies in various species of animals It also varies within the same species according to the type of antigen used for sensitisation. In other words, in this mechanism of sensitisation, two independent and parallel processes take place 1 The lymphoid tissue along the paths of the penetration of antigen is stimulated to produce various antibodies (p 5) 2 The cells with inactive non-specific proteinases are stimulated to produce intracellular specific toxic proteases The physiopathological process in which a normal organism 13 invaded continuously by vanous antigens in ordinary life may be represented by a simplified formula Such a formula illustrates the relationship between the invading antigen and the formation of antibody on the one hand, and the process of sensitisation on the other In this formula AG represents any antigen penetrating a normal organism, AB represents antibody produced locally in the lymphoid tissue around the portal of entry of the antigen, and the numbers attached to these symbols aro arbitrary figures expressing the quantities of the operating antigen and antibody, A-Ar represents antigen-antibody reaction. Each line represents & further stage in the process.

·12 ENZYDIATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY PHYSIO-PATHOLOGICAL FORMULA OF IMMUNITY Initial Stage- AG 10 penetrates organism possessing AB 0, A-Ar 0; AG 10 spreads AG 10 penetrates organism possessing AB 1; A-Ar+; AG 9 spreads AG 10 penetreates organism possessing AB 2; A-Ar+; AG 8 spreads. Further stages of antibody production and sensitisation, until AG 10 penetrates organism possessing AB 10, A-Ar++; no AG spread AG 10 penetrates organism possessing AB 15, A-Ar++; surplus AB 5. (Description in text) In the intial stage of AG 10 penetration the whole amount of AG 10 reaches the interior of the organism and various organs are penetrated by various quantities of AG, the cells with inefficient enzymes cannot proteolyse foreign protein and sensitisation supervenes. If all the cells of an organism possess efficient proteolytic enzymes, sensitisation does not follow despite the entry of antigen. If during the period of sensitisation a sufficient quantity of specific antibodies becomes available, the anaphylactic reaction cannot supervene in spite of uninterrupted invasion by AG 10, because the barrier of antigen-antibody reaction provents further entry of antigen into the sensitised cells. This state of physiopathological equilibrium can be maintained only if the antigenic molecules are permanently neutralised by antigen-antibody reaction either in the regional lymph tissue or in the blood. The production of antibodies in the organism invaded by antigen gives rise to three main varieties of the immunological response 1. The tissue which manufactures antibodies does not produce sufficient antibody to neutralise the whole ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 13 of the invading AG 10. As soon as sensitisation is established, the circulating antigenic protein entering sensitised cells precipitates various forms of anaphylactic reaction, this is the primary anaphylactic reaction. 2 The output of specific antibodies suffices to neutralise the whole of the AG 10. This means that antigenic molecules cannot pass the barrier of antigen-antibody reaction to reach sensitised cells (sensitised in the early stages of AG entry) The quantity of the available antibody is, however, inadequate to form a safety margin Should there be a rapid increase in antigen absorption or a decrease in the amount of antibody, the state of equilibrium may give place to a secondary anaphylactic reaction 3 The production of antibodies goes on until a surplus is obtained, by this action a powerful defence barrier is formed (AB 5 in the example quoted above) This safety margin successfully deals with an accidental increase in the antigen or with a rapid decrease in the titre of antibody This is the state of active immunity The state of equilibrium, mentioned in (2), between the neutralised invasion of antigen and the co-existing state of sensitisation may, in suitable pathological conditions, initiate & manfest anaphylaxis For example, if production of antibodies has become suppressed by some exogenous poison such as barbiturates or iodides or by an infection, the balance between AG and AB becomes upset, this loss of balance leads to the invasion of the interior of the organism by some nonneutralised AG which initiates various anaplıylactic reactions of secondary type This formula gives a simplified picture of how various mechanisms may operate in the anaphylactic reaction Variations are possible, and numerous factors will influence the character of the response The quality of antigen has a decisive influence, thus of the antigenic protein is a virus or belongs to a bacterium, the response to such an antigen will be dominated and modified by the specificity of the living antigen » In this case tso (antigenic and rirus or bacterio-toxic) pathogenetic factors of equal importance initiate at the same ·14 ENZYMATIC CONCEPT OF ANAPHYLAXIS AYD ALLERGY time two different pathological mechanisms which are superimposed one upon the other. Other factors of equal importance in shaping anaphylactic response are the temperature and pH of the reaction. These may modify the yield of antibodies and change the velocity of the chemical and physico-chemical reactions. Sensitisation by the Mechanism of Rapid Spread of Antigen. In the mechanism of sensitisation induced by the rapid spreading of antigen via the blood stream (as in most cases of experimental anaphylaxis) the antigen reaches the interior of the organism without having made contact with the lymphoid tissue Antigen reaches all organs rapidly but the amount which actually enters the cells varies considerably because of the dilution. Those cells which are unable to proteolyse antigenic molecules manufacture a specific toxic protease in the manner described below Thus the difference between these two mechanisms of sensitisation lies in the rapidity of the antigen penetration, without initial contact being made with the regional lymph tissue Lymphoid Tissue in Rapid Spread of Antigen. In the hæmatogenous spread of antigen, the lymphoid tissue all over the body is invaded simultaneously with all other organs by a smaller amount of antigenic protein than it would have received by the (slow) lymphatic route, this difference is due to dilution and dissipation. Antigenic molecules are soon proteolysed by highly active non-specific trypsinases in normal organs as well as in the lymphatic tissue (Drinker et al, 1941; Fruton, 1916). Before being split the antigen stimulates lymphoid tissue to produce various specific antibodies. This short stimulation of lymphatic tissue may give two quantitatively different responses 1. The specific antibodies are produced and stored in lymphatic tissue until they are liberated into the body fluds by its disintegration in the course of normal ageing. The cells with a deficient proteolytic system have been sensitised by the same dose of antigen as entered the lymphoid tissue by hæmatogenous spread. In short, the sensitisation of the shock organs and the ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 15 production and commencing liberation of specific antibodies into the blood have occurred simultaneously. Neither process has interfered with the other. 2. If the lymphoid tissue has been unable to produce specific antibodies despite stimulation by antigen (eg, because of primary deficiency of lymphoid tissue, or because of too rapid disintegration of the antigenic molecules before the stimulus could be exerted), these antibodies do not appear in the body fluids The organs with the deficient enzyme system will, however, have become sensitised Thus the final state of affairs is that sensitisation has been established in the shock organs and that no specific antibodies are present in the organism If the shock-dose of antigen invades the interior of the organism by both hæmatogenous and lymphatic routes, the sequence of events will differ as between (1) and (2) In the type of response shown under (1) some part or the whole of the dose of antigen may be neutralised by the circulating specific antibodies If only a part of the antigen 13 neutralised, the remaining active antigenic molecules will reach sensitised cells at the same time as they enter the lymphoid tissue. Antigenic molecules which make contact with lymphoid tissue are inactivated by one of the five forms of antigen-antibody reaction The part of the antigen which reaches sensitised cells precipitates a somewhat abortive form of anaphylactic reaction, since the shock-dose has been partly neutralised. Anaphylactic shock, like any violent stress, mobilises the pituitary-adrenal response. Lympholytic steroids are released and dissolve lymphoid tissue on a large scale. This large-scale release of antibodies (Dougherty et al., 1944 a), as an emergency measure, comes, however, too late to neutralise the circulating antigen completely and thus prevent the anaphylactic shock. Hence, the finding of a high titre of specific antibodies in the blood, during the manifestations of shock or immediately after they have subsided, does not prove that they were present in equally high titre at the onset of anaphylactic shock. The biochemical events of the early stages of anaphylactic shock are of sufficient magnitude to precipitate the pituitaryadrenal response On the other hand, the velocity of release of ·16 ENZYDIATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY corticosteroids in the pituitary-adrenal response is so high (Vogt, 1950) that the effects of their lympholytic action may coincide with the manifestations of the later stages of the shock. The high titre of specific antibodies in these circumstances, therefore, does not prove that they were available in the same concentration at the onset of the shock and that by interaction with the shock-dose of antigen they precipitated the shock. If the total shock-dose of antigen has been completely neutralised by circulating antibodies, the antigen will not reach the sensitised cells and anaphylactic shock cannot occur, despite the injection of an apparently sufficient shock-dose. An experimental animal showing such a lack of response is commonly regarded as not sufficiently sensilisel. This certainly 18 not the case. The lack of anaphylactic response in these circumstances 18 caused by the high titre of circulating specific antibodies In type (2) (in which specific antibodies have not been produced at all) the injection of a shock-dose is followed by a full-blown anaphylactic shock because the whole dose of antigen reaches the sensitised cells These animals are commonly regarded as highly sensitised, which they are not. The explanation of this high sensitivity lies in a complete lack of antibodies in the blood As a result the injected antigen has free access to the sensitised cells, which, however, are no more highly sensitised than those of type (1). Anti-anaphylaxis. The involution of the lymphoid tissue (with stored antibodies) following the stress of anaphylactic shock (p. 50) raises the titre of free proteolytic and non-proteolytic antibodies. The surplus of circulating antibodies is able to prevent a second anaphylaetic shock should another dose of antigen be administered. This state, known as anti-anaphylaxis or de-sensitisation, develops of the animal survives the anaphylactic shock (Zunz et al, 1915) A similar procedure may be utilised for therapeutic purposes, except that full-blown anaphylactic shock should be avoided by the injection of a sub-toxic dose of antigen. A similar therapeutic measure is the production of non-specific shock by the injection of heterologous protein, colloidal sulphur, etc (P 44).

·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 17 Passive Sensitisation or Passive Anaphylaxis. Passive sensitisation or passize anaphylaxis is a condition in which a normal, never previously sensitised person or animal, if injected with the serum from an anaphylactic person or from an anaphylactic animal, may develop at the place of injection various forms of local reaction (wheal, urtica, etc) when forty-eight hours after the subcutaneous injection the specific antigen is administered into the general circulation An example of passive anaphylaxis is the Prausnutz-Kustner phenomenon (Prausnitz et al, 1921) Coca et al (1925) maintain that the antibody operating in the Prausnitz-Kustner phenomenon has a special character and they designated this antibody as atopic reagin The enzymatic concept of anaphylaais offers the following explanation of passive anaphylaxis The toxic enzyme system" (nuclear factor plus molecule of specific antigen (for details see p 25)) formed in the sensitised cells is continuously excreted into the body ffuds in various quantities, thus the serum obtained from an allergic subject possesses a certain amount of this toxic enzyme system as free protein molecules So long as these large molecules remain in the infiltrated tissue (they remain there by force of adhesion) the local enzymatic set-up is identical with that of the donor-sensitised organism There are, however, the following differences between the donor (sensitised) and the recipient (non-sensitised) organism 1. In the sensitised organism the fozic enzyme system is distributed all over the organism, whereas in the recipient organism the toru enzyme system is only locally accumulated 2. In the donor organusm the toxic enzyme system persists permanently with some fluctuations in its concentra. tion, whereas in the recipient organism the locally accumulated toxic enzymes disappear in a short time as the result of normal proteoly813. Anamnestic Reaction, Anamnestic reaction (Greek: árauseris =recollection) is a reaction to the parenteral administration of a heterologous protein in which various antibodies (not related to the injected protein) appear in the body fluids The enzymatic concept of ·18 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY anaphylaxis explains this reaction in the following way: Injected molecules of the heterologous protein release the pituitary-adrenal response and liberated lympholytic corticosteroids produce rapid involution of the lymphoid tissue. Those antibodies whose production was induced sometime in the past, and which have been stored in the lymphoid tissue, are released into the body fluids in the course of the disintegration of the lymphoid tissue Therefore any factor which is able to release the pituitary-adrenal response is apt to induce anamnestic reaction Histo-chemical and Sub-microscopical Structure of the Cell. Before entering into a discussion of the altered chemistry of the cells and body fluids found in anaphylactic reactions it would be well to summarise some of the established facts of the present-day knowledge of sub-microscopical cellular structure. The next few paragraphs are based mainly on three sources: Frey-Wysgling's (1953) monograph on sub-microscopical biology of the cytoplasm, Monnė's (1948) review of cellular enzymatic structure, and "The Enzymes " by Sumner and Myrback (1950). The data will be presented in summary form; they will serve as a framework and basis for a more detailed review of cellular changes found in anaphylaxis and in allergy. The cytoplasm of a living cell consists of semi-liquid matter composed of proteins, lipoids, carbohydrates, electrolytes, etc., dissolved or suspended in water The framework of this mass is built mainly of proteins whose molecules possess a fibrillar structure and form a fine network. A stream of water (water plus substances dissolved in it is called by some workers enchylema, Monnė, 1948) constantly circulates in the interfibrillar canals thus formed and contacts various parts of the cell by means of chemical, physical, and physico-chemical interactions. The protein fibrils are formed by a variable number of polypeptide chains linked with each other by the end- or side-groups of the amino acids from which the chains are built. This linkage is formed either by direct interaction of their side- or end-groups, or by the formation of salts (bridge formation) by various polyvalent ions (Fig. 1, A, B, and c). The cortical part of the cell (called also the cell membrane) is composed mainly of lipoids whose rod-shaped molecules are in some places pierced by protein molecules The polypeptido ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 19 chains of these protein molecules are continuously coiling and uncoiling, shrinking and swelling, shortening and elongating. In so doing they cause periodical fenestration" in the cell L-Cystine .-Tyrosine CH-5-5-CHE HH CEC CH-NH, CH–NH, HO-C5 -CH, CH–NH, COOH COOH COOH 1Iryptophane H C H HC -C-CH-CH–NH, CH COOH H Fig. la Examples of three main types of amino acids (aliphatro, aromatic, and hetero-cycle) which build polypeptide chains Homopolar cohesive bonds CH CH, CH, CH- Heteropolar cohesive bonds 000.00 OH OH Soooo OK O OH- CI OH *NH, 50;- C1- COOH Cart K Ether bridge Salt formation Ester bridge Amide bridge CO-NH, Com ENHE NH -- Sulphur brides Fra 1B Dragrammatic representation of junction between neigbbouring polypeptide chains in the cytoplasm, 0= water molecules. (From A. Froy ligosling, * Submicroscopic Morphology of Protoplasm," Second English Edition, 1933. Elsevier Publishing Co. Inc, Nor York and Amsterdam) membrane and thus permit a foreign molecule, whether protein or not, to enter the cell and induce various chemical or physicochemical reactions such as enzymatic adaptation, inactivation of cellular lipoids, etc (Fig. 1, 4, B, and c).

·old
Pro
++
Pe
Pro
Pe
Pro
Pro
FIG. IC
Model of a molecule of & simple proten of which eight molecules of proteoga
are sepa
represents linkage between polypeptile molecules (PP); this linkage
18 hydrolysed by 650-peptidases (Stith, 1952)
-.-.-represents linkage between peptone molecules (Pe) by polyvalent
hong, this lumbago 18 hydrolysed by endopeptedases (protasses)
representa lizhage between molecules of proteoses (I'ro), this
linkage is hydrolysed also by endopeptidasas.

·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 21 Antigenic Conditioning of Proteins. For a foreign particle to enter the cell certain conditions must be fulfilled ; a particle of appropriate size must be adjacent to an open cortical fenestra and a force must be available to drive it through the cortex into the interior Two points in this statement require to be discussed in greater detail 1. The size of the particle 2. The force of suction which actively shifts the particle into the interior 1 The importance of the size of the penetrating particles must be strongly emphasised because size is one of the factors determining the antigenicity of a given protein. Foreign protein molecules, whether they have originated outside or inside the organsm, are disseminated all over the organism by the blood and body fluids (Coons et al., 1951) Protein molecules of size larger than that of the cortical fenestra of the bost-cell cannot penetrate the cellular interior, for the fenestræ form the sole means of entry Therefore these proteins cannot exercise any antigenic action, despite the fact that they possess all the other characteristics of an antigenic protein The importance of molecular size in the determination of the antigenic potency of a given protein has been stressed by many investigators (Neuvil et al, 1946, Loiseleur et al, 1947, Loiseleur, 1950, and others), their statement that the antigenicity of a protein varies inversely with its molecular size is consistent with the theory described above, re, that antigenic molecules enter the cell through the fenestræ The difference in the molecular size of various proteins also explains the fact that protein which is highly antigenic when applied to one species may show no antigenicity when applied to another Different species have proteins of different molecular size and that confers specificity on their proteins ; hence their cell fenestræ are also of different sizes 2. The force which moves the foreign particle from the outside to the inside of a cell through the fenestræ in the cortex may be desenbed as “suction." This svetion is due to periodical movements of the protein molecules in the cortical canals of the cell. Protein molecules periodicaly shrinking and extending in the cortical canals exercise movements which ·22 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY may be compared with the piston moving in the cylinder whereby the fuel mixture is sucked into the cylinder of a motor engine. By this mechanism the foreign particle is passively aspirated into the interior of the cell. Antigenic potency depends also on the chemical and physico-chemical properties of the protein, For example, protein molecules deprived of their lipoid conjugation acquire strong antigenicity, whereas if the lipoid-free protein moiety is covered with lipoids such as sodium oleate it loses some of its antigenicity (Jobling et al , 1915 a) These alterations in antigenic power, which are related to the presence of the lipoids in the protein molecules, depend on a twofold action of the lipoid-free proteins in the interior of the invaded cell: 1 By surface-acting forces they remove the cellular Lipoids from the lipo-protein molecules of structural proteins (55) 2 They augment the existing toxic enzymatic system, built in the course of sensitisation (p. 28); by this action they initiate toxic proteolysis by specific proteases In short, a protein, vf it is to have antigenic potency, must fulfil the following three conditions. 1 The size of its molecules must be smaller than those of the structural proteins of the invaded cell 2 Its molecules must escape proteolysis and be brought in unsplit form to the surface of cells possessing an inadequate enzymatic system, they must be placed sufficiently close to an open fenestra to be sucked through it 3 Its chemical or physico-chemical structure must be such as to induce the formation of toxic enzyme system and to initiate its activity in the sensitised cells Terminology and Classification of Proteinases. The term profernases is applied to all types of proteolytic enzymes The terminology of the intracellular proteinases accepted in this essay is based principally on Bergmann's classification (1912). His earlier work with Fruton and Irving (1941) provided him with the framework of this classification.

·EYZYBLATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 23 He distinguishes, amongst others, two types of Cathepsin (intracellular proteinases) which he extracted from various bovine and porcine organs. Their proteolytic action on synthetic substrates resembles either 1. That of gastric pepsin, and these he called pepsinases or Cathepsin I, or 2. That of intestinal trypsin, and these he called trypsinares or Cathepsin II. However, he found differences between the action of intracellular proteinases and that of the proteolytic enzymes of the alimentary canal. For example, the optimal pH for the proteo lytic action of gastric pepsin 18 1.5 to 2, whereas for beef spleen pepsinase it 18 56, for intestinal trypsın it is 7.8 and for beef spleen trypsinase it is 4 9. This demonstration of two different intracellular proteinases, each splitting proteins to a different level of degradation, is of fundamental importance to the enzymatic concept of anaphylaxis and allergy The terms toxic proteinase and toxic enzyme system are used In this essay for those enzymes which by their hydrolytic activity reduce the protein molecules to the level of toxic catabolites For example, a proteinase which splits protein molecules to the level of proteose and which liberates proteoses intracellularly or extracellularly is called a toxic protease Specificity of Intracellular Proteinases. Intracellular proteinages were regarded at one time as specific, and this belief formed the basis for the Abderhalden reaction (Abderhalden et al, 1909, 1910, a and b, 1911). Jobling with his co-workers (1915 a) proved this theory to be not entirely correct They separated various proteinases from serum and demonstrated that the tryptic proteinases po-sessed the capacity to proteolyse any protein provided that the lipoid "antiferments" of the serum had been removed The present view 18 that only some proteinases Bro specific. The proteolytic enzymes classified in this essay as trypsinases or Cathepsın II are non-specific in character and are normaally abundant in all living tissues Lymphatic tissue is particularly well provided with highly active, non-specific proteinases (Drinker et al., 1941; Fruton, 1946). A very brief summary of two theories, illustrating the ·24 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY specificity of enzyme-substrate protcolysis, is presented here. These are 1. The binding planes theory (Bergmann, 1936). 2. The bridge formation theory (Smith, 1948, a and b). 1. The binding planes theory holds that enzyme and substrate molecules cannot interact unless the configuration of atoms and atom groups located on the interacting planes 18 such that they fit spatially into one another. In other words, the specific combination between enzyme and substrate depends on the pattern in space of the interacting molecules ? The bridge formation theory holds that the molecules of SIDE- AND END-GROUPS OF POLYPEPTIDE CHAINS PROTEIN OF ENZYME ION+ PROTEIN OF SUBSTRATE FIG. 2 Description in text an enzyme and a substrate cannot start the enzymatic process until the reaction between the end- or side-groups of the interacting molecules is completed. One or more of the endor side-groups cannot react with the corresponding group of the opposite molecule unless a bridge is formed between them by one polyvalent metal ion Thus the required ionic bridge enables the initiation of the enzymatic procoss, provided the polyvalent ion itself can react with both enzyme and substrate molecules In this way the polyvalent ion selects chemically those molecules which can initiate the enzymatic process By this action the ionic bridge confers the specificity upon the enzyme and protein This theory is presented above in diagrammatic form in Fig. 2 The distinction between specific proteolytic enzyme and specific proteolytic antibody has been discussed on page 8.

·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 25 Reorganisation or Adaptation of Intracellular Proteinases. Sufficient evidence has been adduced that an unsplit protein molecule or a colloidal particle of suitable size can enter a normal cell (Wonne, 1948), this penetration takes place during the periodical fenestration of the cortical part of the living cell (p. 18) Protein molecules, marked with fluorescent dye, were demonstrated by Coons et al (1951) in the cytoplasm and nuclei of cells which they had entered by this route The foreign protein molecules remaining intact inside the cell upset the intracellular equilibrium by their chemical and physicochemical actions, thus delivering a toxic stimulus to the nucleus In response to this stimulation the nucleus produces an organising factor for a new enzyme, and the nuclear element enters the cytoplasm (Monné, 1948) The nuclear organising factor may be looked upon as a gene which, having reached the cytoplasm as a cytoplasmatic gene (Spiegelman et al, 1946), interacts with the foreign protein molecule and so creates a new enzymatic system This process, leading to the formation of a new proteinase, may be set in motion of the existing intracellular proteinases are unable to split the foreign protein (see next paragraph). The formation of a new proteinase in these circumstances must be regarded as a counter-measure taken by the nucleus against the toxic action of the foreign molecules remaining unsplit inside the cytoplasm. If the toxic impact made by the foreign molecules on the biochemistry of the nucleus is deep enough, or if the constitutional biochemistry of the nucleus is such that it cannot form a new normal enzyme in response to the toxic stimulation, then the nucleus may manufacture a pathological or toxic proteinase The toxicity of the new enzymatic system may be characterised by only partial proteolysis and by action solely on molecules of the same protein which participated in the formation of the toxic enzyme system, se, this toxic enzyme may be specific. The reaction of the invaded cell to a foreign protein may tako a different turn. The new adaptive enzyme may be able to carry out complete hydrolysis of the invaded protein. Such on enzyme, according to Bergmann's suggestion (1942), may, in suitable conditions, be bi-phasic. In the catabolic phase the large protein molecule 13 split to its primitive components amino acids. In the anabolic phase of the same proteinase, the ·26 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY ro-synthesis of the protein molecule may be accomplished. If the new adaptive enzyme has catabolised the intruding protein it may, in its anabolic phase, re-synthesise it. Ultimately this replica of the forcign protein may be incorporated into the structural proteins of the primarily invaded cell. The future of these cells is influenced by many factors. They have acquired at least two new features, namely, a new proteolytic enzyme and a new structural protein. These two characteristics completely alter the nature of these cells. The character of these cellular units will entirely depend on : (1) The character of the adaptive enzyme ; (2) The character of the reconstituted protein. 1. If the anabolic and catabolic activities of the adaptive enzyme are well balanced, and if the reconstituted protein molecules have no pathological features, the cellular metabolism will be little altered If catabolic activity predominates in the cellular metabolism, these cells soon die out and the whole incident is closed More pregnant in consequences is the alternative in which the anabolic phase of the adaptıre enzyme predominates in the metabolism of the affected cells: the subsequent pathology will depend to a great extent on the type of reconstituted protein. 2. The primitive catabolites derived from hydrolysis of the foreign protein are at first the sole protein material for the reconstitution of its molecule by the anabolic phase of the adaptive enzyme. A further supply of the required material is later derived from the organism's own resources. The newly synthesised protein molecule is a replica of the invading protein since it was built by the adaptive enzyme This replica next becomes incorporated into the structural proteins of the invaded cell. Thus the foreign protein is incorporated into the organism's own structural proteins, by this action a fundamental and deeply rooted alteration has been implanted in the affected cells. If the character of the adaptive enzyme is of anabolic typo and the self-renewing protein possesses antigenic power, te., gives rise to toxic protease, a vícious circle may develop. The invading protein may be pathogenic not only by virtuo of its antigenic power, for example, it may be a virus protein. If so, the vicious circle is doubly harmful, since the reconstituted ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 97 protein has two distinct pathological effects. But this aspect of the problem cannot be discussed further in this essay, for such discussion would lead us into the vast fields of inflammatory and neoplastic pathology. The process of new formation or reorganisation of the existing intracellular enzymes under the action of a new substrate is called by some enzymologists enzymatic adaptation (Karstrom, 1938, Spiegelman, 1950, and others). This theory is built on observations made on lower organisms, but there is a strong suggestion that the same process may take place also in higher organisms (Burnet et al., 1949). This concept is, however, not universally accepted in its original form Those who object to it argue that the new substrate (e, the penetrating antigen) must be chemically closely related to the existing substrate (t.e., the cellular structural proteins) for adaptation to be possible This argument implies that the new substrate stimulates the existing enzymatic system in such a way as to bring out the activity of an enzyme already present in the cell (Sevag, 1946) Toxicity of Enzymes, Somo enzymes possess brological toxicity which they exercise in various pharmacological ways (Warrack, 1950) Some of the toxicity of the toxic proteinases is inflicted by the end products of the hydrolysis carried out by them (Peters, 1952 a) For example, proteoses resulting from this proteolysis may appear in the tissues or in the body fluids and be highly toxic, as, for example, in anaphylactic shock, the protease responsible has acquired the features of a toxic enzyme. Peterman (1912) has suggested that there may be two types of enzymatic proteolysis . (1) the all or none type, in which large protein molecules are broken down rapidly to small dialysable fragments, (2) the precemeal type, in which lughly dialysable fragments are split off, leaving heavy particles but no unchanged protein The pattern of precemeal hydrolysis may be the one which occurs in the anaphylactic reaction Partially altered protein molecules which have been reduced in size by the splitting-off of a certain number of light dia. lysable fragments (amino acids) are, of course, proteoses--the first breakdown products of proteins In a later discussion it will be pointed out (p. 39) that Menkın (1950) was able ·28 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY tu reproduce some of the manifestations of anaphylaxis by parenteral injection in non-sensitised animals of substances which he had isolated from inflammatory exudates; these isolated substances were products of high-level proteolytic degradation. It is therefore reasonable to assume that the toxic substances isolated by Jenkin are identical with the proteoses liberated by toxic proteases Similar toxic proteolysis may occur in other types of inflammation The formation of the specific toxic enzymatic system takes place during the period of sensitisation which runs the following course the nucleus invaded by the antigenic molecules is stimulated by the toxic action of the unsplt protein (Branchet, 1050), this results in the formation of an organising factor for the formation of a new enzyme (Monné, 1948) The organising factor, when it enters the cytoplasm, reacts with other molecules of the same (sensıtısıng) dose of antigen by the mechanism of couple reaction The newly formed toxic enzymatic system can start proteolysis only when another molecule of the same protein augments the coupled enzymatic system (Bergmann, 1942), this augmentation takes place when antigenic molecules enter the sensitised cells, as, for example, during the administration of the shock-dose. The process of sensitisation and anaphylactic response can be presented in the following formula AG+ TE →AG'TE toxic proteolysis AG TE + AG-AG TEAG? →AG’TE +proteoses > AG1 represents unsplit antigenic molecules of the sensitising dose in the interior of a cell to be sensitised, AG? represents unsplit antigenic molecules of the shock-dose; TE represents toxic enzyme inside a sensitised cell, AG'TE represents toxic enzyme system inside and outside the sensitised cells AG’TEAG? represents augmented toxic enzyme system which immediately splits the augmenting antigenic molecule (AGP) to the level of various proteoses At the end of this hydrolysis toxic enzyme system (AG'TE) remains intact inside the sensitised cell ready to be augmented again if another shock-dose of specific antigen enters the sensitised cell. Ability to produce non-specific proteinases and inabihty ·EXZYBIATIC CONCEPT OF ANAPHYLAXIS AXD ALLERGY 29 to form toxic proteases appears to be, in some cases, organ and species specific. For example, the connective tissue surroundling the bronchioles in guinea-pigs responds to the injection of a toxic dose of specific antigen by forming gross edema (Warren et al., 1948); hence, according to the enzymatic concept of anaphylaxis, the cells of the connective tissue around the bronchioles in guinea pigs furm toxic specific protease during the course of sensitisation Why only certain tissues respond to antigenic stimulation by forming specific proteases in species specific anaphylaxis is difficult to say In the case of hereditary sensitivity the root of this inability les probably in a genetic deficiency of the biochemical constitution of the nucleus (Bourne, 1951), whereas in the case of acquired sensitivity the inability may be caused either by a constitutional debility of the enzymatic system (in the gwen organ or given species) or by the ageing of cellular proteolytic enzymes in the course of normal or pathological reear and tear Protein Catabolism seen in the Sub-microscopical Structure of the Cell. The cytoplasmic proteolytic enzymes are proteins whose fibril-like molecules are formed of polypeptide chains just as are those of substrate proteins The enzymatic proteins are, however, separated from the cytoplasmie substrate proteins by an insulating barrier of lipoids as shown in Fig 3 This insulating action of lipoids depends on a homopolar cohesive linkage between the end- and side-groups of the polypeptide chains of the substrate and those of the enzyme proteins This blocking action of lipoids has been called by various Authors the antifermenl polency (Jobling et al, 1915 a) Any action which can disrupt this intracellular lipo-protein linkage 19 apt to proteolysi The velocity and co-ordination of the proteolysis depends on the manner in luch the lipoid molecules are removed from the lipo-protein conjugation. For example, surface-acting substances, such as molecules of proteins or piroteoses (or any other colloidal particles) can initiate proteolysis by rapid alteration of the dispersion phase of lipoids. Colloidal molecules reaching the intenor of a cell may start a chantic proteolysis in this way. If the cells involved possess & specific toxic enzymatic system (ie, if they are ·30 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY sensitised), the specific antigenic molecules can initiate an uncontrolled toxic proteolysis by the mechanism described on page 28. The chemical reactions resulting from the substrate enzyme union lead to the removal of the peripheral amino acids or polypeptide chains from the substrate proteins (Fig. lo). Temporary inactivation of the cellular lipoids may also be achieved through other mechanisms, as, for example, by a purely chemical action (p. 53). It has been proved (Jobling et al., 1914) that the lipoid molecules can exercise their blocking action only if they possess unsaturated carbon atoms in their fatty acids. The saturation of these carbon atoms by various chemical methods (p. 108) may be another mechanism by which intracellular lipoids are inactivated It is not the mechanism, however, by which proteoses inactirate lipoids. These operate in all probability by & physico-chemical means, eg., by altering the dispersion phase of the lipoid molecules (Jobling et al., 1915 a). For further discussion on this problem see Chapter II ANAPHYLACTIC SHOCK AND THE ANAPHYLACTIC REACTION : Peptone Shock, It has long been known that so-called peptone shock and anaphylactic shock are two identical reactions so far as their manifestations are concerned (Biedi and Kraus, 1909; and others). The resemblance of these two syndromes is so marked that most of the early work concerned with the fundamental problems of anaphylaxis were studied on peptono shock. The main argument against the peptone-shock theory of anaphylaxis was that blood withdrawn from a dog in anaphylactic shock contained no transferable toxic agent (peptone) by which peptone shock could be reproduced in another non-sensitised dog (Weil, 1917). Grosjean in 1892 found that intravenous injection of Witte's peptone in a dosage of 0.15 to 0.30 gm. per kilo into a normal dog produces a reaction shown by later workers to be characteristic of anaphylactic shock (Table 1). The basic difference between these two syndromes is as follows. in anaphylactic ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLEROY 31 shock the peptone molecules (or any other proteoses) are liberated inside a previously sensitised cell, whereas in peptone shock the peptone molecules invade a non-sensitised cell from outside. In order to produce peptone shock in a non-sensitised dog one must inject a relatively high dose of peptone, because TABLE 1 Comparison of the Manifestations of Anaphylactic and Peptone Shock with their References Manifestation, Anaphylactic Shock. Peptone Shock. Hyperbeparinæmia Jaques et al, 1910 Quick, 1936 Hyperbistaminæma Code, 1939 Dale, 1929 Leucopenia Widal et al., 1921 Widal et al, 1921 Thrombocytopenia Dragstedt, 1941 Rocha e Silva, 1946 Increased titre of non. Jobling et al, 1915c| Jobling et al, 1915 c specific protewase in blood Increased titre of pro- Pfeiffer, 1915 tease in blood Increased NPN in blood Jobling et al, 1915 c) Jobling et al, 1915 d Hyperglycæma Zunz, 1924 Zanz, 1924 Hypoproteinemia Petersen et al, 1923 Petersen et al, 1923 Increased proteins Petersen et al, 1923 Petersen et al, 1923 lymph Increased lymph flow Petersen et al, 1923 Heidenhain, 1891 RBC's in lymph Petersen et al, 1923 Heidenhald, 1891 Increased permeability of Calvary, 1911 Calvary, 1911 capillares Decrease of plasma Simonds, 1923 Simonds, 1923 volume with bæmoconcentration Fall in blood pressure Simonds, 1923 Simonds, 1923 in (1) the dose is diluted by the body fluids and (2) the amount of peptone which reaches the interior of the cell must be well above the proteolytic capacity of the invaded cell Injected peptone molecules penetrate all organs, and those cells which possess highly active intracellular non-specific proteinases split the peptone molecules immediately to the level of non-toxic catabolites. Cells possessing insufficiently activo proteinases cannot proteolyse the whole shock-dose of ·32 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY peptone; the portion which escapes proteolysis inflicts its toxic action upon the intracellular lipoids in a manner resembling that of the proteoses produced intracellularly during the anaphylactic reaction (see next paragraph) Peptone in a dosage lower than that required for shock is immediately proteolysed and there is no surplus of peptone left to initiate rapid and inco-ordinated intracellular proteolysis. This point is worth stressing, because it explains why in the blood of a dog in anaphylactic shock Weil (1917) could not discover any transferable agent capable of reproducing peptone slock in another non-sensitised dog Anaphylactic Shock, Anaphylactic shock and the anaphylactic reaction are phenomena which differ from each other only quantitatively. Both take place in sensitised cells, both result from the invasion of the interior of sensítı ed cells by antigen, and in both the intracellular specific toxic proteases hydrolyse the antigenic protein to the level of peptone-like proteoses The main difference lies in the amount of antigenic protein penetrating the sensitised cells, and in the rate of its penetration By detailed analysis of the histo-chemical events involved in anaphylactic shock, four stages of shock may be distinguished This separation into stages is somewhat arbitrary because each phase of the reaction runs uninterruptedly into the next with a very high velocity. However, for purely practical purposes of presentation of the chemical events, a slow-motion picture of the biochemical sequence in anaphylactic shock has been formulated Its value is solely descriptive First Stage of Anaphylactic Shock The shock-dose of antigen enters sensitised cells which already contain a toxic enzymatic system as a result of sensitisation The antigenic molecules augment tlus enzymatic system and thereby set in motion the toxic proteolysis of those antigenic molecules which have augmented the enzymatic system with the production of free proteose molecules in the cytoplasm At the end of this hydrolysis the toxic enzymo system is ready again to be augmented (and start proteolysis) by another molecule of antigen if it is available in the cell ·ENZYDLATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 33 (p 28), the turnover of this reaction will depend on the dosage of the applied shock-dose of antigen. Second Stage of Anaphylactic Shock Proteose molecules which have been born in the cells disrupt the lipo-protein molecules by altering the dispersion phase of the lipoid component The cytoplasmic structural proteins, deprived rapidly of their lipoid protection, are chaotically exposed to the hydrolytic action of the cellular non-specific proteinases which have been activated by the same action (lipo-protein disruption) Uncontrolled proteolysis of the structural proteins of the sensitised cells begins in this way and eventually leads to the disruption of the cellular structure and to the release of the cellular enzymes and toxic catabolites into the body fluids These first two stages may be regarded as the intrinsic phase of anaphylactic shock because they occur inside the sensitised cells Third Stage of Anaphylactic Shock The toxin released into the blood during the first two stages of anaphylactic shock exerts its pharmacological actions in organs hitherto unaffected and so gives rise to the various constitutional manifestations of anaphylactic shock The third stage may therefore be looked upon as the extrinsic phase of anaphylactic shock ar Fourth Stage of Anaphylactic Shock In this stage the final 188ue of anaphylactic shock takes place either recovery or death. In the recovery period the organism may acquire immunity against further invasion by the specific antigen of the organism Such immunity is due to a massive liberation of specific and non-specific antibodies by lyınphoid tissue as a result of the lympholytic action of the pituitary-adrenal response (p. 49), (2) pathological lesions in the shock organs or in the organs not primarily involved in the anaphylactic reaction (ectopic lesions): both of these changes may ultimately heal or may establish (3) secondary chronic anaphylactic conditions (see below).

·34 EXZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY Chronic Anaphylaxis. The severity of the anaphylactic shock depends on the amount of proteose released in the cells. This in its turn depends on two conditions: 1. On the amount of the antigenic protein which has entered the cells 2. On the kinetics of the intracellular toxic proteases and non-specific proteinases If the velocity of the protease hydrolysis is rapid enough and if the amount of antigenic protein is adequate, a full-blown anaphylactic shock results If, however, only a small amount of antigenic protein has entered the cells and this has been peptonised, the amount of released proteose is also small and the damage done to the lipo-protein structure is relatively slight. The symptomatology of this mild shock is therefore less dramatic But this mild degree of shock, if continually repeated, is apt to precipitate cellular alterations (see below) of sufficient magnitude to produce a pathological response called primary chronic anaphylaxis or the primary chronic allergic condition. If an acute shock precedes the onset of the chronic manifestations, the chronic condition is called secondary chronic anaphylaxis or the secondary chronic allergic condition. The acute develops into and merges with the chronic stage. The manifestations of both types of chronic anaphylaxis may be localised either in the shock organs or in the distant tissues (ectopic reactions) or in both The description of the local and constitutional manifestations of the anaphylactic reaction is deferred to the next few paragraphs Hepari in Anaphylactic Reaction. Heparın is present in all living cells in variable amounts, either free or conjugated with proteins (Chargaff, 1941). If * protein salt of heparin is exposed to the proteolytic action of intracellular enzymes, the heparin molecule may be split off. Free heparin is liberated in and eliminated from the affected cell. The remaining cellular proteins, deprived of heparin, undergo reversible gelation (Fisher, 1931). This change in the physico-chemical structure of the substrate proteins may ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AXD ALLERGY 35 influence the affected cell in various ways, a mobile cell, for example, may be immobilised An example of such action is the arrest of moving eosinophils in the lymphatic barrier in the lamina propria of the intestinal wall (p. 74) If the amount of the tissue involved in the anaphylactic reaction is great enough, so much heparin may be released that, entering the blood, it will cause its incoagulability. This actually occurs in the early stage of both peptone shock (Quick, 1936) and anaphylactic shock (Jaques et al, 1940) In the stage of recovery from the shock, heparin re-enters the interior of the gelated cells and restores the semi-liquid state of their cytoplasmatic proteins (Fisher, 1931), if the cells were immobilised, it restores their mobility (Godlowskı, 1952 a). The re-entrance of heparın into the cells leads to rapid reduction of the heparin level in the blood This in turn may create conditions favourable for spontaneous clotting, especially where there is a physiological slowing of blood flow, as, for example, at the junction of a collateral with a main vein (eg., the junction of saphenous and femoral veins). This fall of heparin concentration may explain the tendency to thrombotic accidents in the clinical conditions associated with various types of anaphylaxis (Gauthier-Villars, 1951) Another consequence of the fall in heparin concentration is capillary thrombosis with some extension proximally and distally into arteroles and venules. The mechanism of this capillary thrombosis may be as follows in tho recovery stage from acute or chronic anaphylactic shock, the focus involved in the anaphylactic reaction rapidly absorbs heparin from the passing blood, and so promotes clotting in the capillaries of the affected tissue This in turn causes an acute ischæmia of the tissue involved in the anaphylactic reaction, leading finally to ischamic necrosis The releaso of heparin and its sequela are the mere consequences of the intracellular proteolysis initiated by the inactivation of the intracellular Lipoids by proteose molecules. The release of heparin is thus secondary, having no direct causal connection with the primary mechanism of the anaphylactic reaction, therefore heparin injected in large doses should not influence the initiation of shock This con. clusion was confirmed by an experiment in which the injection of a very large dose of heparin (2,000 units per kilo) into sensitised rabbits and guinea pigs did not lessen the lethal ·36 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY effect of an injection of antigen thirty minutes later (Godlowski, unpublished data) Histamine in the Anaphylactic Reaction. If the uncontrolled catabolic activity of the anaphylactic reaction splats off the amino acid histidino from the polypeptide chain it may be rapidly converted into histamine by histidinedecarboxylase (Holtz, 1937, Blaschko, 1945) Histamine may be released also by a different mechanism In conjugated form it exists in almost all tissues (Gaddum, 1948) In the course of toxic proteolysis such conjugated histamine may be liberated in the cells, where it exerts an intrinsic action, and may escape into the body fluids, where it exerts its extrinsic action, precipitating a great variety of manifestations in various tissues By its extrinsic action it is responsible for most of the dramatic symptoms of anaphylactic shock (Gaddum, 1948) and of syndromes resembling anaphylactic shock ( r40) Its powerful action on epithelium and capillaries, on the smooth muscles of various organs and on the secretory action of various glands, is the chief agent responsıble for most of the symptoms of shock, so that at one time the anaphylactic reaction was regarded as histamine poisoning (Dale et al, 1910-11, Dale, 1929, Schild, 1939, Dragstedt, 1941, and others). The violent symptoms of the anaphylactic shock produced mainly by the release of histamme involve the pituitary-adrenal response (Sayers and Sayers, 1917) with its own train of effects (for details see" Hormonal Involvement in Anaphylaxis ") Heparin and histamine should therefore not be regarded as taking an integral part in the anaphylactic reaction, they may or may not do so The liberation of these substances will depend on the chemical constitutions of the split proteins and on the kinetics of the available enzymes in the affected tissues If the proteins involved in the toxic proteolysis contain a sufficient quantity of heparin salts, then heparin may reach the body fluids in detectable amounts If the affected proteins contain enough histidine and if this histidine is set free by uncontrolled proteolysis, then listidine-decarboxylase, if available, may convert it into histamine. But if histaminase is present in sufficient concentration at the place of the reaction it may destroy the released histamine and so prevent it from exerting any extrinsic action (Gaddum, 1951). In other words, ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 37 an anaphylactic reaction may take place without the release of either heparin or of histamine or of both. The leading symptoms of anaphylaxis may therefore vary from case to case although the principal pathogenetic mechanism remains the They may vary in different species and in various syndromes within the same species same, P108) Proteolytic Enzymes and Proteolytic Catabolites in the Anaphylactic Reaction. Human and dog sera normally contain tryptic enzymes whose activity is blocked by the blood lipoids. When the dispersion phase of these poids is altered, the proteolytic enzynies become active in vrro and in vitro against both homologous and heterologous proteins (Jobling et al, 1914) Extraction of a normal serum with chloroform activates these enzymes, when the lipoids extracted by the chloroform are added to the treated serum its tryptic enzymes become inactive again. Simular effects may be obtained by extracting with other fat solvents, by saturating the unsaturated carbon atoms of their fatty acids, or by altering the dispersion phase by means of surface-acting substances (Jobling et al, 1915c, see also A similar mechansm of proteolytic activation may operate in intracellular and extracellular proteolysis. Thus proteolysis in the blood and in the tissues is controlled by the availability of active lipords for the insulation of the substrate proteins against normally present non-specific proteolytic enzymes. In the anaphylactic reaction an alteration occurs in at least three enzymes in the blood I A quantitative change in the titre of non-specific proteinases. 2 The appearance of toxic proteases 3. An increased titre of serum esterase 1. During anaphylactic shock the non-specific proteolytic power of the serum rises rapidly This leads to a significant elevation of non-protein nitrogen (NPN) in the blood in eito (Abderhalden et al, 1911; Jobling et al, 1915c, and others). Increased tryptic actirity of the blood obtained from an anaphylactic dog has also been demonstrated in vitro ·38 ENZYBIATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY (Abderhalden et al, 1911, Zunz et al., 1915; Pfeiffer et al., 1915). The rise of the tryptic power of the serum varies with the severity and the duration of the anaphylactic shock (Jobling et al, 1915 c) Since the increase of NPN in the blood of an anaphylactic dog exceeded the total N content of the applied shock-dose of antigenic protein, Jobling and his co-workers (1915c) rightly concluded that the excess of NPN in the blood is derived in these circumstances from the hydrolysis of the organism's own proteins In other words, during the anaphylactic reaction a rapid toxic proteolysis of antigen and increased normal proteolysis of the organism's own proteins elevate the NPN in the blood. on the other hand, the increased and accelerated non-specific proteolysis depletes the organism's stores of proteins The increased proteolysis in anaphylaxis is caused by two factors (a) Inactivation of lipoids which protect substrate proteins. (6) Increased output of non-specific proteinases. (a) The inactivation of lipoids in intracellular and extracellular lipo-protein molecules is due to the release into the body fluids of free molecules of various proteoses during the second stage of anaphylactic shock, the proteose molecules disrupt the lipo-protein linkage by surface-acting forces (P 55) (6) The elevated titre of non-specific proteinases is due to the massive dissolution of lymphoid tissue caused by the lympholytic steroids released during the pituitary-adrenal response This is put into operation by the stress of the anaphylactic shock itself. 2 The appearance of toxic proteases in the blood of a sensitised dog, and the rapid increase in its titre during anaphylactic shock, has been independently established in various laboratories (Vaughan et al., 1913. Jobling et al., 1915€, Zunz et al, 1915, Pfeiffer, 1915, and others). The titre of the serum proteases gradually rises during the period of sensitisation and reaches its peak at about the fifteenth day. According to the suggestion of Jobling el al. (1915c) the initiation of protease-hydrolysis in anaphylactic shock is made by the inactivation of the blood and tissue lipoids which block its action. The toxic proteolysis carried out by various proteases takes ·EXZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 39 place mainly in the sensitised cells but partly also in the blood during the second stage of anaphylactic shock (p 33) The toxicity of this proteolysis lies in the release of toxic end products of protein breakdown (proteoses) The toxicity of the proteoses has a double mechanism (a) Proteoses are, per se, toxic, causing various constitutional manifestations, and (6) their molecules exercise a surface action whereby the dispersion phase of lipoids is altered As a result the substrate proteins (of both primarily and secondarily involved cells) are exposed to energetic proteolysis by non-specific proteinases The toxic protease-hydrolysis in the affected cells starts in the first stage of anaphylactic shock and goes on through the whole of the second stage, proteoses released into the blood are found there in detectable amounts soon after the injection of the shock-dose of antigen (Jobling et al, 1915 c) The whole amount of the free proteoses released into the blood throughout the period of shock is, however, not big enough to produce shock the whole blood of an anaphylactic dog, transfused into a non-sensıtısed dog, does not give rise to shock in the recipient (Weil, 1917) This fact has the following explanation. The main source of proteose production is the tissue involved in the anaphylactic reaction Although damaged by the anaphylactic storm, these cells are not totally destroyed and a small quantity of proteose 13 spilled over into the blood This relatively small quantity of proteose, however, is sufficient to inactivate the kpoida of cells not primarily affected, and to precipitate manifestations which intermingle with symptoms derived from the anaphylactic reaction The total amount of free proteose, however, does not reach the quantity required to produce peptone shock (Weil, 1917), and this fact is the reason for Weil's (1917) being unable to reproduce the anaphylactic reaction in a non-sensitised dog using as a shock-dose the whole blood of an anaphylactic dog The toxicity of various proteoses must be discussed in the light of recent work by Jenkin (1950) on inflammatory exudates. He extracted from the inflammatory exudates of serous membranes and tissues various protein derivatives, which he designated according to their pharmacological actions. Thus pyrexin, leucotaxin, leucocytosis promoting factor, leucopenic factors, necrosin, etc, have been isolated in pure forms and their respective toxicities hare been identified in normal ·40 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY animals. The toxic actions of these compounds can be traced in any form of inflammation; the anaphylactic reaction histologically represents one form of inflammation whose main distinctive feature lies in the mechanism of its initiation. Menkin rightly expressed the view that these toxins operate in anaphylactic inflammations as well. In other words, the anaphylactic reaction, from the histological point of vieur, represents tissue inflammation localised in certain shock or certain ectopic organs, on this local reaction there may or may not be superimposed manifestations derired from the release of histamine and heparın, as well as from the release of the pituitary-adrenal response 3. High titres of serum esterase have been found in the anaphylactic dog by Jobling and his co-workers (1915 e). The meaning of this finding has not been clearly explained. It is possible that inactivated cellular lipoids, derived from lipoprotein molecules, escape into the blood from the cells damaged by anaphylactic shock, and that the high level of lipoids in the blood, in turn, mobilises tissue esterase. Thus the increased titre of blood esterase may represent a secondary measure whereby the increased blood Lipoids may be restored to their previous state. Syndromes similar to the Anaphylactic Reaction. The chief incentive to revision of the theory which accepts an antigen-antibody union as the basic pathogenetic mechanism of anaphylaxis was the discovery of the fact that intravenous injection of human gamma globulin into humans, or into intact or adrenalectomised non-sensitised dogs, produced manifestations similar to those of anaphylactic shock (Godlowski, 1952b) In none of these conditions is the existence of specific antibodies admissible, an antigen-antibody reaction, therefore, can form the pathogenetic basis for none of these conditions By analogy, the reaction whose manifestations are identical with the above-mentioned syndromes should not be linked pathogenetically with the antigen-antibody reaction. In a review of the literature which has some bearing on the subject, conditions were found in which the presence of antibodies could certainly be excluded but whose manifestations possessed a striking resemblance to those of anaphylactio shock. These were peptone shock, trypsin shock, and the ·ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 41 reaction due to the intravenous injection of chloroform ; to the same category belong reactions following intravenous injections of colloidal and highly emulsified suspensions, or parenteral administrations of heterologous sterile protein solutions, etc. In order to find a common mechanism for all these syndromes it is essential to discover the primary factors operating in each reaction, and then if possible to link them all to a single mechanism To be able to do this one must first abandon the theory of antigen-antibody reaction as the basic factor of anaphylaxis and allergy One must then discuss in detail the mechanism of each syndrome individually and find a common denominator responsible for the resemblance of syndromes of such diverse ætiology The mechanism of peptone shock has already been discussed on page 30. The presence of specific antibodies is inadmissible in a dog never previously injected with peptone Yet such a dog, if injected with the proper dosage of peptone, usually develops symptoms identical with those of anaphylactic shock This fact alone should rule out the antigen-antibody reaction as the pathogenetic mechanism of both anaphylactic and peptone shock Close analysis of biochemical events operating in both types of shock will elicit the fundamental factors at work in both conditions The basic difference between anaphylactic and peptone shock 19 the period of sensitisation its presence in anaphylactic and its absence in peptone shock In the first stage of anaphylactic shock the toxic enzymatic system yields free intracellular molecules of proteose In its second stage these proteose molecules disrupt lipo-protein linkage, thus exposing structural proteins to non-specific proteolysis. In peptone shock, injected molecules of peptone (which chemically belongs to the proteoses) initiate chaotic, non-specific proteolysis of the substrate proteins by a similar mechanism In other words, peptone shock runs a biochemical course similar to that of anaphylactic shock from its second stage onwards. There is no sensitisation period in peptone shock and therefore no formation of a toxic enzymatic system; the role of proteose molecules has been taken over by the injected poptone molecules. The biochemical events and the symptoms produced by them are those of the second and third stage of anaphylactic shock, and hence the similarity of these two types of shock ·42 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY In the reaction follow ing intravenous injection of human gamma globulin the molecules of gamma globulin, deprived of their conjugation with lipoids by ether fractionation, enter various cells where they display the properties of surfaceacting substances (Godlowskı, 1932 b). Such action leads to the adsorption of cellular lipoids on their surface, thus disrupting the lipo-protein conjugation. This action, in turn, exposes the substrate proteins in the blood and in the invaded cells to an uncontrolled, non-specific proteolysis with the liberation of the various toxins described previously. Thus the reaction following intravenous injection of human gamma globulin into a man, or into an intact or adrenalectomised non-sensitised dog, starts biochemical reactions of the second and third stages of anaphylactic shock; the manifestations which follow are identical with those of anaphylactic and peptone shock Trypsin shock can be produced by intravenous or intraperitoneal injection of an active or inactivo trypsin solution into a non-sensitised dog (Kirchheim, 1911, 1912, 1913 a and 6); these observations have been confirmed by Jobling et al (1915b). The symptomatology thus produced 19 identical with that of peptone and anaphylactic shock. The differentiating features between trypsin shock and anaphylactic shock are the absence in trypsin shock of a sensıtısation period. Chemically trypsin is a protein If active trypsin us administered it may display a twofold action : (1) as tryptic enzyme and (2) as protein molecules Action (1) cannot start unless protein molecules in the blood and cells are made accessible to the tryptic action by removal of the lipoid fraction from the lipo-protein molecules (e.g, by action (2) Normally the blood, and all tissues, possesses enough pon-specific trypsinases to proceed with hydrolysis, provided that the lipoid barrier in the lipo-protein conjugation is removed. Therefore action (1) of the injected trypsin plays an insignificant role. The main factor responsible for the production of trypsin shock is action (2), in which molecules of trypsin disrupt the lipo-protein conjugation by a physico-chemical mechanism. Hence the results are the same whether the trypsin is administered in its active or inactive form The disruption of the lipo-protein linkage by trypsin molecules exposes the organism's own proteins to non-specific proteolysis identical with that of the second and ·ENZYMATIC COXCEPT OF AXAPHYLAXIS AND ALLERGY 43 third stages of anaphylactic shock. hence the identity of the manifestations of trypsin shock with those of anaphylactic shock. The intravenous injection of chloroform into a dog produces manifestations which are similar to those of anaphylactic shock, but they are intermingled with symptoms derived from the direct toxic action of chloroform on the liver cells (Ratner, 1913) In this case there is a chaotic and rapid disruption of the lipo-protein linkage by the fat solvent The biochemical events which follow are similar to those of the second and third stages of anaphylactic shock, and this similarity is the cause of the resemblance between anaphylactic shock and the reaction following intravenous injection of chloroform Non-protein colloidal solutions (eg, sulphur), highly emulsıfied non-colloidal suspensions (eg, fat, kaolin, Jobling et al., 1915 f, iodine compounds, Pendergrass et al, 1949, Ramsay, 1953, and others), and heterologous protein solutions, when applied parenterally, are apt to precipitate reactions similar in their mechanism and symptomatology to those of anaphylactic shock These reactions may take place if the injected particles possess the features required for the penetration of the cell (p 21) Once in the cell, they display their disrupting action on the lipo-protein linkage and initiate the type of proteolysis found in the third stage of anaphylactic shock In the anti-anaphylactic state the high titre of proteolytic and non-proteolytic antibodies released into the blood during the shock is maintained thereafter by a higher output of antibodies by the lymplioid tissue (p 33) This countermeasure prevents the penetration of the antigenic protein into the sensitised cells should another invasion of antigen occur. It operates me in the body flwds. Although in the antianaphylactic state proteases are present inside the sensitised cells, these cells have recovered from the previous shock and are as yet undamaged For this reason an escape of proteases into the blood on a large scale cannot occur (Pfeiffer, 1915). If, however, proteases are present in the body fluids after the symptoms of acute anaplıylactic shock have subsided, one may conclude (a) that there is an active anaphylactic focus in the body, and (6) that the anti-anaphylactic mechanism is ·44 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY inadequate. Had it been adequate, all the antigen would have been neutralised by free antibodies and a chronic state of anaphylaxis could not have developed To summarise. the factor common to anaphylactic shock and to the syndromes which resemble it is a rapid and uncontrolled proteolysis of the organism's own substrate proteins initiated, in the case of anaphylactic shoch, by the augmented toxic enzyme system, in the cases of the anaphylactic-like syndromes by the pseudo-antigenic actions (p. 3) This is followed by symptoms characteristic of the second and third stages of anaphylactic shock; hence the similarity betueen all these reactions The ultimate outcome of the non-specific reactions of anaphylactic type may be different in different circumstances. For example, if an organism suffering from a chronic inflammatory lesion is subjected to one of these reactions, it may even benefit from it The metabolic storm may exercise its beneficial action on inflammatory lesions by (a) the engagement of the pituitary-adrenal response with release of C1-oxygenated corticosteroids (p. 50) and (b) the inutiation of a proteolysis of great relocity, which causes a rapid involution of the inflammatory exudates containing highly active, but temporarily blocked, proteolytic enzymes The Enzymatic Concept versus the Concept of Antigen-antibody Reactions in Anaphylaxis and Allergy. The present orthodox view on the mechanism of anaphylaxis and allergy holds that an antigen-antibody reaction takes place in the sensitised cells and in the body fluds, with release of various toxins (heparin, histamine) having intrinsic and extrinsic actions. In other words, the presence of specific antibodies is essential for the initiation of the anaphylactic reaction This theory cannot explain the identity of the manifestations of anaphylactic shock with those of peptone shock, with reactions following the intravenous injection of human gamma globulin or fat solvents, or with " tryptic shock”-in all of which the presence of specific antibodies is inadmissible The enzymatic concept could be adapted to the theory of antigen-antibody reactions as the basic phenomenon of anaphylaxis if all proteolytic enzymes were looked upon as antibodies. The formation of a specific toxic enzymatic system inside the sensitised cells could then be regarded as the formation ·ENZYSTATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 45 of a pathological antibody which by union with the specific antigen carried out a pathological proteolysis Such an approach to intracellular proteolysis cannot be accepted for the following reasons 1 Hydrolysis carried out by this enzyme liberates end products of high toxicity, whereas antibody was defined (p 4) as a substance which neutralises antigen and by such action eliminates its toxicity 2. The manifestations of anaphylactic shock are closely related to the appearance of proteoses in the body fluds and are not always related to any known antigenantibody reaction (precipitation, agglutination, etc), furthermore, antigen-antibody interaction is very often devoid of any noxious activity in the affected organism 3 The antigen-antibody reaction is a defence measure by which the invading antigen is neutralised and immobilised, and it cannot, without alteration, be both a protection against damage and a cause of damage or both defender and aggressor If all proteolytic enzymes are regarded as specific or nonspecific antibodies the whole process of protein catabolism should logically also be regarded as an antigen-antibody reaction Existing views on protein anabolism would then need modification, for in suitable conditions anabolism and cata. bolism are carried out by the same enzymes (Bergmann, 1912) Similarly, enzymatic metabolism of lipoids and carbohydrates would also have to be included in the category of antigenantibody interaction. Thus adherence to the theory of an antigen-antibody mechanism in anaphylaxis would throw all present views on metabolism into confusion, giving in return confused picture of anaphylaxis and allergy. It is therefore suggested that one should abandon altogether The riew that an antigen-antibody reaction is the basic mechanism in anaphylaris and allergy and shoull accept the riew that two independent reactions are concerned-antigen-anhborly reaction and sensitisation process, the imperfection of the first reaction causes the second Thus the anaphylactic reaction must be clearly distinguished from the prerequisite failure of the antigen-antibody reaction, ·46 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY The sequence of events leading to anaphylaxis and allergy may be formulated in the following way: 1 An antigen-antboily internction, a physiological defence measure, preserves the balance between the organism and the various antigens which normally invade every organism. This defensive mechanism, whether it operates inside or outside normal cells, neutralises the antigens and protects the organism from harm. If this defence fails, a pathological process begins. 2. If the invading antigen overpowers the antigen-antibody defence barrer, rt induces the formation of a specific proteolytic toxic enzyme system, this process constitutes the primary mechanism of sensitisation A later invasion of antigen (1.e, a shock-dose) inituntes toxic proteolysis and liberates various proteoses, these actuate normal non-specific proteinases and thereby start a chaotic proteolysis of the organism's own proteins The various organic toxins which result from this proteolysis are responsible for the loral and constitutional manifestations of anaphylaxis and allergy. 3 un ·CHAPTER II HORMONAL INVOLVEMENT IN ANAPHYLAXIS INTRODUCTION (N the preceding paragraphs attention has been concentrated mainly on the primary proteolytic inability of cells liable to be sensitised. As a result of this inability the invading antigen molecules cause a reorganisation of the intracellular proteinases In this reorganisation a specific toxic protease 19 formed within the cell, and this newly formed protease splits the antigenic protein to the level of proteose during the next invasion of the sensitised cells Proteose molecules produced in small amounts in the cell in the initial stages of the anaphylactic shock (intrinsic phase (p 33)) disrupt cytoplasmic lipo-protein molecules whereby the substrate proteins become exposed to a rapid and chaotic proteolysis exerted by the intracellular proteinases This proteolysis results in the release of various toxic intermediate protein catabolites which are responsible for various manifestations of anaphylaxis Hormonal participation in the anaphylactic reaction may be evoked through various mechanisms In the present discussion, attention will be focused mainly on the mobilisation of the pituitary-adrenal response in acute and chronic anaphylaxis, and on the interrelation between lipoids and adrenocorticosteroids PITUITARY-ADRENAL RESPONSE Mechanism of Pitaitary-adrenal Response. The pituitary-adrenal response operates through five independent but closely co-ordinated mediators : 1. Peripheral chemo-receptors and neuro-receptors of excitatory or inhibitory stimul: 2 Pathways transmitting these stimul: from the peripheral receptors to the central receptors. 3 Central receptors in the hypothalamus relaying the impulses towards the adenohypophysis. 47 ·48 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 4. The adenohypophysis producing hormones which act upon the cortex of the suprarenal. 5 The suprarenal cortex producing a great variety of steroids which participate in the metabolic activities of all tissues 1 The Chemo-receptors and Neuro-receptors—The stimul acting on the peripheral tissues from the external or internal environinent may be of physical, chemical, or emotional character (p. 78) They are recorded by chemo-receptors and neuro-receptors, and are eventually transmitted to the autonomic nervous centres 2 The Pathways Transmitting Recorded Stimuli -The pathways transmitting stimuli from the periphery are neural and humoral in nature Stimulation of the autonomic nervous system may affect directly the relaying centres in the hypothalamus (Vogt, 1951) High cervical transection of the spinal cord, however, does not prevent the mobilisation of the pituitary-adrenal response, in these circumstances this response is mediated by a humoral pathway, ie, by adrenaline liberated from the medulla of the adrenal glands (Long, 1947), functioning through its intact secretory nerve supply (Harris, 1948) Physiologically, the central receptors may be stimulated simultaneously through both these pathways 3 Hypothalamus as Relaying Organ.--The neuro-humoral stimuli brought to the hypothalamus are relayed in the region of the median eminence, this results in "... the liberation of a chemo-transmitter which is transported by the portal circulation to excite or inhibit secretory action of the adenohypophysis” (Quotation from Harris, 1951, reported previously by Harris, 1914, and Green et al, 1947.) 4. Secretory Action of Adenohypophysis There is TLO evidence that the adenohypophysis (in humans) possesses neuro-secretory fibres (Harris, 1951). In rabbits, Vaques-Lopez (1949) had demonstrated neural fibres connecting the hypothalamic area with the adenohypophysis It is at present believed that anterior pituitary secretion can be stimulated by three mechanisms which may operate simultaneously or independently These are. (a) Release of adrenaline by the sympathetico-adrenal system.

·HORMONAL INVOLVEMENT IN ANAPHYLAXIS 49 (0) Fluctuations of the concentration of adreno-cortical hormones in the body fluids (c) Direct hypothalamic stimulation (a) The adrenaline and adrenaline-like compounds released from the adrenal medulla and from other chromaffin tissues may influence the secretory activity of the adenohypophysis (i) via the hypothalamus (Gershberg et al, 1950), or (1) by directly stimulating the cells of the adenohypophysis to secrete adrenocorticotropic hormone (ACTH) (McDermott et al., 1930), or (iii) by promoting the excessive utilisation of corticosteroids by the peripheral tissues, thus lowering the steroid level in the blood, which in turn stimulates the release of ACTH (Sayers, 1950 b). (6) The fluctuation of the concentratron of cortxosteroids in the body fluids has been experimentally demonstrated to be an inhibitory or excitatory stimulus for anterior pituitary action The repeated administration of cortisone caused depression of ACTH secretion (Sayers and Sayers, 1947), whilst repeated stress stimul (which caused increased utilisation of the corticosteroids) exercised a stimulating effect on the secretion of ACTH (Sayers and Sayers, 1918) (c) Direct stimulation of the hypothalamus in the region of the tuber cineroum or mammıllary hodies produces circulating lymphopenia (de Groot et al, 1950, Hume et al, 1950) This is considered to be the result of the release of lympholytic corticosteroids through ACTH action (Dougherty et al, 1946, a and b, Whyte et al, 1946) 5 Response of the Suprarenal Cortex to ACTH-The exact mechanism of ACTH action on the suprarenal cortex is unknown. It is known that the administration of ACTH stimulates all known functions of the adrenal cortex, and this is evidenced by (a) hypertrophy and hyperplasia of the cortex (reviewed by Sprague, 1931), (b) decreased content of ascorbic acid (Sayers et al, 1945), and cholesterol (Long, 1947) in the cortex of the adrenals, (c) increased urinary output of cortical steroids (critically reviewed by Sayers, 1950 b), (d) atrophy of lymphoid tissue and thymus (Selye, 1946), and fluctuations in circulating eosinophils and lymphocytes (Thorn et al, 1948). The practical value of the “eosinopenic test" (Thorn et al, 1948) in assessing adrenocortical activity seems somesliat 4 ·50 EXZYSTATIC COXCEPT OF AXAPHYLAXIS AND ALLERGY diminished by new light thrown on the mechanism of hormonal eosinopenia and lymphopenia. Relation of Pituitary-adrenal Response to Anaphylaxis. The stimulus of a chronic or acute anaphylactic reaction erokes the pituitary-adrenal response, and the release of cortical steroids precipitates symptoms which are superimposed existing manifestations derived, directly or indirectly, from the anaphylactic reaction There is strong suggestion that Cil.oxygenated corticosteroids exert a powerful influence on the catabolic phase of intracellular protein metabolism (Engel, 1949 and 1951; Hoberman, 1950) This hypothesis was corroborated, both in vitro and in riro, in experiments in which these steroids exhibited their destructive action on white blood cells (Godlowski, 1952, Muehrke et al, 1952) The histological evidence of this action is a rapidly progressive involution presenting in the nucleus as pyknosis, karyolysis, and karyorrhexis, and in the cytoplasm as vacuolisation, degranulation, and loss of staining affinity In its extreme forms, such destructive action causes complete disintegration of the cell, leaving in its place an amorphous mass including the débris of the nucleus and granules (P 90) Since this leucolytic action of corticosteroids occurs also in vitro (Godlowski, 1952 a; Muehrke et al., 1959), it is reasonable to postulate that under these experimental conditions corticosteroids exert a direct action on all white blood cells. Dougherty et al (1914, a and b) and White et al. (1916) showed that ACTH injected into intact animals causes a gross involution of lymphoid tissue by lympholysis, and that such an effect cannot be obtained in adrenalectomised animals. From all these experimental data it is reasonable to assume that ACTH enhances the catabolism of protein not only in lymphocytes but in all white blood cells, by stimulating the release of leucolytic cortical steroids It has been conclusively proved that in association with the cytolytic action of corticosteroids the NPN in the blood rises significantly (Pearson et al., 1949, Engel, 1919, 1951, a and b). These findings may be interpreted as showing that the intracellular proteolysis enhanced by corticosteroids splits proteins to the level of NPN. It is known, on the other hand, that such an action is carried ·HORMONAL INVOLVEMENT IN ANAPHYLAXIS 51 out by proteinases of the tryptic type (Holman et al, 1917, and others) Therefore one can further assume that corticosteroid cytolytic action may result from the direct or indirect stimulation of the intracellular catabolic phase of the metabolism of protein INTERRELATION BETWEEN LIPOIDS AND CORTICOSTEROIDS Influence of Exogenous Corticosteroids on Blood Lipoids. In connection with the mediation of corticosteroids in the proteolysis whose results have been observed in white blood cells it may be of some value to discuss the interrelation between lipoids and corticosteroids in the light of present knowledge in this field There are contradictory reports on the response of lipoids to the administration of leucolytic suprarenal lormones In a short experiment in normal humans Conn and his associates (1949) showed that injection of ACTH (30 to 100 mg ) caused a marked decrease in total serum cholesterol, due chiefly to the reduction in the ester fraction Their explanation was that in this experiment cholesterol served as a material from which cortical steroids were synthesised Bloom et al (1952) found that ACTH and cortisone given in dosage sufficient to ameliorate various allergic conditions did not significantly affect serum cholesterol, they found, however, that in normal rabbits treated with ACTH severe lipæmia and cholesterinæmia resulted Further experiments on animals (Kobernik et al, 1950, Katzin et al., 1950, Rich et al, 1951) and prolonged observations on humans (Aldersberg et al., 1950, Wolfson et al, 1950) have demonstrated definite increase in serum phospholipoids and in both fractions of cholesterol after the administration of ACTH and cortisone The discrepancy in the reports on fuctuation of blood lipoids in various species of animal and m men following the administration of cortisone and its alled substances has its source probably in the technique used in these experients. The main site of chemical activity of corticosteroids is in the cells, therefore lipoid fluctuations in the blood may or may ·52 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY not reflect the actual cyto-chemical alterations. Hence one may expect to obtain more information on this problem from the quantitative and qualitative husto-chemical analysis of the tissue lipoids after the administration of corticosteroids. In the following paragraph an attempt has been made to adduce evidence that CW-oxygenated corticosteroids participate in protein metabolism by saturation of the unsaturated carbon atoms in the fatty acids of the lipoids in lipo-protein molecules In such a concept the elevation of the blood lipoids which accompanies prolonged administration of these corticosteroids may be referred to the mobilisation of intracellular lipoids by the corticosteroids themselves, by this action corticosteroids disrupt the lipo-protein linkage of the cytoplasmic molecules (by chemical mechanism, p108), thus initiating protein hydrolysis, and liberated lipoids escape from the cells into the blood This contention derives valuablo support from the co-existing slight elevation of the proteolytic power of the serum (Holman et al, 1947, Sayers et al, 1949, Godlowski, unpublished data) Mechanism of Lipoid Inactivation by Corticosteroids. Before entering a discussion on the theoretical and practical applications of the above-mentioned hypothesis it would be advisable to discuss the facts which led to the suggestion of the mechanism of elevation of the blood lipoids during prolonged administration of the Cul oxygenated corticosteroids 1 It is well established that if normal blood serum is treated with fat solvents such as chloroform, ether, acetone, etc , tryptic hydrolysis of its own proteins begins, both in ritro and on tivo, this proteolysis is caused by the removal of the lipoids (Fig. 3) which protect the blood proteins against tryptic enzymes normally present in the blood (Schwartz, 1909, Sugimoto, 1913, Jobling et al., 1914) 2 It has been proved that the antitryptic action of lipoids is directly connected with the presence of unsaturated carbon atoms in the fatty acids of blood lipoids, any oxidising agent (such as hydrogen peroxide or iodides, etc.) can destroy this antitryptic action of lipoids by breaking the double bonds of the unsaturated carbon atoms in the fatty acids (Jobling et al, 1914 and 1915a).

·HORMONAL INVOLVEMENT IN ANAPHYLAXIS 53 3. Desoxycholic acid, which structurally is closely related to the corticosteroids (reviewed by Jacobsen et al, 1931), under sutable experimental conditions (Wieland et al., 1916) can form" a co-ordination compound ” with fatty acids. This newly formed compound of eight molecules of desoxycholic acid and one molecule of fatty acid acquires new physico- CH, CH, CH, CH, 000 COHO CH, CH, CH, Water CH, lecithin polypeptide chain lecithin far FIG 3 Relation between polypeptide chain and 0=water molecole (From A Frey-Wyssling, ** Submicroscopir Morphology of Protoplaşın," Second English Edition, 1933 Elsevier Publishing Co Inc, New York and Amsterdam) chemical features such as altered solubility, changed melting point, etc If one may be allowed to speculate in human biochemistry the following mechanism of the chemical inactivation of lipoids by the corticosteroids might be accepted The oxidation potential of cortisone and its allied steroids (Anderson et al, 1951) Les in the oxygen atoms or hydroxyl groups attached to Cs, Cai, and C17 It has been proved (Kendall, 1949, and others) that C11 and C17 possess a key position guaranteeing the known biological effects of these steroids (effecting protein metabolism) Therefore one can further assume that the Oxygen attached to the C'11 and C17 is the element which breaks the double bonds of the fatty acids, thus inactivating cellular and blood lipoids The reduction potential of corticosteroids lies in the reducing group attached to the (17, saturation of the double bonds in the fatty acids by hydrogenation can safely be excluded for two reasons (1) In the living cell oxidation is more easily carried out than reduction of the fatty acids, and it has been slown Joung aal'., 1914) chat ontation is the process ohiak destroys the insulating action of bpoids ; (2) corticosteroids in which the reducing group is attached to C1 and which possess no oxygen in the positions Cll and C" (eg., desoxycorticosterone).

·54 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY do not have the biological action connected with increased proteolysis (critically reviewed by Fieser, 1950) The quantity of Chl-oxygenated corticosteroids required for the inactivation of one molecule of fatty acid depends on the number of unsaturated carbon atoms, which varies in different fatty acids Therefore the degree of protein catabolism would vary not only with the quantity of hormone given but also with the chemical composition of the lipoids which insulate the protein Effects of Lecithin on Biological Action of Corticosteroids. These theoretical speculations have been tested in an experiment in which ovo-lecithin was used as phospholipoid. The rationale of this experiment was as follows. If C11. oxygenated corticosteroids inhibit the protecting action of lipoids against tryptic proteolysis by saturation of fatty acids, then, vice versa, excessive amounts of lipoids (ovo-lecithin) should exhaust the oxidation potential of Cu-oxygenated corticosteroids, thus blocking their biological activity, In one group of experiments cortisone acetate (15 mg.), incubated for one hour at 37° C, was tested for its eosinolytic and lympholytic potency on adrenalectomised dogs. A few days later the same dogs were injected with 15 mg, cortisone acetate which had previously been incubated at 37° C. for one hour with 150 mg of ovo-lecithin suspended in distilled water (5 per cent ) The eosinolytic and lympholytic potency of cortisone was completely eliminated In another group of experiments, adronalectomised dogs were intravenously injected with 20 ml. of a 5 per cent highly emulsified ovo-lecithin suspension in distilled water, fifteen minutes afterwards 15 mg of cortisone acetate was injected subcutaneously During the next four hours the dogs were injected intravenously every half-hour with 3 ml of the same suspension of ovo-lecithin The eosinolytic and lympholytic action of cortisone acetate was grossly reduced The data so far obtained in the experiments in luck the blocking action of lecithin on the biological activity of cortisone has been assessed support the theoretical speculations The number of the experiments, however, is too small and their nature too limited to draw any definite conclusions ·HORSIONAL INVOLVEMENT NN ANAPHYLAXIS 55 Physico-chemical, Physical, and Chemical Factors Inactivating Lipoids. Cellular and blood lipoids possess the capacity of insulating the substrate proteins against the tryptıc action of proteolytic enzymes in the manner shown in Fig 3 The destruction of this insulating barrier may be achieved either by the disruption of the lipo-protein conjugation by physical and physico-chemical factors, or chemically by saturation of the unsaturated carbon atoms (Jobling et al., 1915c), the exact role played by the unsaturated carbon atoms in the insulating action of lipoids 18 not known The factor which inactivates the lipoids' insulation of substrate proteins against proteolytic action may operate through at least three mechanisms 1. Physico-chemically, by the alteration of the dispersion phase of the lipoids in the lipo-protein molecules colloidal substances whose particles possess high surface-acting forces disrupt the lipo-protein conjugation by adsorption of the lipoid molecules on the large surface of their molecules. In this way protein or proteose molecules which have escaped proteolysis and have penetrated various cells (eg, in human gamma globulin reaction or in peptone shock) or have been produced inside the cell (eg, in anaphylactic shock) disrupt the upo-protein equilibrium, thereby exposing substrate protein to proteolytic action. 2. Physically, by intravenous injection of fat solvents which remove lipoid molecules from the lipo-protein molecules, this action leads to the same disturbance of lipo-protein equlibrium as described in (1). 3. Chemically, by saturation of double bonds of the unsaturated carbon atoms in the fatty acids by various methods such as oxidation, hydrogenation, or halogenation (Deuel, 1951), whereupon the lipoids aro rendered incapable of protecting the substrate proteins The first and the third methods of inactivation of lipoids take place in different stages of anaphylactic shock: tho ·56 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY physico-chemical method operates in the second stage of anaphylactic shock and the chemical method, which operates in the third stage, is carried out by corticosteroids released through the pituitary-adrenal response ; the latter type of the initiation of proteolysis continues proteolysis of a physiological pattern at a rapid rate which is characteristic for the third stage of anaphylactic shock Each of the above mechanisms results in the disruption of the lipo-protein conjugation in a different manner and with different consequences The fundamental result of all these mechanisms is, however, the same: the breakdown of the organism's own substrate proteins and the dissociated lipoids from the lipo-protein conjugation may leave thereafter the interior of the cells. This may explain the elevated lipord concentration in the body fluids found after the administration of Cu-oxygenated corticosteroids Kobernik et al., 1950; Katzın et al, 1950, Adlersberg et al, 1950, Wolfson et al., 1950, Rich et al, 1951 Bloom et al, 1932) The flooding of the blood with lipoids may, in turn, induce the liberation of tissue esterase to dispose of the excess blood lipoids This may be the cause of the high level of lipoids and of esterase in the blood which is found in anaphylactic shock. The inactivation of blood and cellular lipoids, leading to the initiation of proteolysis, is a process the velocity of which gradually increases This action of corticosteroids may be regarded as a physiological mechanism for the initiation of protein catabolism, whereas the physico-chemical (by surfaceacting forces) and physical (by fat solvents) inactivation of lipoids may be looked upon as examples of pathological initiation of proteolysis. The rapid and disorderly proteolysis initiated by the last two factors leaves behind partially catabolised protein breakdown products of high biological toxicity (proteoses) and liberates physiological substances which, because of the rapidity of their release, acquire a pathological potency (histamine and heparin) The chemical method of initiation of proteolysis (by the release of corticosteroids) may be considered in these circumstances as & counter-measure whereby the toxic catabolites are disposed of by normal proteolysis.

·HORDIONAL INVOLVEMENT IN ANAPHYLAXIS 57 Tentative Explanation of the Action of Cil-oxygenated Corticosteroids in Anaphylaxis, It has been conclusively proved in animal experiments that cortisono and its allied substances have no effect on the course of anaphylactic shock itself (Leger et al, 1948, Dworetzky et al , 1950) The improvement in allergic conditions obtained by the administration of these corticosteroids must, therefore, operate through a mechanism other than a direct effect on the anaphylactic shock which is a basic pathogenetic factor in allergy. It is suggested that the mechanism of the therapeutic action of corticosteroids in allergy is through chemical inactivation of the lipoids in the lipo-protein whereby physiological proteolysis is initiated The velocity of this protcolysis will depend primarily on (1) the kinetics of the proteolytic enzymes available in the tissues involved and (2) the rate of the disruption of the lipo-protein molecules by the corticosteroids The cellular and extracellular anaphylactıc inflammatory exudates contain large quantities of lughly active proteolytic enzymes (Weiss et al, 1935, Jſenkın, 1950, and others) If the C11-oxygenated corticosteroids administered reach the quantity required for the chemical inactivation of lipoids in lipo-protein molecules, a rapid proteolysis begins The accelerated physiological proteolysis which follows eliminates exudates accumulated in the anaphylactically inflamed tissues Thus C11-oxygenated corticosteroids exert their beneficial effects through the elimination of the secondary histopathological reaction due to the biochemical activities of the third stage of anaphylactic shock (p. 34), leaving the primary pathogenic mechanism of toxic proteolysis of the first and second stages of the anaphylactic shock unaffected. In other words, CH-oxygenated corticosteroids initiato physiological proteolysis by a systematic and co-ordinated inactivation of lipoids in lipo-protein molecules, according to the proteolytic capacity of the available proteolytic enzymes. Hence the therapeutic potency of cortical hormones depends on the kinetics of the proteolytic enzymes available at the time of the administration of the cortical hormones Granulation tissue, cellular and fluid components of the inflammator exudates, all possess a high content of rery active proteinase:

·58 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY and therefore all these elements are very susceptible to the proteolysis initiated by cortical hormones. Lymphoid tissue containing as it does a large amount of highly active proteolytic enzyme (Drinker et al., 1941; Fruton, 1946; and others), is also highly susceptible to hormonal proteolysis, which leads to involution (Selye, 1946). Eosinophils, cells which have recently reorganised their intracellular enzymatic system in the process of cosinophilisation (Godlowski, 1952). possess highly active proteolytic enzymes, and are also highly susceptible to hormonal proteolysis Neutrophil leucocytes containing highly kinetic proteinases (Weiss et al, 1935, Drinker et al, 1941, Rich, 1944) respond equally well to the proteolysis induced by steroids , their destruction, however, is not reflected in the blood, because the co-existing hyper-mobilisation of neutrophil leucocytes from the bone marrow masks the rapid leucolysis Polymorphonuclear leucolysis, however, has been clearly demonstrated in vitro and in experiments in viro in which a perfused organ was separated from the general circulation and the perfusing blood was subjected to the cytolytic action of corticosteroids (for details see pp 88-91). The ability of CH-oxygenated corticosteroids to induce proteolysis in a tissue containing proteolytic enzymes may lead to the dissemination of a quiescent infection, eg: tuberculosis (Michael et al, 1950, Hart et al., 1950, and others). A focus of infection in its inactive stage is cut off from direct circulatory contact with the rest of the organism by a wall of tissue which consists mainly of epitheloid cells, lymphocytes, polymorphonuclear leucocytes, and other cells characteristic of young granulation tissue , all these cellular elements are abundantly provided with proteolytic enzymes (Weiss et al, 1935, Rich, 1914) Henco rapid hormonal proteolysis induced by corticosteroids destroys the barrier surrounding the focus, thereby facıhtating the hæmatogenous spread of infection Green, on the ground of experimental work on mitosis done in association with his co-workers (Green, 1950, Green et al, 1951, a and 6), came to the conclusion that cortisone inhibits the anabolic activity of cells. Short-lived mesenchymal elements which manufacture various antibodies die out in the course of the normal ageing process, and they are not replaced because of the inhibition of the anabolic phase by corticosteroids.

·KORJIONAL INTOLYEJEST IX AYAPHYLAXIS 59 Our knowledge about protein anabolism is very scanty There is, however, good reason to believe that the same proteolytic enzymes are engaged in both anabolic and catabolic action (Bergmann, 1942) Reversion of metabolism from one action to another depends on the contemporary requirement of the chemical environment in which the metabolic activity takes place. Therefore the inhibition of mitosis seen by Green and his co-workers in tissue cultures in utro and in mto may result from both actions ; proteolytic enzyme engaged in catabolic activity is automatically excluded from the anabolic action The final morphological results are therefore due to two co-existing components aplastic (due to the inhibited anabolism) and destructive (due to the stimulated catabolism) ·CHAPTER WII THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS INTRODUCTION TE Hæmatopoietic and Non-hæmatopoietic Formation of Eosinophils. CHE origin and function of eosinophils are as yet not conclusively established. There are two opposing theories of eosinophil formation 1. Leucoblastic in the bone marrow (Ehrlich, 1878-79), 2 In non-haematopoietic tissues. The protagonists of the first theory maintain that the presence in the bone marrow of eosinophilic myelocytes as well as all other forms of maturation of eosinophil, including those seen in the circulating blood, strongly supports the view that eosinophils are formed in the bone-marrow In other words, the formation and maturation of eosinophils takes place in the bone marrow, and the mature forms of eosinophuls are discharged into the circulation by the same mechanisin as that by which the rest of the white cells formed in the bonemarrow are released into the blood The non-hæmatopoietic formation of eosinophils is less widely accepted , none the less, arguments adduced by its supporters possess also a sound basis. Many cytologists (Dominici, 1909, Ringeon, 1921, Gauthier-Villars, 1950; and others) have observed eosinopoiesis similar to that seen in the bone marrow in various non-hæmatopoietic tissues in experimental animals, without an increased eosinopoiesis in the bone marrow. In other words, in some organs (intestine) of these animals, a constant eosinopoiesis takes place physiologically, pathologically, eg, in some inflammations, early and mature forms of eosinophils are seen in the vicinity of inflamed foci (Maximow, 1927, Gauthier-Villars, 1950) If the presence of early, transitional, and mature forms of eosinophils is a valid argument for the medullary theory, it should also hold good for the formation of eosinophils in non-hæmatopoietic tissues. 60 ·THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS 61 The truth, therefore, seems to be that eosinophils are formed in the bone-marrow as well as in non-hæmatopoietic tissues, wherever the conditions for their formation may exist Therefore the elucidation of the mechanism of eosinophil formation Is more important than the dispute about the place of their origin MECHANISM OF EOSINOPHIL FORMATION Factors Conditioning Eosinophil Formation. The main principles of eosinophil formation may be summarised as follows 1. Any cell in the living organism invaded by molecules of antigenic protein may become eosinophilie, provided 2. The enzymatic structure of the cell which is about to be cosinophilised bears features of an insufficiency in intracellular proteolysis Such a cell bears, therefore, the features of an inadequacy as regards the performance of normal functions 3 An unsplit protein molecule possessing antigenic capacity invading such a cell exerts a stimulus for the reorganisation of the intracellular proteolytic enzymes This reorganised enzyme system is ahlo not only to catabolise the invading molecules but also, in the anabolic phase, to reconstitute the antigenic protein and to incorporate it into the structural proteins of the cell (for details see p 73) Classification of Eosinophils. From the point of view of the existing embryological classification, such a concept is inadmissible because all morphological elements of the blood hare been proved to bo of mesodermal origin In view of the evidence of the formation of eosinopluls in tissues of non-mesodermal ongin (eg, epithelial hining of the intestinal crypts), a formula has to be adopted which admits the existence of two types of cells with eosmophilic granulations in the blood. 1. True blood eosinophils formed in the bone-marrow or in other hæmatopoictic organs.

·02 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 2. Pseudo-eosinophils formed physiologically or pathologically in various non-hæmatopoietic organs, and subsequently mobilised and shifted into the blood. Genetically, the latter type of eosinophil does not belong to the blood elements The “true blood eosinophils" are descendants of immature white blood cells (promyelocytes or still carlier forms) which became eosinophilised in the bonemarrow by the same mechanism as that of the eosinophilised cryptal epithelial cells. The protein molecules which invade the immature leucoblastic cell may be of endogenous origin, eg., hæmoglobin, which was found normally to be free in the bone-marrow (Ringeon, 1938) The protein which initiates eosinophilic transformation of the leucoblastic cells in the bone-marrow may well be also of exogenous origin, eg, protein absorbed in the alimentary tract and disseminated all over the organism by the hæmatogenous spread. This physiopathological alteration, viz., eosinophilic transformation, deprives the eosinophylısed cell of its original morphology and primary function Effects of Antigenic and De-antigenised Diets on Circulating and Tissue Eosinophils. The tissue eosinophils in the lamına propria of the intestinal mucosa in dogs have been seen by many workers (Kultchitzky, 1897 · Simon, 1905. Biggart, 1932, and others), and all of them agree that the number of eosinophils is somewhat related to the ingestion of proteins The early forms of eosinophils which were also found there were described as eosinophilic myelocytes (Simon, 1905) In an attempt to explain this phenomenon it is advisable first to review briefly the literature which has some bearing on this subject. Absorption of unsplit protein from the intestinal tract takes place physiologically, but only in quantities which are insignificant so far as the nutritive value of proteins is concerned. Alstyne et al (1911), by using a very sensitive precipitin test, have clearly established the fact that unsplit protein is absorbed from the alimentary tract of the guinea-pig Sussman et al. (1928) and Brunner et al (1928) confirmed these experiments ·THE ROLE OF EOSINOPITILS IN ANAPHYLAXIS 03 on human material in various anaphylactic conditions In intestinal disorders associated with impaired digestion, the absorption of unsplit proteins is greatly increased, the ingestion of various antigens may give rise to the symptoms of anaphylaxis (Verzår et al, 1936) From the experimental Forks of Hardenhain (1888), Opie (1904), Simon (1903), Biggart (1932), and others, done on guinea-pigs, rabbits, and dogs, there appears to be a close correlation between the tissue eosinophils of the intestinal mucosa and the ingestion of proteins. Various forms of eosinophilised cryptal cells are described in the literature under various terms, eg, Paneth cells or Globular leucocytes (quoted by Moximow and Bloom, 1948), the origin and function of which are not clearly defined Kulichutzky cells (Kultchitzky, 1897) are those elements in the intestinal crypts which are nearest in their histology and suggested function to the "transitional eosinophils” described elsewhere in this essay. Duran-Jorda (1943) suggested that eosinophuls may be formed in the mucosa of the alimentary canal from lymphocytes passing through the stage of Paneth In an attempt to elucidate the connection between intestinal eosinophilia and the ingestion of proteins, two separate experiments were carried out for the reinvestigation of this problem : feeding animals with a highly antigenic diet, and alternatively with a de-antigenised diet Tables 2A, 2B, and Fig. 4 show the data of the experiment in which two normal dogs were kept for five days on a raw-meat diet. On the sixth day both were laparotomised and biopsies taken from the stomach, duodenum, jejunum, leum, and colon, biopsies from the bone marrow (rib) and from a mesenteric lymph node were taken also at the same time For the next five days (post-operative catabolic phase) the dogs Tere kept on a light mixed diet In the succeeding ten days both animals were put on amino acids diet, described by Silber and his associates, which consists of ten essential amino acids and corers the N requirement (Silber et al, 1919) This diet Was supplemented with a yitamin mixture, salts, lard, dextrin, and water, and it supplied S0 calones per kilo, according to the recommendations of Allison et al. (1945). On the eleventh day of this diet the dogs were laparotomised again, and biopsies cells.

·64 ENZYDIATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY taken from the same areas. In the next seven days the dogs were kept on normal diet and, at the end of this period, they were killed and specimens of all organs taken for histological TABLE 2A circulating Dag DO Changes of Protein Content in Diet reflected in Circulatory Eosinophilia Day-to day circulating Day to day or Day to day or Boeinophils/cum altamata days alternata dAVO Day to day circulating during 5 days Circulating Gondophila/eu mm Light diet of Bobinophulscu rom dolinophila cu mom during 5 days poet-operative during 10 days during 7 days raw meat diet period Atdino acid diet mixed diet 504 230 870 63 684 415 517 67 730 620 299 119 810 885 99 196 900 IOTS 144 328 265 30 880 0 713 163 642 99 1040 399 158 604 359 850 214 275 480 978 520 309 B38 133 200 2 TABLE 2B no Changes of Protein Content in Diet reflected in Tissue Eosinophilia of Ahmentary Tract Tuue bobinopbut countja PZ Tissue nosinophil counts Tissue notinophit counter after 5 days raw meat diet after 10 days amino acide diet after 7 days mixed diet (mean ESD) (mean tsb) (moan +8D) Fominophula Eosinophile Eonidophilus Dog Trans Trot Tran Orge tiona! Matum Organ tional Mature Organ tional Mature Stomach 1+0 LO Stomach 0 1+006 Stomach 0 Duodenum 24 40 50 38 20 40 Duodonura 3+0 21 12 70 60 Duodenum 10 20 330 20 Jaunum 140 20 48+06 Jejunum 320 19 14 +011 Jejunua 070 11 89 +03 Toum IB+041 75 + 0 52 lleum 40-19 20 +0 46 Ilown 6 + 0 72 49+00 Colon 810105+015 Colon 11008 21015 Colon 2001 Stomach 0 4+02 Stomach O 05+010 Stomach 0 Duodequr 20+01 50+ 0 62 Duodenum 8+003 147022 Duodenuro 10+0 31 38+0+ Jujunum 24+0 22 520 35 Jejunan 320 10 JA 2038 Jejunium 150-48 33+0 28 Lleum 25 1051 65+0 44 Ileurd 5004 28+0 44 Louro 11 +063 45 +0.78 Colon 8+0 08 25+ 0 90 Colon 9+012 3+0 40 Colon The slean values are of 39 PT Radia 3 *2015 6+042 D 30 10 9+03 analysis All these periods of various diets were preceded, accompanied, and followed by the “circulating eosinophil count" (p. 79).

·THE ROLE OF EOSINOPHLS I AXAPHYLAXIS 6.5 11 days amino-acids 3 days 5 days raw meat mixed diet Transitional forms/ HPFI Intestinal mucosa 8 35 15794douisoa anst HPF la intestinal mucosa LR 939 Circulaning cosina phils: absolutt number/cu mm 484 165 FIG. 4 The effect of dietary changes on eosinophula From the data shown in Tables and 2B and (uagrammatically presented in Fig. 4 the following conclusions can be drawn : | In a normal dog a ras-meat diet creates contutions in which the formation of transitional eosinopluls in the intestinal crypts is greatly increased; during this stage a very high blood eosinophila and high intestinal eosinophilia develops 2. & subxequent complete elimination of proteins from the det, and their replacement by amino acids, reduced the eosinophilisation of the cryptal cpithelium. Ten days of such a diet, honerer, cannot completely suppress eosinopliìlisation 3. Circulating eosinopenia during the penod of protein-free diet may be regarded as resulting from the suppression of eosinoplul formation in the cryptal epithelium.

·GO ENZYMATIC CONCEPT OF ANAPHYLAXIS AXD ALLERGY Effects of the Diet with De-antigenised Proteins on Intestinal Eosinophilia, In an attempt to elucidate the influence of the nature of the ingested proteins on the eosinophilisation of the cryptal cpithelium an experiment was carried out on mice. Mice were chosen for this experiment because, when kept on ordinary diet (rat-cubes manufactured by the Agricultural Co-operative Society Ltd, Aberdeen), they do not possess eosinophils in any form in the mucosa of the intestine, nor do they develop an intestinal lymphocytic barrier to any great extent, they do possess, however, at the bottom of the intestinal crypts, numerous zymogen secreting cells (Paneth cells). These cells in mice differ markedly in their histological appearance from the transitional eosinophils (if these forms of eosinophilised cells appear in the mucosa) The rat-cubes are prepared from various cereals, salts, vitamins, fish, meat, and dried milk. In the course of preparation these ingredients are subjected to a temperature of 100° C or more, and to a pressure of 25 lb) per sg in or greater, both for over one hour. By these procedures the proteins of the rat-cubes became denatured, and they lost their antigenicity (Ratner et al, 1935) but retained their nutritive value In this experiment thirty mice of both sexes, of Swiw strain, kept on the above diet, and thirty mice on denatured milk both served as control The experimental series consisted of four groups of ten mice each, and all of them were fed on fresh milk only, for various periods as shown in Table 3A and 3B At tho end of each period, the whole group was killed The intestinal tract, spleen, and bone-martow of each animal were then luistologically analysed. The results of these experiments wero as follows: I Normal white mice kept on a de-antigenised diet do not derelop a lymphocytic barrier to any great extent in the lamina propria of the absorptive part of the alimentary canal. Eosinophils are completely absent from their intestinal mucosa , numerous Paneth cells are at the bottom of the intestinal crypts closely grouped one beside the other 2. A moderate number of early and mature eosinopJutla is present in the subcapsular and trabecular areas of the spleen, and also in the bone marrow.

·Fc 5 Jejunum of a mouse for thirty days on fresh milk diet In the bottom of the crypt numerous columnar cells (Panpil cellu) filled with large cosinophilic granules formed by zy mogenic materiel partly discharged into the lunen of the crypt Staining, azo cosin Magnication x 800 FIO 6 Jejunum of tho same ntou as in Fiz 5 with numerous transitoral enrinophilic cpithelial cells with no semblance to l'arseth cella. Inmer matute erwinnphais and lymphostes in Lumim propiru taining, azo ermin Magnification x M. (To face page 66

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·TABLE 3 Disffects of Denaturel (De-antigeniserl) Proteins in the Diel on Circulating Eosinophils. }}ood painontals of Dlood Donnaplule of Blood cosinophils of Blood Bohinophile of Blood cominophils of Blood onohde of 3D : fermanent diet 30 m 30 daze on 10 mice on fresh milk IO mica on fresh milk 10 mico on freth milk 10 mica en fresh milk of denatured protein denatured milk for 10 daye for 50 Jaya for 30 day. for 40 days meanD) (tanto) (meen :D (mean 3D (mean tebi (nean :D) 11/16 19245 189410 66215 21001 TABLE 30 Effects of Denaturel (De-anngenised) Proteins in the Diel on T'issue Eosinophils. Timu mmophila of T68 wunople of Tud posinophuis or TIRUOosinophule of Tave poesophila of TIMU winophils of 30 mce permanent 30 muro 30 day on 10 mies on frosh mik 10 msco on fresh milk ID co on fresh naik 10 mice on fresh milk depatured protein danaturot milk for 10 days for 20 dayo for 30 days for 60 days (ran D) Imenn IIDI (mean I (moen D) (mean ISD men SD) THE ROLE OF EOSINOPHILS SAYAPIYLAXIS Valuro Mature Paneth coils 2:021 Transitional) Transitional Transitional) Small Mature Paneth cells 3018 Vature 1035 Mature 0 2010 Transitional Lone Transitional Transitional Smail Mature Paneth cells 30 36 Nature 720 20 sfature 1610 28 Transitional Transitional Mature 31016 Siature Paneth cell TirkiGal 21031 Transitional Espell Paneth cells 10+ 0 IT Natung пҙте + bisa o + o ++ 0 The mean repare talen from 90 I readings of daklenana. Bwnym, len 30 fark TH Try wllx In call (tu! chest frWIE Beneturi pilk 1 fat I lr unler 5 ih pre ·os ESZYSTIC CONCEPT OF ANAPHYLAXIS AYD ALLERGY 3 A great number of Paneth cells is present in the crypts in the control and in the experimental series. they are histologically different elements from the transitional eosinophils of the cryptal epithelium (Figs. 5 and 6). + After the first ten days of the fresh milk diet, mature eosinopluls begin to appear in the villi of the small intestine, thereafter their number gradually increases. 3. Between the twentieth and thirtieth days of fresh milk feeding, transitional forms of eosinophils begin to appear in the lower parts of the intestinal cripts (Fig G) and they persist in unchanged number from the thirtieth day to the end of the erperiment (sixty days). on Influence of Large Dosage of Heparin Circulating Eosinophilia. A certain amount of light may be thrown on the problemi of the release of eosinophils from the sources of their origin by the study of the effects of large doses of heparin administered prior to the injection of eosinolytic hormones (horinones which precipitate circulating eosinopenia) This experiment was carried out on four men and fise dogs Each individual experiment was repeated twice (Table 4). In the first part of the experiment the eosinopenic hormone was given alone, and this served as a control In the second part of the experiment, carried out four days later, heparin, in dosage much higher than required for the prevention of clotting, was administered intravenously ten minutes before the injection of eosinopenic hormone. Three dogs were injected with heparin alone. In tho eliminary experiments (two dogs and one man) a low dosage of heparin, 2.e., sufficient to prevent coagulation of the blood (100 units per kilo), either did not influence the eosinopenia following the administration of adrenaline or gare no constant results The amalasons from these experiments are 1. The number of circulating eosinophils is associated in some way with clotting (for details see pp. 75-76) The clotting process exercises a less marked influence on the circulating lymphocytes.

·TABLE + Culting No 1 ban (a) 9,337 2,038 % Van 1) ) 3 Man (0) 16) 4 Nen (a) 10) 8 Dog (a) (b) 0 Dog (a) (b) 7. Dog (a) (0) 7 Inkimtion of llormonally inducel Eosinopenia by Large Doses of lleparin. Valuus stond of 4t!) hour Initial values Circu. titulating Circulating Circulating Heparin (lotting lating Circulating የሄነከሳ γημένο ol 17) Ant me 10 min Mormont Bono ty poho Circulating morpha time coagulant for begasin treatment płaley cylen! polymorpha Clotling cu mm cu mimi cu im (min) ou) injection (mg) cu mm cu mm cu min. tune 241 1,840 2,700 14 0 ACTII, 30 100 1.240 9,020 733 2,375 10 min. 12 125,000 >4 hr ACT, 30 214 3,760 12,760 > 4 hr. 115 2,300 2,200 26 5 D ACTII, 45 63 7,738 118 1,640 2.000 16 125,000 > 4 hr 10 min ACTH, 45 152 5,390 9,570 >>4hr. 100 2,700 -.385 66 0 Adrenal, 5 10 2,780 134 2,385 3,264 4 min 75,000 Adrenal, s 108 3.132 7,320 729 3,888 3,480 0 Adrenal, 5 260 1,824 3,900 3.450 20,280 7 125,003 Adrenal, 5 293 3,160 18,000 ><hr 521 1,806 5,500 6 0 Cortisone, 20 390 304 1.380 6,714 1,300 7 15,000 10,400 > 4 hr Cortisone, 20 759 1.312 9,840 4,600 4,100 16,100 > 4 hr 739 Insulin, 50 units 483 4,235 11,300 1,539 30,000 S 13,680 > 4 hr 3 min Insulin, 50 units 858 3,042 090 3,255 5,183 18.450 >> 4 hr 9 0 609 3 910 Insulin, 50 units 279 2,011 5 6,500 25,000 > 4 har Braun Ineuhn, 50 units 545 3,640 647 2,384 13.280 8,158 7 25,000 > 4hr 0 847 32 2,140 2,914 7 918 5 204 > 4 hr 8 25 000 > 4 hr 0 243 3,870 522 5,220 5,400 7,973 9 20 000 > 4 hr 0 860 6,332 10,492 >> 4 hr Row (A) represent control and rows() esperimental serie * The figures of the last font column were chtrine after the end of 24th hour. TILE ROLE OF EOSIXOPIS IX ANAPHYLASIS 5 TL 3,200 9 Dog 9. Dog 10 Dog 69 ·70 ENZYBIATIC COXCEPT OF AXAPHYLASIS AYD ALLEROY 2. Circulating cosinopenia is prevented by heparinisation of the blood in rito (in proper dosage) when this precedes the injection of the eosinopenic hormones. In some cases heparinisation may produce even an increase in the number of circulating eosinophils. 3. The polymorphonuclear leucocytosis which follows the administration of these hormones is not affected by heparinisation. 4. The normal clotting time is slightly shortened by the administration of eosinopenic hormones. Origin of Eosinophilisation of the Cryptal Epithelial cells. An understanding of how the cryptal epithelial cells of the intestine become eosinophilised may illuminate the problem of eosinophil formation in general For this reason various sources of this phenomenon are cuscussed below There are at least four possible explanations of the appearance of cosinophilic granules in the cryptal epithelial cells. 1, Zymogenic Material as Source of Eosinophilisation of Intestinal Epithelial cells ---It is well established that cryptal epithelial cells actively participate in the intestinal secretion (Verzår et al, 1936, and others) There nre suggestions that this activity may be the source of the eosinophil granules in tho epithelial cells, they might result from the retention of the zymogenic material in the resting stage of these cells, or the enzymatic material itself might form the eosinophilic granules inside the cytoplasm before it is eliminated during the active secretory stage In an attempt to show the relationslup between the secretory stages and the appearance of eosinophilic granules in the cryptal epithelial cells the following histological sections were prepared. Figs 7, 8, and 9 show the epithelial cells of a dog's jejuniin in the resting phase, The dog. previously fed on raw meat for five days, was started for forty-eight hours prior to the experi. ment. Fig 9 presents an intestinal crypt slightly clistended, with large columnar epithelial cells containing lig vacuolex of retained secretory material in their cryptal ends cells of the resting stage show no resemblance to the eosinophilic granules seen in Figs. 7 and 8 of the same dog ; Pere eosinophilic epithelial cells in vamous stages of maturation are scattered Theo ·FC 7 Biopsy of dog's duodenum on mixed det forty eight hours on water and destroge. Plane of section cuts tangentially thmugh the wall of the crypt, lumen of the crypt 19 not seen: artificial stratification created Vanou stages of maturation of eosinophilie epathelium Two neighbouring cells in central crypt in early stage of coinophilisation. Two other celle are about to lose the etyptal wall Staining, azo CODUL Magnification x 100) FIC & Biopay of dng's jejunum on mw-meat det fanting con. trons Specimen taken dunng third bopay operation in control animal Numeroua manophulse cryptal epithelm cells in various stages of maturation with variable amounts of eosinophilic granu- Lition in cytoplasm Two neighbouring cells in latera) crypt in later stage of jej otoping esaltanphilisation In lamina propria numerous lymphocytic cell, and few Rinophus Staining. At eNeLn Magnification sin கம் ·In 9 Dog's jejunum, raw meat det for five dass, in fastang con dition, with secretory action in form of large vacuoles located at the end of the cell directed towards the lumen of the crypt Yo resemblance to the development of eosinophilisation, Staining, azo ecsin Magnification * 1000 [To face page 71 ·THE ROLE OF EOSIYODLS 18 AXAPHYLAXIS 71 irregularis throughout the whole length and thickness of the cryptal wall. The erample of intestinal epithelial cells in the secretory pha-e, shown in Figs. J and , taken from & non-fasting mouse. The mouse has been chosen as the expenmental animal because in the mouse the secretory cells and connophilic cells of the intestinal crypts are diferent types of cells bearing no resemblance to one another The secreted zymogenic granules are seen in the cryptal end of the secretory cells as well as in the lumen of the crypt, they are large, coarse granules localised ala aj s at the end of the cell nearest to the lumen of the crypt The cells containing these granules are grouped close together et the very bottom of the crypt. these are the Paneth cella, concerned probably in the zymogenic acinity of the cryfstal epithelmm; they are, however, distinctly different from the eosinophilised epitlielial cells seen in Fig o in the same non-fasting mouse Thus the suggestion that the secretory material of the intestinal cryptal cells is the source of the cosinophilic granules In the eosinophilsseel epithelsal cells may reasonably be Considered as invalidated 2. Blooil Eosinophils as Source of Eosinophilisation of the Intestinal Epithelial Lining --The eosinophiled cryptal epthelial cells may be derived from altered blood eosinophils which have invaded the cryptal wall and subsequently adapted themselves morphologicalls to the local conditions This contention cannot be accepted for the following reasons fa) Circulatang eosinophils have completed theus maturation ; a morphological transformation of the mature blood enuinophil into an early or transitional form of eosinophil, of size and share rimilar to those of the adjacent columnar ethelial cells, 19 histologically inadmissible Yumernus immature eosinophuls of mono-lobular tyje Ate present in the lamina propria outside the cryptal lining. at a time when the peripheral blood entirely free from these comme If cryptal eosinophilised cells Ars served from mature blood eosinophil it would le difficult to explain why they legin their structural transfonna. On tie tion before entering the cry pta} kring.

·72 ENZYMATIC COXCEPT OF AXAPIYLAXIS AND ALLERGY otlier hand, early eosinophils, formed locally and learing the cryptal lining in their immature forni, may become arrested in the lamina propria by the mechanism pieviously described (pp 35.74). The arrest of eosinophils moring from the crypts towards the interior explains the presenco of immature eosinophils in the lamina propria while the circulating blood is entirely free from them. (6) The nucleus of the eosinophilised cells in the epithelium in the early stages 18 always large, vesicular, and regularly rounded, containing threads of coarse chromatın By contrast the nucleus of the blood eosinophils is very densely filled with chromatin and characteristically bi-lobular. A transformation of bi-lobular into a mono-lobular nucleus cannot be casily accepted (c) Figs 7, 8, and 10 shon two adjacent eosinoplulised epithelial cells. It is difficult to believe that these two cells could have penetrated to such a position in the cryptal wall by amoboid movements froni outside the crypt, whereas the process of eosinophilic transformation may affect two neighbouring cells of the conditions for cosinophilisation exist in two adjacent cells. Thus the myelogenous origin of the cryptal eosinophilsed cells appears to be highly improbable 3. Argenta fine Cells as Source of Eosinophılısanon of Intestinal Epithelial cells –Argentaffine cells can easily be excluded as a source of eosinophilisation of the cryptal epithelial cells, since the gastric mucosa contains a great number of argentaffino cells and is absolutely free from transitional eosinophils (Figs 12 and 13). 4. Local Formation of Eosinophils as Source of Eosinophilisation of Intestinal Epithelial cells - Another explanation of the appearance of the eosinophilic granules in the cryptal epithelial cells is a local formation of eosinophils in the intestinal mucosa. To substantiate this contention it is essential to discuss intestinal digestion of proteins in the light of the experiment with de-antigenisation of proteins in the diet (p. 66) and in the light of the theory of enzymatic adaptation of intracellular enzymes initiated by the invasion of the affected ·Fin. 10 Drundenum of a dog on mine) duet, forty etght hours on Tater and dextrose in the wal of central crypt two early transitional enamojihula an bs drated condition but retanung columnar shape In the lateral cryp, partly been. two splghbouring epithelial relle in later stage of the development of coxínophilia. In lumen of central crypt, PRI epainoph! at different stages of maturation Fairly numerous lymphoid cella anil *thophils with mono lobular and bilobular auclet in Lama mina, Staining, azo-in. Magnification x 1000. oporation are two Fra 12 Jejunor brows of dog No 13 (Tahle 5) on hired diet, forts eht hours on water and der trou only Порҹ геmоtеd tun weeks after the usual Intestinal crypto cut transverseli, in fuocrypee different stages of epithelial extrophil forma t1071 11) The earliest stage of coesophuration of the columnar epithelium with mund nucleus and chromatin thread ww.nophilae mass begin to granulate, very fine seules are presents are the of hydration () An Pokinn. epitheliom in the next rypt in 4 latet stape, nuclius more condenseil, deeply stayed. palo cytoplasm with tery numerota lymphoid fentiana cheophil uthmano lobular and bi-lobular nucler in lumina The shape of the cella 14 irregular *ith one poinnelaum- Tropria Staining, 121) Mein Vagnification < [To fare piger 12 VAC phuis Afax Courtero inopule granulee ·Fr 12 Dog's stomach on mured diet: forty eight hours on water and dextrose Two argentafline cella stained with azo-ensin showing fine reddish brown granulition in cytoplasm, different from eosinophilic granulation of epithelium in intestine. Nodevelopment of Bosinophilication in stomach gland epithelium Magnification 1000 F10. 13 Stomuch of the same dog as in Fig. 12, stained by Jacobsen's methol. Serm argentaffine cells in close provimity Mignification ! To trene 73 ·THE ROLE OF EOSLYOPHILS IN ASAPHILEXIS cells by the unsplit protein molecules possessing antigenic capacity. l'nsplat protein molecules can be absorhed from a normua! of pathological intestinal tract and cau-e vanou anaphylactic reactions (Verzar el al., 1936) In Part A of the letetic experiment described on page 63, ten days of an amino acil det rapidly reduced cryptal eosinophilisation In Part B of the saine experiment with the de-antigenused protein riet. transitional and mature eosinophils remained absent from the intestinal mucosa. They appeared there only when the same protein in its native form (not de-antigenised) had been surpledd From this experiment it may le deduced that the main factor which precipitates posinophilisation of the cryptal epithelium 19 the antigenicity of the ingestei proteins The physico-chemical equilibrium of these cells is gererely disturbed by the entry of foreign protem with antigenic power It is suggested that this disturbance leads to the appearance of eosinophilic granules in the cytoplasm. Enzymatic Adaptation as Working Theory of Eosinophilisation. The intracellular changes leading to cosinophilisation mar Le better understood in the light of the theory of enzymatic adaptation (Karstrom, 1938 Spiegelman 1950, and others) Details of this theory hare been discussed in the chapter on "Sensitisation." The nucleus, stimulated by the intruding protein molecules, produces, as an adaptive enzynie, a new proteinase which can catabolise the foreign protein molecules (Fruton et al, 1941) In its anabolic phase the same adaptive enzyme can re-synthesise a new protein molecule (Bergmann, 1942) u luich in its structural pattern is identical with that of the invading molecule The newly formed replica of the foreign protein in next incorporated snto the structure of the primarily inrades cell, because its molecules syntjiesised by the cells own enzyme acquire the structural patter of the cell's onn proteins. In thy way a cell inraded by antigenic molecules lecomes a new biological entity, losing its original structure and function, One of the structural and functional trans. formations is the acquisition of amaboid moults. Ultimately thue cell mores away from the cryptal lining erther into the lumen of the crypt (Fig. 10) or, niom often, into the interior of ·74 EXZYSTATIO CONCEPT OF ANAPHYLLIS AND ALLERGY the villus (Fig 11) When it thug migrates, its antigenic property is carried with it (Godlowski, 1948 b). Function of Lymphoid Barrier in the Lamina Propria Mucose of Intestine. On the way into the interior of the organisin this newly formed eosinophil must pass through the lymphocytic barrier of the lamina propria mucosæ. This lymphocytic barrier has been hult gradually in the course of repeated invasions by various antigens from the lumen of the gut (Kuczynski, 1922). This barrier, through the vanous antibodies which it produces, nerves as a defence wall agaist penetrating antigens from the alimentary canal (Knezynskı, 1992, Howell, 1928, VaVaster el al, 1933, Ehrich et al, 1942) It may operate also by means of an antigen-antibody reaction in one of the forme previously described (p 5) The eosinophils containing & replica of antigenic protem in their cytoplasm enter the larına propria, where they encounter a high local concentration of specific antibodies These antibodies penetrate the interior of the passing eosinophils and interact with the antigen therein As a result of this interaction the eosmophils become temporarily zu mobilised Thus those eosinophils which carry antigent are arrested in the lamina propria and remain immobilised there until the immobilising mechanism of the antigen-antibody reaction becomes exhausted Mechanism of the Arrest of Eosinopbils in the Intestinal Lymphocytic Barrier. Blood eosinophils are erther of true or of pseudo tyle (p 61), true cosinophils (1 € , those formed in the bone marrow) pass unaffected through the lamına propria mucose, causing no histo-chemical repercussions, because the intestinal lymphoid barrver possesses no specific antibodies against antigen carried by them. The true eosinophils may, on the other hand, posses an antigen uluch was incorporated in their structure in the Bone-arrox, which, however, was brought to the lone-marrow by hæmatogenous spread from the absorptive surface of the alimentary canal. In this case the lymphocytic hartier of the Jamina propria mucosæ may possess specific antibodies and, if these eosinophils incidentally are pasemg the intestinal mucosa ·73 THE ROLE OF EOSINOPIILS IX ANAPHYLAXIS with the circulating blood, they may be arrested there by antigen-antihody reaction. This explains the appearance of mature eosinophils in the intestinal nucosa before the transitional forms of eosinophils were formed in the cryptal lining in mire which were fed on antigenic proteins (Tables 34 and 3B) The pseudo eosinophils (e, those formed in the nonhæmatopoietic organs--eg., intestine), which were initially immobilised in the intestinal lymphocytic barrier and which subsequently regained their mobility in the final stage of the antigen-antibody interaction (p 76), can be rearrested in the zone which contains a high titre of locally accumulated antibodies in lymphoid tissue, provided that the eosinophils have in the meantime resynthesised the antigenic protein in their cytoplasm. The process of clotting appears to play an important part in the immobilisation of eosinophils That this is 80 is suggested by the fact that heparınısation of the blood, in concentration higher than that required for the abolition of clotting in rivo, prevents or significantly diminishes eosinopenia and lymphopenia, or in certain cases causes a rise in the circulating eosinophils and lymphocytes (see experiment with heparin on p 68). These fluctuations in the numbers of eosinophils and lymphocytes cannot be attributed to a technical error, since they were consistently found under the same experíniental conditions, and they were also observed in similar conditions by Samter (1949) The experiments with de-antigenised proteins in the diet and those with heparinisation in viro suggest 1. That the primary mechanısın which immobilises eosinopluſs in the intestinal mucosa 14 closely related to the antigen-antibody reaction : That the arrested eosinophils are in that stage of the tigen-antibody reaction in which cellular proteins Are deprived of heparn The process of de-heparınısation in the initial stage of antigen-antibody reaction) resembles blood coagulation (precipitation), but it takes place in the cytoplasın of the allected cells, The mam difference, however, hetueen intracellular clotting and blood coagulation lies in the reversibility of the former process (Monně, 1948). One can therefore postulato that the arrest of eggshile in the lymphatic Larrier of the ·76 ENZYMATIC CONCEPT OF AXAPHYLAXIS AD ALLERGY intestinal tract is due to the precipitation of the cytoplasnic proteins which follons the elimination of heparin; this in turn leads to loss of mobility of eosinophils As soon as thuis phase of the cellular reaction ceases, the cytoplasmic proteincoaguldım within the eosinophils may become re-heparinised. The cell, thus regaining its movihty, is able to leave the lymphoid barrier Such a mechanism would also explam the mobilisation of eosinophils br a lugh concentration of heparin Role of Heparin in Mobilisation of Eosinophils. Heparin conjugated with proteins (Chargaff et al., 1941) is present normally within the cells of all tissues (Charles el al., 1933), and it ensures their semi-flmd state (Fisher, 1931), During anaphylactic shock the lerel of heparın in the blood is abnormally hugl (Jaques et al, 1941). The excess heparin is probably released from its conjugated form in the cello involved in the anaphylactic reaction The mobilisation of cosmophils by a high concentration of heparin in the blood has been confirmed in an experiment in which an isolated loop of gut was perfused with highly heparinsed blood Heparin in lower dosage, but in amounts sufficient to present coagulation, gace snconstant and rariable figures for circulatıng eosinophils (Table 4) It is asquwed, therefore, that heparın can penetrate the arrested eosinophils and restore the Auidity of the cytoplasmic protens only when in high concentrations. The relatively small rise of eosinophils in the perfusion experiment (described on p. 89) was due to the perfusing of only a small area of intestine If. howerer, the whole organism was properly heparinised (dogs 7, 8, and 9 of Table 4), so that eosinophils were mobilised from the whole surface of the intestine, the increase in the circuating cosinophuls was high enough to be statistically significant. Thus eosinophils regain their uobility and, reaching the blood, elevate the circulating eosinophil count. Further details of the heparin action on hormonally induced eosinopenia is discussed on page 99 Place of Origin of Eosinophils. The above experiments suggest that edullary and extra. medullary eosinophil formation may normally take place ·THE ROLE OF EOSLYOPHILS IX ANAPHYLAXIS simultaneously. They also lead one to regard the mucosa of the small intestine as a normal source of eosinophilic cells in the blood ; these, as pseudo-eosinophıls, may contribute to the blood eosinophilia. The absorptive surface of the intestinal tract acts physiologically as a wide portal of entry for various Antigens. During this physiological absorption, minute amounts of unsplit protein penetrate the cryptal epithelial cells and transform them into eosinophilic cells These cells thus carry various antigens whose invasion of the organism on a laige scale is prevented by the defence mechanism of the intestinal lymphoid barrier (p 96) The formation of pseudo-eosinophils in non-hæmatopoietic organs other than alimentary canal 1s conceivable on theoretical grounds, provided that a focus containing antigenic protein exists in the body Eosinophils which differ in origin possess two features in common their histological appearance and the biochemical mechanism of eosinophilisation by which they were formed. The type of protein which initiated the eosinophilisation may differ in eosinophils formed in different sources Thus there are at least two types of eosinophils in the circulating blood under normal conditions, namely, those formed in the bone marrow and those formed in the alimentary canal, each type 19 genetically different and each type may carry a different antigen in its cellular proteins THE FATE OF EOSINOPHOLS IN EOSINOPENIA HORMONALLY INDUCED INTRODUCTION The problem of circulating eosinophil fluctuations lias in recent years aroused a great interest in many research laborstories and in clinical departments mamly because these fluctuations have been observed to be closely correlated to alrenocortical activity, similar eosinophil fluctuations have been found to accompany the administration of those specific (ACTH) and non-specific (eg, insulin) stímuli which induce endogenous liberation of adreno-corticosteroids Tlie closo association of the circulating eosinopenia with the administra. tion of exogenous ACTH led Thom and his associates (1948) ·78 ENZYMATIC CONCEPT OF ANAPHYLAXIS AXD ALLERGY - to the elaboration of the eosinopenic test for the assessment of the physiological activity of suprarenals. Others suggested that the eosinopenic test may well be used for the assessment of the biological potency of various brands of corticosteroids with assumed eosinopenic action (Randolph, 1930, Speirs, 1952). Before accepting eosinophil diminution in the circulating blood as a morphological expression of cortical activity it must first be proved conclusively that this phenomenon results directly and solely from the action of corticosteroids If the eosinopenic activity involves intermediate secondary and tertiary factors, the conclusion from the eosinopenic test niay be fallacious, because the intermediate factors may be primarily affected (for details see pp 75-99) It is an undeniable fact that the injection of potent C1l-oxygenated corticosteroids is normally followed by eosinopenia, this eosinopenia lasts for so long as the other acute effects which resulted from the increased level of these corticosteroids are present After administration of a potent preparation of ACTH similar effects can be noticed plus other signs of adrenocortical stimulation, eg., increased urinary output of 17-ketosteroids (Sprague et al, 1950, and others) Stimulı such as injection of adrenaline (Bertelli et al., 1910, and others), insulin hypoglycæmia (Godlowski, 1946; and others), intraperitoneal injection of ammonium chloride in saturated solution (Josey et al., 1932 b), intramuscular injection of sodium bicarbonate (Lawrence et al, 1932), injections of turpentine or nuclein (Staubli, 1910), as well as a great variety of infections (Zappert, 1893), parenteral administration of foreign protein (Staubli, 1910), starvation (Opie, 1901), intravascular hæmolysis (Meyer et al, 1909), internal hamorrliage, hypertensive crisis, acute anoxia, colicky pain, and various chemical agents (Domarus, 1931), surgical procedures (Laragh. 1948), traumatic hæmorrhage (Recant et al., 1950), and febrile conditions (Gabrılove, 1950) are among those stimuli which have been observed to cause eosinopenia. Finally, physical stimuli such as exposure to rapid changes of temperature (Josey et al, 1932 a), and various emotional reactions such as anger, fear, excitement, etc. (Speirs et al., 1949), can also precipitate eosinopenia, it seems reasonable to describe all these influencing factors as stress met with by every living organism during everyday life (for details see * Hormonal ·THE ROLE OF EOSIYOPHILS IX ANAPHYLAXIS 79 Involvement") In other words, these pharmacological, immunological, nutritional, physico-chemical, and emotional stimuli which act upon an organism influence also circulating eosinophils and they do so by adrenocortical hormones liberated through the pituitary-adrenal response These complicated biochemical actions reflected in eosmophil fluctuations do not explain the role, the mechanism, and the meaning of the eosinopenia which follows. METHOD The following technique was appled in an attempt to elucidate the fate of eosinophils in hormonal eosinorenia Eosinophil Counting. An eosinophil count was made simultaneously by two methods 1 Randolph's counting-chamber method (1914). 2 Differential count and total white blood cells (\'B(') count Method (1) gives the white blood cells count and the absolute eosinophil count. Method (2) gives the number of eosinophils per 100 white blood cells The absolute eosinophil count by method (2) was determined by plotting the white blood cells and differential count The circulating eosinophil count was calculated by taking the average of the two figures obtained by these two This modification was called for because the other white blood cells reported in the present communication were calculated also in absolute numbers per cubic milimetre from the differential count and white blood cells count experiments were preceded, accompanied, and followed 15 such a circulating eosinophil count, otherwise it is specified which count was determined. methods Alost Assessment of Tissue Eosinophilia. Tissue eosinophilia during hormonally induced cosincopenia was investigated both in human and in animal material at the end of the fourth hour after the corinoxnie stimulus was admmistered. The Jiuman material was obtaiunt from gurmal ·SO ENZYMATIC CONCEPT OF ANAPHYLAXIS IND ALLERGY individuals during the administration of an adrenaline drip, or four hours after the symptoms of hypoglycæmia had appeared. Tissue from lungs (Godlowski, 1949), liver, spleen, and bone-marrow was obtained by needle aspiration biopsy An inguinal lymph node (with the adjoining subcutaneous tissue and skin) as well as a specimen of skeletal muscle was surgically removed. Experiments were conducted on dogs wherever material for histological analysis of human organs was unobtainable Seventeen dogs were divided into four groups of these, three groups were treated and one group served as control The eosinopenic action of ACTH * was investigated in the first group (comprising three dogs), of adrenaline in the second group (comprising three dogs), and of insulin hypoglycæmia in the third group (comprising two dogs) The last group of nine dogs served as a control (Table 5) Two other dogs were given adrenaline and killed four hours later, specimens of all organs were taken for sections to detect whether the eosinophils had been arrested during the circulating eosinopenia, and if so, where It was found that the intestinal mucosa was the only organ showing tissue eosinophila, attention was therefore directed to this tigue. For this reason biopsies in survival experiments were taken only from the alimentary canal, mesenteric lymph node, and rub bone-marrow, the eosinophil count in rib bone-marrow films was made arcording to the recommendations of Best and Samter (1951) The findings in the mesenteric nodes and in the bone-marrow were persistently negative, therefore tfiese results are not presented in the tables, and in further references to biopsies these organs are not mentioned The iemaining organs were not obtained for histology until after death of the animal All dogs, after a few days of preliminary observation and routine treatment for intestinal vorins (Dicestal, N & B), were subjected to an initial laparotomy. During laparotomy biopsies were taken from the stomaclı, duodenum, jejunum, ileum, and colon Two or three weeks later the dogs of the experimental series were injected with eosinopenic hormones. At the end of the fourth hour the animals were either + ACTH, Irmour Standard L.14, and Cortisone acetate in 13 per cent benzyl alcohol, Merck & Co., Inc, n-ed in the present investigations, sere supplzet by the Medical Research Council, London, without charge ·THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS S1 (1) re-laparotomised and alimentary canal specimens taken, or (2) killed and specimens of all organs removed for section. The animals which were re-laparotomised were kept for seven clays, in order to recover from the effects of the injected hormones and from the operation itself Then they were killed and specimens of all organs were taken The control group was treated in the same manner except for injection of eosinopenic hormones, thus the effects of the operation itself on the tissue eosinophilia of the intestinal mucosa was assessed Laparotomies were performed under general anæsthesia (sodeum thropentone, Boots) In the first group two hypophysectomised dogs were injected intramuscularly with 10 mg. ACTH on the sixth day after hypophysectomy, and at the end of the fourth hour both dogs were killed All dogs of treated and control groups were kept on water and dextrose for forty-eight hours prior to biopsy This measure was adopted to eliminate the influence of protein intestinal digestion on the histology of the mucosa whilst minimising starvation effects on the hypothalamicopituitary-adrenal system (Opie, 1904) The tissue specimens removed were fixed in Helly's modification of Zenker's solution, embedded in paraffin, sectioned and stained with azo-eosin. In cases treated with ACTH some sections were stained with azo-eosin and some by Jacobson's method for argentaffine cells (Jacobson, 1939) Effects of Eosinopenic Hormones on White Blood Cells. Direct action of eosinopenic hormones on wlute blool cells was investigated in vivo and 212 vitro. In vivo. inferior vena cara (iv c) way ligated below the openings of the renal veins (Ligature 1). Both common iliac veins (C1T) were also ligated at their ongin (Ligatures 2, a and b) Blood in one civ. was separated from the rest of the vessel by another ligature placed at its junction with the i ve (Ligature 3) and served as a control. Through the wall of the other civ a needle was inserted and all the blood fioin this care-zliae pocket was aspirated into a siliconnel Aytinge the desired amount of eosinopenic hormone was added to it Through the same needle the blood was reinjected into the vessel and another ligature (Ligature +) placed before the 1 ·82 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY needle was withdrawn, the coagulation factors liberated from the damaged venous wall were thus isolated The cara-aliac pocket remained in the peritoneal cavity of the anæsthetised dog for two to three hours and the white blood cell analysis of both control and treated blood was made. 2. In vitro.--Heparinised blood (0.9 ml. of venous blood + 100 anti-coagulant units of heparin) was mixed with eosinopenic hormone Another sample of the same heparinised blood served as a control Both samples were placed in the incubator Inf v cava v renalis -Ligature 1 Ligature 3 Ligature 4 Ligature 2a com iliacy Ligature 26 Ext diac Int tacy FIG 14 Cara ilunc pockel, description in text at 37° C for three hours and white blood cell analysis was made of both samples. The correction of the dilution factors in all calculations was made by the following formula. Number of cells found x total blood volume (blood +hormone) Original blood volume This formula was applied to all experiments. Intestinal Perfusion. Intestinal perfision was performed on four dogs under sodium thiopentone general anesthesia A loop of jejunun was removed from the peritoneal cavity (rein and arters ·THE ROLE OF EOSINOPHILS IX AXAPHYLAXIS 83 ligated, leaving untouched nerve supply in the mesentery). and a segment of about 15 cm of it was perfused sith the dogs own blood (200,000 anti-coagulant units per 200 ml of blood) at the rate of 100 drops per minute As soon as perfusion was established the perfused loop was returned into the peritoneal cavity. The perfusing blood in polythene tubing was warmed to 38° C in a water bath. The perfusion lasted two to four hours and was made in an ordinary transfusion set Oxygenation of the perfusing blood took place in the air through wluch the falling blood drops from the venous outlet of the tubing passed (a distance of 1 m.) to the collecting vessel, the change of colour of the venous blood to bright red proved that orygenation took place In two dogs the effects of heparinised blood alone on intestinal eosinophilia was studied In another two dogs perfusion consisted of 200 ml of heparinised blood to which at the beginning of the perfusion 2 ml Eurortone (Allen & Hanburys Ltd ) was added as eosinopenic hormone. a further 2 ml Eucortone was added at the end of the second hour of perfusion A biopsy from the jejunal loop outside the perfusion area was taken before the perfusion was started At the end of the perfusion biopsies were taken from the perfused area and also from the area outside the perfused loop The collection of samples of perfused blood for white blood cell analysis was made at hourly intervals from the venous end of the tuhing, at the same time systemic blood samples were collected The morphological discrepancies between these two blood specimens proved the complete isolation of the two circulations Hypophysectomy. Hypophysectomy * carried out transpalatally was followed by electric cauterisation of the stalk After the death of the animal the stalk area was investigated by serial sectioning to discover whether the destruction of the pare tuberalis was complete (Pickford et al., 1931). Clotting Time. Clotting time was determined by the glass capillary method. Hypophysectomy was performed by Dr Pichford in the Department of Physiology, Edinburgh Linserity. $ ·84 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY Adrenaline Infusion and Insulin Hypoglycaemia. Adrenaline infusions and insulin hypoglycæmia were used in men according to the technique described elsewhere (Godlowski, 1946). Adrenaline (0-3 mg per kilo of 0.1 per cent. aq. sol) was injected subcutaneously into dogs and supplemented by 1 mg of adrenaline in oil. Insulin Jypoglycæmia was induced in dogs by subcutaneous injection of j units of soluble insulin per kilo. Hypoglycæmia has been terminated by intravenous injection of 20 to 40 ml of 20 per cent glucose solution followed by high carbohydrate diet. RESULTS Clinical Experiments. Clinical experiments were carried out on five normal persons; three were put on adrenaline drp and two were given insulin hypoglycæmia Four hours afterwards biopsies were taken from the bone marrow, lung, skin with subcutaneous tissue, lymph node, skeletal muscle, liver, and spleen. None of these biopsies showed eosinophils in sufficient number to explain the co-existing eosinopenia. These findings confirm the previously reported fact that eosinopenic hormones possess no eosinopenic action on the bone marrow eosinophils (Godlowski, 1918 a). Tissue Eosinophilia in Animals. In the experiments with eosinopenic hormones the influence on tissue eosinophilia was studied in eight dogs, nine dogs served as control The result of the eosinopenic action of ACTH, adrenaline, and insulin are presented together, since they all work through the same mechanism, re, the liberation of adreno-cortical steroids. At the time of circulating eosinopenia the lamina propria mucosæ of the small intestine was riddled with mono-lobular and bi-lobular eosinophils (Fig 16). The data presented in Table 3 show the mean values of ninety High Power Field (HPF) counts of the tissue eosinophils with their Standard Deviation (SD). The term early or transitional forms refers to mono-nuclear cells of various shapes and sizes which contain ·8. masses THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS variable amounts of eosinophilic granules and eosinophilic These cells are, as a rule, localısed in the lower parts of the crypts of Lieberkuhn The most immature forms of these cells have a columnar shape similar to the neighbouring epithelial cells (Figs 10 and 11) They are, however, usually larger than the adjacent cells in the epithelial lining They are four to five times bigger than mature blood eosinophils The nucleus of such a primitive cell is, in its initial stages, large and round, and contains coarse chromatin threads, in the later stages of maturation the nucleus becomes more compact (Figs 7, 8, and 11). The presence of many vacuoles in the cytoplasm gives this primitive cell an appearance of being hydrated (Figs 10 and 11), it thus stains more lightly than the adjacent cells of the epithelium Later still such a cell shrinks the eosinophilic granules condense and the cell approaches the pattern of a circulating eosinophil (Figs 7 and 8) The original columnar shape is lost as it acqures anicebord motility, and the nucleus gradually assumes an irregular shape (Fig 11). These transformations may occur in the cryptal epithelium, in the lumen of the crypt (Fig 10), or, most commonly, in the lamina propria mucosa, therefore the lanına propria may contain mono-lobular and bi-lobular eosinophils The columnar pattern of the deepest parts of the crypts is Usually lost owing to the crowding of epithelial cells at the semicircular base of the crypt the part of the cells directed towards the lumen of the crypt may become very thin If the plane of the section cuts tangentially through this part of the crypt, certain cells--by pure artefact--may become separated from the cryptal lumen and so produce an artificial stratification Whenever this happens to an eosinophulised cell, one gets the erroneous impression that it is situated at the periphery of the crypt and that it has no direct contact with the cryptal lumen (Fig 7) This may be misinterpreted as being penetration of the cryptal wall from outside This point is worth stressing because in the discussion on the eosinophilisation phenomenon at plays an important role. Argentaine Cells in Alimentary Tract. Abundant argentaffine cells (ac) (showing real-tone granulations when stained with azo-eosin as seen in the 1 are scattered throughout the whole thickness of the Pazine ·S6 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY TABLE 5-Eosinophil Lerels (ceris 5 mg 1100 +12 Pod or 3rd First biopat #bek Inítsal eosinophli Tissue eosinophile toeloopbi1 tevel, level. PUUD mean Don ESD SD Borbone Iog Trade (coile adolai- 20 cu um Organ tional Mature cu mm) tered 1 200 +10 Stoutach 0 19 30 350 +06 ACTE. Iutestine 5 +060 19 +070 10 mg Colon 1 + 99 40 41 176 15 Ştomach 0 1 +0 25 200 +10 Асти. Iotestite 9 +0 30 28 30 68 Color 0 10 mg { 220 +10 Stomach D 0 2807 Intestine 3004 ACTE 14 2006 10 mg Colos 0 +0 02 4 19013 Stomach 0 0 2015 Adrenaline, Iotestlae 52002 18 +0 07 Colou 11001 30 04 5 24210 Stomach 0 O 260 £15 Adrenaline, Intestine 7 +001 41 = 60 Colon 5 +0 02 6 206 27 Stomach 0 3*003 789 +5 Adrenaline, Intestlat 1+002 24009 3 mg Coloa 1001 7 960 +12 Stomach 0 0 Taalla Intestipe 7+008 41 +0 09 50 Dito Colon 2 +004 5 +004 8 190S Stomach 0 0 336 +7 Inmulia. Intestine 18 +0 06 30 units Colon 1+1 3 +003 9 2517 Stomach 0 140 02 110 +6 None Intestino 43005 20+0 09 control Colon 23 02 4005 10 2193 Stomach o 2001 1903 Nono, Intestine 3+0 03 32 +0 07 control Colon 13003 4+008 11 1006 Stomach 0 0 131 3 Nope, Intestine 2003 530 08 control Colon 1004 100S 12 240 +7 Stomach 10 01 635+10 Nope. Intestine 6+003 20 £0 30 control Colon 1+0 07 90 08 13 3003 Storpach 0 1001 3039 None Intestino 6 +0 08 50 +0 03 control Colon 1004 43002 1930 +15 Stomach 0 1005 1500 310 None, Intestine 12 +0 06 86009 control Colon 11001 1+0 02 15 6309 Stomach 0 D 5268 None Intestine 8 2007 33 +0 09 Colon 2 tooi 12002 16 520 38 Stomach 0 0 900 34 Node Intestane 60 OS 36 +0 09 control Colon 15001 940 02 17 684 +7 Stomach 0 9 +002 454 Nope, Intestine 13 +008 63 +9 36 control Colon 18+0 20 16 +009 1009 Control The mean values of intestinal tissue eosophilia were obtained from the readings of no Flute Power Fields (HPS) of tr-sue sections of daoletym, ieludum and lleval, 30 h P T of eack. mucosa Fig. 13 shows argentaffine cells in a section from the same tissue block of the same animal as shown in Fig 12 stained by Jacobson's methodl) but no transitional eosinophils in the gastric mucosa In the small intestine in two adjacent sections of the same tissue block the number of argentaffine cells per high power field is +2, whereas the number of transitional eosinophils is +8.

·ind after Hormone Treatment Cantine TIE ROLE OP EOSLXOPHILS IN ANAPHYLAXIS $7 Eorlog. phil level 7 days Eoknaphut 4br tar bormone treatment atser 7 days after second binpsy Setel 4th borzone bt etter Tiarue eotrophilst action Tissue eosinophus/ hotenopa APUD mese RY+8D (Oost ct to Trans (cells! Traped Organ tional Brature cumin Organ tional Mature 05 stomach 0 +39 Intestine 32013 Slobineb 1+000 6+004 18 +0 09 Intestine 9 +08 38 +0 09 Colon 2006 6 ŁO 14 Coton 3 +0 09 8 +0 20 69 Somach D 1 $105 Intestige 110 37 509 38 Colon 4+0 40 T 06 100 ſtomach 0 fotextile 1+005 IR +007 Colop I 2004 102 Stomach 0 O Intestine 3+0 09 36 +0 07 Colou 1+0 07 7 10 DS G stomach © 0 lutestine Colog 6093 10 10 19 Ftomach 1005 Intestine Colon 130 Stomach 0 0 803 17 Stomach 009 Intestine B+Opn SIE! fotektide 250 13 70 70 16 Color 6 +012 8 +005 Colon 1006 30 10 61 10 stomach Intestine 7 ODS 33 10 09 Colon i to 0 4 + 38 stomach o 3+1 06 3009 28 + TO 1 2003 Stomach 0 0 Intestine 6. OS (3 +010 Colon +008 3 Ł0 20 Stomach 0 1 +0 06 Talestin 18 2004 Colon 1 2010 1 +0 09 Stamark O 1 +004 Intentiae 38 +9 19 410 19 Colon 2009 2010 Stomach O 5 +0 09 Intestine 10014 70 +0 50 Colon 0 26 +014 Stomach 0 0 Intestin 29 O SO 55 +0 09 Colon 1+062 30 UG Stomach 0 1004 660 +22 1007 Stomach Intestine 12 +0 06 45 071 lattie 14 : 99 59 0 23 Colon $DOT 1 +006 Colon 21004 JTD2 Stomach 0 1 + 01 60018 11006 Stomach Intentine 48 016 8010 Tastine 37 +014 $1001 90 od Colon 10 20 Colpo 1 +007 Stomacb 0 Stomach 576 +12 O 10 *010 Inmatige Istesting 29009 91 3033 11 +0 17 55 0 27 11 010 40 $013 Colon Colon 3+9 06 10 2009 - - - +004 1+003 Falues for stomach ani calon were obtained in the wme way so-standard deviation Dogs 2 and 3 Here hypophysectored, latestinal Eosinopbilla related to Eosinopenic Hormones. Tissue eosinophilia of the small intestine, at the time of blood eosinopenia, shows a significant rise in the number of eosinophils when compared with the initial biopsics (Figs. is and 16 and Table 3). The cryptal transitional eosinophils in the pre-experimental and experimental periods showed no ·88 EXZIBATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY significant change. The intestinal eosinophilia of the postexperimental period was of the same degree as in the experimental period (dogs 1 and 7 of the treated series). The control animals showed an equal rise in intestinal eosinophilia during comparable periods (Fig. 17) Furthermore, the cryptal transitional cosinophils in the post-experimental periods of the treated and control series showed a significant rise in number per high power field despite the fact that there was no concomitant circulating eosinopenia The conclusions from these experiments may be summarised as follows -- 1. In the epithelium of the intestinal crypts there are cells with eosinophilic granules which morphologically can be regarded as early forms of blood eosinophils. 2 The increased eosinophilia of the intestinal mucosa in the experimental and in the control periods should be considered as a result of repeated trauma of the operation itself These traumatising stimuli intensify the process of local formation of eosinophils and mobilise the wechanism whereby eosinophils, which are moving from the cryptal lining towards the interior of the organism, are arrested in the lapina propria mucosa The arrest of eosinophils in the lamma propria, however, does not prevent the return of circulating eosinophils to the previous level. 3. Eosinopenic hormones which exhibit their action on the circulating eosinophils do not maintain their eosinolytic activity on the tissue eosinophils 4. Argentaffine cells are histological elements unconnected with the transitional eosinophils of the cryptal epithelium Intestinal Perfusion. The perfusion experiment was carried out in two groups of two dogs each The data obtained are presented in Tables GA and B. In Part A a loop was perfused with 200 ml. of the dog's own heparinised bloocl. In the systemic blood the white blood cell count ras significantly increased in tro hours, the increase being due to circulating polymorphonuclear leucocites.

·mm FIG 15 Jejunum Lopay of dog Xo ! (Talle 5) removed three weeks before the experiment. O'rcu- Liting eosinopbulla, 179 per cu!) Two intestinal crypts in one crypt an early ectrophil with mono-lobular nucleus, dende endophilic monolulular exinoplast in Franulationsincytoplanm One intcnor of the vilius and unc mono-lobulir eosinophil in the lumen of the crypt I'sur cobinophils and a few lymph. od cells m lamına propria mucona Staining, azu eosin. Jagnification x 800 crypt four transitional I'71 16 Jejunum bop of the Kitill dong remove three weeks after first loons at the end of fourth bour after Injetion of 10) my of ACTH, circulating eenino O percuh mm In the pranulated cosinophulk cell with munu lubular nus k propa lymphonil cells and numereits cemophils with mono lobular as well as likular nuk Sting and mamification as in Fig 15 03 Lumina אנול וחזנות (To fare pw*

·F1 17
Jejunum bropy of the same dog taken sesen days after the
etperiment presented in Fig lo Circulating cosinophilia,
30 per cub mm Very numerous eosinophuis with mono
tobular and ba lobular nucler anıt very mumerous lymphoni
cells ja lapina poprz Staning and magnification as 15
Fig 16
{To face page 89

·תות G0 120 min 180 JIR 110 2.290 2,565 3.618 9,300 1,043 111 O min #40 MIL Dog I Dog 2 Dog 1 Dog 2 Dog 1 Dog 2 Dag 1 Dog 2 Dog 1 Syetemio blond Dog 2 We feu mm 9 300 11,500 13,268 11,600 18,000 Fosinophudajcu mm 020 1,015 230 536 232 480 Polymorphieu mm 3,030 5,700 8,303 8,710 8,180 11,960 Iymphocytestu inn. 2,330 2,784 3,520 Monocytajcu mm 240 190 345 402 484 040 l'erfusing blood, wc feu mm 8,110 4.600 8,800 6,358 8,440 9,500 9,900 Eosinophilicum OXO 460 1,020 701 1,385 1,233 1,104 ralymorphafsu mam. 3 030 3,700 2,070 4.930 2,600 4,505 3,413 Lymphocytraleu mm. 2,990 2,565 1,840 2,295 2,795 2,465 2,660 2,376 Monoeytentu mm 240 190 230 255 280 85 100 184 Tiau. cominopul count(* Y Jejunal mucosa oforo perfusion $1-61 (60) 40-87 (53) Jejunal mucos afor perfunion 23-42 (30) Control non perfused jejunum 18-32 (23) 44-75 (58) 76-84 (80) TABLE 48 --Perfuseil Jepun un of Dog with lleparınseil Bloodl+? ml Eucortone (1 per cent) Dog 3 Syster blood Dog Dog 3 Dog 3 Dog Dog 3 Dog 4 Dog 3 Dog Wc/cumin 8,060 10,083 5,000 Eominophulafeu minn 12,612 7,881 12,809 240 183 10,400 80 19,970 Polymorphacu tom 173 4,140 3B 37 8,200 4,050 0 20 10.414 Lymphocytealeu min. 1,260 1.315 9,288 1.400 450 14,188 18,000 Manoc leu mm 1,771 164 120 2,709 616 300 Torfuning blood 975 450 204 184 775 618 975 wic leu mm 0.000 8,800 2,760 Eominophilafru mra 7,898 2,100 240 8,000 114 2,100 SI 7,94B 108 2,300 Polymorpha/rum 7.923 3.840 7,216 80 2,174 21 17 7,216 23 Lymphocy loa/ou um 1,848 23 1,500 0,880 1,149 282 1,890 8,952 738 2.001 105 Manny Leu/eu mm 7,031 880 420 328 293 105 711 ICB 138 105 Tue cominophil count/r: 790 100 84 238 138 79 Jojunal mucous before ferunion 32-16 (31) 33–83 (35) Jejunal toucose ate perfunon Contrul non perluoed jejunum 40-54 (60) 53-93 (62) 17-29 (25) 32-40 (36) THE ROLE OF EOSTYOPHILS IN ASAPHYLAXIS Dog Son ·90 ENZYMATIC COXCEPT OF ANAPHYLAXIS AND ALLERGY Within the same time eosinopenia developed. Both these phenoinena resulted from the stress of the operation and general anæstesia (Laragh, 1948). In the perfusing blood a slight eosinophil increase was worth noticing, even though by itself this elevation was too small to be significant. The tissue eosinophilia of the perfused jejunal inucosa showeci a significant decrease during that same period. In Part B a jejunal loop was perfused with 200 ml. of the dog's own heparinised blood to which Eucortone had beon added The systemic blood showed the same findings as in Part A In the perfusing blood the total white blood cell count was significantly decreased only in ono dog (No 3) This was the result of a great reduction in eosinophils, lymphocytes, polymorphs, and monocytes (Tables 6s and GB). All granular groups of whute blood cells showed conspicuous degranulation and loss of staining affinity, with racvolisation of the cytoplasm The nuclei showed marked regressive changes in the form of pyknosis and karyorrhexis. The lymphocytes and monocytes exhibited similar changes. The red blood cells did not show any significant signs of damage due to the altered osmotic pressure At the end of the perfusion the eosinophil count per high power field of a section from the perfused area of the jejunui was significantly reduced. The conclusions drawn from the perfusion experiment are I The stress of the operation itself precipitates changes in the systemic blood which are characterised by polymorphonuclear leucocytosis and eosinopenia, 2. During the perfusion the heparinised blood shows a slight eosinophil increase. This rise in number of eosinophils per cubic millimetre is not significant per se, but taken in conjunction with the significant climinution in the tissue cosinoplulia of the perfused jejunlim it may be regarded as a result of the mobilising effect of heparin on the tissue eosinophils; the smal. ness of the perfused area from which oesinophils were mobilised explains tlie slight increase in number of eosinophuis in the perfusing blood. 3. Tlie perfusing heparinised blood with Eucortone added causes & great diminution in the circulating posinophil, polymorphonuclears, lymphocytes, and monocytes.

·THE ROLE OF EOSINOPHILS L AXAPHYLAXIS 91 All white blood cells in these expenmental conditions show regressive changes in both cytoplasm and in nucleus, and these changes take place in the blood stream, 4. The final quantitative and qualitative changes in the white blood cells in the perfusing blood containing eosinolytic hormone result from the independent actions (a) The mobilisation of eosinophils hy heparin from the perfused area (6) The cytolytic action exerted by eosinopenic hormone on all white blood cells in the perfusing blood The Direct Action of Eosinopenic Hormones on White Blood Cells, in titro The direct action of eosinopenie hormones on white blood cells was investigated in two separate experiments in riro and In vivo (Table 7)—Eoynophils were the leucocytes most affected by the leucolytic hormones , next in susceptibility came the monocytes and lymphocytes, and the least affected were polymorphmuclear leucocytes Many of the white blood cells showed the features of disintegration described in the perfusion experiment. Blood samples mixed with desoxycorticosterone acetate (DCA) did not exhibit similar quantitative or qualitative changes in their white blood cells The effectiveness of the cytolytic action in the isolated blood in arvo was related to the concentration of the administered hormone and to the In vitro (Table 8) --Eosinopenic hormone maed with heparinised blood also caused significant diminution in eosinophils, whereas lymphocytes, monocytes, ani polyntorplionuclear were only slightly affected. Tho cytolytic action Was also related to the concentration and the length of time of the action of the hormone ariministeret?. The conclusions from these experiment, am 1. Eosinopenic hormione' pousess a direct cytolytic action on white blood cells under the erysinental conditions apple. duration of its action, leucocytes ·TABLE 7 Venous Blou isolated in Bloral l'essels in vivo not treated with anticoagulant + llormones CAUS07 Fosinopenia Troluminary differential count, absoluto values/eu am Time of expe72. ment (hr.) Differontial count in experimental Differential count in experimental period of non treated blood after period, absolute and corrected 3 hr , absolute valuesfeu mun IR values of the treated blood isolated blood vessel Poly. Eosino- Lym. Mono Leuco. Poly EAN- Lyra Mono Louco morpha phils y hocytes cytes rytobus morphs phile phocytes cytes cytogia 6720 576 2016 288 9600 4134 1014 2418 234 7800 Poly Eosino Lym Mono. Louco morbu phils phocytes les cy10818 G000 1155 2639 273 9067 Na 1. Dog 92 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 2. Dog 5320 501 304 7569 2 6806 275 1452 426 7958 5220 435 2436 009 8700 3. Dog Amount of hormone ml blood Eucottone, 0 16 rol. Eucortone, 0 15 ml Eucortono, 020 ml Cortione Beetalo, 1008 DC4, 9 50 mg 3770 348 1276 408 5800 2 2080 52 364 10$ 2600 3212 290 748 170 4426 4. Dog (a) 3055 783 2916 34B 8700 2 2900 290 2552 58 5810 400$ 847 2618 232 7700 4092 614 1716 198 6620 ·Eucortone, 5,025 Ilorinone m vatro Praiuminary differential count, Diflerential count of created blood Difforontial count of beparized Absolute values/eu mm Jeept for 3 hr in 37°C blood kept for 3 hr in 37* C. Adrenal Poly Eoanno Lyo Mono- Leuco hormonefral Poly. Eosino. Lym Mono Louco. Poly. Eosino. Lyn Mono Louco NO morpho phils phocytes cytes cytosis blood morphs phils phocytes cytes toas morphs phils phocytes cytos y tosis 1. Dog 3,074 348 2.030 336 5,788 Fucortone, 4,173 112 1,400 224 8,907 3,250 250 1,300 200 6,000 0 05 mol 2 Dog 10,428 474 4,108 790 15,800 Eucortone, 11,000 145 2,610 725 14,500 11,076 384 1,536 284 13,300 0 05 ml 3. Dog 8,460 1,170 3,686 705 14,001 5,094 4,440 223 11,000 5,405 849 4,945 315 11,544 0 1 ml • Man 300 1,875 300 7,500 Eucortono, 3,680 126 644 230 4,690 4,624 72 1,572 340 8,808 015 m 8 Dog 8,470 333 1,430 770 11,003 Eucortono, 7,030 95 2,280 95 0.500 8,280 360 3,000 360 12,00) 02 mnl 6 Man (a) 3,717 135 1,888 177 5,917 Cortisone 3,096 B3 989 129 4,297 3,328 124 1,664 10+ 5,220 Acetate 5 1-8 (6) Cortisone 3,024 18 1,536 172 4,780 Acolate 50 MB Cortisono 2,030 0 1,026 114 4,070 scolate, 100 48 3530 151 1.432 104 8,222 Cortisono 3,055 17 1,410 188 4,700 3,536 134 1,352 104 acetate, 1 mg 5,126 1) Cortisono 3,312 05 1,218 114 4,800 cetate, 2 mg Cortisono 2,484 10 972 108 3,574 Bcetate, 3 mg Cortarono 2,015 18 030 124 3,097 () Cortisone 2,400 21 910 119 3,450 135 177 3,917 DCA, 1 mg 4,012 118 1,543 (0) 236 5,909 3,328 124 1,664 104 1,015 5,220 101 1,210 220 5,546 1) 3,432 101 1,080 104 6,617 3,276 108 1,812 208 {m} 3,204 3,020 168 1,344 168 3,600 THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS acetate, 4 mg acetate, 5 mg 93 DCA, 2 mg DCA, 3 mg DCA, 4 ng DCA, 5 mg ·94 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY 2. The destructive action of these hormones affects all white blood cells, polymorphonuclear leucocytes, lymphocytes, and monocytes being less susceptible to this action than eosinophils. 3. Involutionary changes in all white blood cells were present in both experiments described. DISCUSSION Physiological Fluctuations in Circulating Eosinophils, In recently published reports (Rud, 1947 ; learling articles, Lancet, 1950, a and b; and others) doubts have been expressed about the accuracy of the methods at present in use for the counting of eosinophils The mam objection stressed is the great variation in counts of circulating eosinophils, even in normal conditions, which makes it impossible to establish a base line for their evaluation These difficulties, which doubtless exist, result chiefly from the fact that the counts of circulating eosinophils fluctuate significantly even in physiological conditions, such fluctuation is one of the basic phenomena of eosinophil function, this phenomenon has been termed endogenous eosinopenia (Halberg, 1952) Adrenaline (in suitable dosage and administered in an appropriate way) precipitates eosinopenia by mobilising the pitutary-cortical response, the chemical and morphological results can be detected in from ten to twenty minutes after intravenous injection, or a little later after subcutaneous injection (Godlowski, 1948 a. and others) Adrenaline liberated from the adrenal glands of the organism itself acts far more quickly (Vogt. 1950) On the other hand, dissipation of adrenaline from the suprarenal medulla takes place easily during everyday life by the run and fight mechanism described by Cannon, which in recent terminology is designated as sympathetico-adrenal discharge Speirs et al (1949) have shown that a drop in eosinophils occurs after simple handing of experimental animals, and that this fall is caused by liberation of adrenaline from the adrenal medulla, thís eosinopenia is as great as that following injection of ACTH or adrenaline itself. It has been shown (Halberg, 1952, Higgins, 1952) that endogenous eosinopenia occurs physiologically and is characterised by a twenty-four-hour periodicity cycle. Thuis eosinopenic ·THE ROLE OF EOSIYOPHILS IN ANAPHYLAXIS rhythm is species specific, and this species specificity depends on the initiation of vital activities as well as on the action of light Thus in humans and in dogs (diurnal type of life) the lowest count of eosinophils occurs in the early hours of the morning; in mice and rats (nocturnal type of life) it occurs in the early hours of the night This physiological rhythm in the circulation of eosinophils is controlled primanly by the action of the autonomic nervous system, the endocrinological response being secondary. It is worth mentioning that a post-adrenaline eosinopenia in hypophysectomised or adrenalectomised animals does occur, but its origin is not clearly understood It has been found that in adrenalectomised dogs (Erdstein et al 1945) and in hypophysectomised rats (Fry, 1952) and mice (Speirs, 1952 and others) a transitory eosinopenia takes place after the injection of adrenaline, this type of post-adrenaline eosinopenia 13 non-specific in character and raries greatly from one species to another. The suggestion that adrenaline in these circumstances causes sequestration of eosinophils in the capillary bed of various organs (eg, lungs, liver, spleen) by its action on the autonomic nervous system has been disproved in men and in dogs by the experiments previously described (P84) The post-adrenaline ensinopenia of adrenalectomised or hypophysectomised laboratory animals may be explained by the following coincidental factors (1) The existence of additional adreno-cortical tissue elsewhere in the body. (2) regeneration of the remored organs, or (3) stimulation of other organs by adrenaline to produce steroids with mild eosinolytic action (eg, gonads . Fry, 1952) In other words, the great variations circulating eosinophils found in physiological conditions are the result of a very sensitive and rapidly operating eosinopenic reaction in which the autonomic nervous system, glandular part of the pituitary gland, and suprarenals are involved. the autonomia nervous system is, however, the primary link in this chain of physiological reactions. Hormonal Eosinopenia and Tissue Eosinophilia. In the experiments already described, in which eosinoni stimuli were applied in the form of three different formalina, the results obtained mere almost identical One could say in ·36 EXZYJIATIC CONCEPT OF ANAPHYLAXIS AND ALLERGI anticipated this, since all three lormones stintulate (directly or indirectly) the secretion of those adreno-cortical steroids which are responsible for circulating eosinopenia. The mechanism and the meaning of this action is not clear and needs elucidation ACTH does not exert its eosinopenic action unless the cortex of at least one adrenal gland is able to secrete steroids in sufficient amount to eliminate eosinophils from the circulation (Thorn et al, 1948). But there is no general agreement is to which steroids possess an eosinopenic action. Compounds E And F, the amorphous fraction of cortical extracts (Well et al, 1910, Engstrom, 1918, Fourman et al., 19.30 Higgins, 19.72; and others), and whole gland extracts unquestionably possess this activity. Desoxycorticosterone acetate exhibited very weak eosinopenic as well as leucolytic action in the experimental conditions described elsewhere in this essay The histological results of the series of experiments presented here point to the mucosa of the small intestine as the only tissue in the dog which contains a significantly high number of eosinophils during hormonal circulating eosinopenia Thie question now arises as to whetlier circulating eosinophis are arrested in the intestinal mucosa and thereby removed from the circulation causing eosinopenia The answer to this question Jies in a closer analysis of the fluctuations in tissile eosinophilia of the small intestine Eosinophils were found in the intestinal mucosa in the experimental period's in much higher numbers than during the pre-experimental periods On the other hand the readings of tissue eosinophilia in the post-experimental periods and in the non-treated controls hele either equal to or higher than those of the experimental perìoris, while the number of circulating eosinophils remained within normal limits (Table 5) In other words, the high intestinal eosinophilia of the experimental period cannot be regarded as resulting from the arrest of the blood eosinophils, with consequent eosinopenia in the blood The local arcumulation of eosinophile in the mucosa and the contemporary blood eosinopenia uje tro unrelatert phenomena; the increasing intestinal cosmoplastia is caused by accelerated formation of eosinophils in the mucosa and their arrest in the lymphatic barrier of the lamina propria while moving from the crypts into the interior of the organisin {for Inechanism see p. 75). Both these factors, ie, the forma- ·THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS 97 tion and the arrest of eosinophils, are aggravated by the repeated traumatisation of the intestinal mucosa by biopsies The blood eosinopenia, coinciding with the tissue eosinophila, cannot therefore be regarded as resulting from the arrest of circulating eosinophils in the intestinal mucosa , its cause must be sought in the direct action of the corticosteroidy on eosinophils by the mechanism which is described in detail on page 58, The increasing tissue eosinophilia in the traumatised organs during the coexisting hormonal eosinopenia in the blood points to the difference between tissue and blood eosinophils so far as their susceptibility to the eosinolytic action of corticosteroids is concerned. The tissue eosinophils are persistently resistant to the eosinolytic action of corticosteroids at a time when the circulating eosinophils are highly susceptible (Godlowski, 1952a) This observation has been confirmed also by others (Gordon, 1952; Vuehrke, 1952, Speirs, 1952) In an attempt to explain the resistance of tissue eosinophils to the hormonal lytic action at will be well to examine the arrest of eosinophils in the intestinal mucosa more closely (for details see P 75) The arrest of eosinophils in the lymphoid tissue which surrounds the portal of entry of the antigen (carried by eosinophils) is performed by an antigen-antibody reaction in the following manner the gelation of the cytoplasm of eosinophıls, which occurs during the antigen-antibody reaction (antigen of eosinophils and antibody released from the lymphocytes of the lymphoid barrier, for details see p 74), immobilises the eosinophils and makes them impenetrable by the corticosteroids Until the cytoplasm of the eosinophils has been made flud again by heparin (p. 76) the corticosteroids cannot enter them and are therefore unable to mitiate protein catabolism, which is ultimately responsible for the destruction of eosinophils. But when their cytoplasm becomes flud again the eosinophils regain their motility and move away from the tissue in which they were arrested; at the same time they become penetrable by the corticosteroids and may be destroyed by the accelerated protein catabolism which follows this penetration Direct Action of Eosinopenic Hormones on White Blood Cells. Experiments to test the direct action of CH-oxygenated corticosteroids on white blood cells in time and in miro 7 ·98 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY (Tables 6 A and 6B, 7, and 6) have shown that a very high dosage of cortisone acetate is required to perform the destructive action. These experiments have also confirmed the finding that corticosteroids destroy eosinophils in the blood stream but not in the tissues (discussed in the preceding paragraph). The concentration of cortisone in these experiments was much higher than is ever found under natural conditions. It seems that such massive dosage is necessary to compensate for the artificia) milieu of the experimental conditions described. Since the involution of all white cells caused by leucolytic corticosteroids is both marked and rapid (confirmed also by Padawer et al., 1952), one can reasonably postulate that the involution is due to the catabolic phase of intracellular protein metabolism The alternative suggestion that the anabolic phase is inhibited (Green, 1950, and others) seems less attractive because anabolic inhibition would require a much longer time to produce the degree of destruction seen in the presented experiments. The susceptibility of white blood cells to this action under experimental conditions diminishes in the following order eosinophils, monocytes, lymphocytes, and polymorphonuclears It is suggested that eosinopenia following the applicatron of a potent eosinopenic steroid is due to the disintegration of eosinophils in the blood as a result of mediation of the eosinopenic steroids in the protein metabolism leading to a rapidly progressing protein degradation. Disintegration of the nuclei and cytoplasm of all types of white blood cells resulting from the accelerated protein catabolism can be seen in a blood film. The coexisting mobilisation of polymorphonuclear leucocytes may mask the cytolysis of these cells (see next paragraph). By analogy one can expect acceleration of the catabolism of cellular proteins in other tissues whose enzymatic structure is similar to that of white blood cells (for details see "Anaphylactic Reaction"). This contention finds valuable support in the negative nitrogen balance seen in clinical cases of hypercorticism and in treatment with a high dosage of cortisone or ACTH. Muehrke et al. (1952) find that heparin mixed with the blood. in ritro inhibits the destruction of eosinophils caused by Cil-oxygenated corticosteroids They do not report the effects of these steroids on other white blood cells, nor do they offer any explanation of the inhibiting action of heparin in their experimental conditions, ·THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS 99 The inhibiting action of heparin on the eosinolytic activity of corticosteroids may be alluminated by discussing two findings reported independently (1) Inhibition by heparin of the hydrolytic action of trypsin at pH 73 through the formation of a trypsin-heparin complex, this complex may be dissociated by acidifying to pH 30 (Fisher et al, 1937, Rocha e Silva et al, 1945). (2) The destructive action exerted by corticosteroids on eosinophils by means of a rapid proteolysis of their cellular proteins (Godlowski, 1952 a) In the experimental conditions applied by Muehrke and his co-workers, heparin inactivated the tryptic enzymes in the blood, thus corticosteroids could not initiate proteolysis in eosinophils despite the disruption of the lipo-protein molecules (p 51) and despite the exposure of their cellular proteins to hydrolysis, because the proteolytic enzymes were blocked by heparin This mechanism is unlikely to operate in vizo unless and until a very high dosage of heparin (P 68) has temporarily inactivated all the available active proteinase in the organism This independent observation of Muehrke and his co-workers is very valuable because it supports the suggested contention that eosinolysis, when caused by corticosteroids, is primarily a proteolysis In the prevention of hormonal eosinopenia by heparin, two independent factors are involved. 1. The inhibition of proteolytic enzymes which are actively destroying the eosinophils of the circulating blood 2. The mobilisation of the tissue eosinophils. Heparin when injected intravenously in high dosage causes a significant increase in circulating eosinophils (dags 8, 9, and 10 (Table 4)) When in a perfusion experiment it is added to the perfusing blood a significant diminution of the tissue eosinophil count of the perfused organ results, together with a simultaneous but slight increase in the eosinophil number in the perfusing blood (Tables 6 A and 6B) In the light of this discussion the eosinopenic test (Thorn el al., 1948) must be regarded as an insufficiently reliable test for the biological activity of Cat.oxygenated corticosteroids ir it is not accompanied by evaluation of the hoparin level in the blood. Heparin in high concentrations prevents hormonal eosinolysis even in the presence of fully active corticosteroids Hence the conclusion that corticosteroids and the suprarenal ·100 EXZYNATIC CONCEPT OF AXAPHYLAXIS AND ALLERGY glands are not the only factors which may significantly influence the eosinophil count in the cosinopenic test. Mechanism of Polymorphonuclear Leucocytosis following Injection of Corticosteroids. lfenbin's (1950) work on the biochemical composition of inflammatory erudates explains (in some measure) the leuco. cytosis liich follows the administration of (11.oxygenated corticosteroids, a leucocytosis which masks tlie sinuultaneous destruction of polymorphonuclear leucocytes (see also pp. 58, 88). A close analysis of the fluctuations which occur in the polymorphonnclear leucocyte count during perfusson experiments (Tables A and GB) strongle suggests that Sfenkins findings are applicable in these conditions. In the experiment in which a dog's intestine tras perfused with his own blood to which Eucortone had been added, the polymorphonuclear leucocytes (in dog No 3) were rapidly reduced in number The masking action could not take place in the perfusing blood because the perfusing circuit was cut off from the source from which polymorphonuclear leucocytes rould hare been mobilised But the systemic blood, at the same tune, showed a steady rise of polymorphonuclear leucocytex (this blood remained under the action the corticosteroni liberated from the dog's own suprarenals by the stress of the operation and general anæsthesia) Comparing at Jyourly intervals the leucocyte figures of the perfusing blood with these of systemic blood, one finds a reduction of polymorphonuclear leucocytes in the former and a rise in the latter. Menhin isolated from inflammatory exudates a substance sluch he designated leucocytosis promoting factor which is derived from the cells participating in the inflammatory reaction. Since polymorphonuclear leucocytes are the most characteristic and numerous cells participating in inflammation, it is conceivable that they contnbute snustantially to the production of the leucocytosis promoting factor These two independent findings can be correlated with one another and may esplain the mechanism of polymorphonuclear leucocytosis follon ing the administration of Chl-oxygenated corticosteroids. As a result of the catabolic action of these corticosteroils, polymorplionuclear leucocytes desintegrate and ·THE ROLE OF EOSTOPHILS IN ASAPHYLASIS 101 they release the leucorytosis promoting factor; this in turn mobilises polymorphonuclear leucocytes from the bone marrow to such an extent that the coexisting polymorpholysis is completely masked and leucocytosis superrenes FUNCTION OF EOSINOPHILS Summary of Eosinophil Formation. Any living cell, whose intracellular enzyme system has become changed in such a way that invading protein molecules are no longer split, may turn into an eosinophil This change seems to be related to the physiological or pathological ageing of the enzyme system The foreign protein molecules remaining unaltered in the interior of such a cell stimulate the nucleus to produce a new proteinase or to reorganise the existing debilitated proteinases (by a process of adaptation of enzymes) in such a way that the foreign protein can then be catabolised. This reorganised enzyme in its anabolic activity may resynthesise a protein molecule which in its pattern is a replica of the original invading molecules (Bergmann, 1942) Thus newly reproduced protein molecule, as manufactured by the cell's own enzymes, is incorporated into the structure of the cytoplasmic proteins Such a possibility in the formation of eosinophils is supported by the recent histo-chemical study of eosinopluls by Vercanteren (1951) In thug way the debilitated cell becomes a new biological and histological unit By the formation of a new structure this cell loses its original function and former appearance, acquiring at least two neu character. istics • motility of amæbord tyre (Kabin, 1923) and the ability to transport antigens in its own cytoplasmic proteins (Godlowski, 1948 b) In such circumstances the cell loses the functions of the tissue in which it onginated and it migratey from its place of origin hy its newly acquired motility. Relation of Eosinophils to Lymphoid Tissue. The further fate of such a cell depends on whether it is intercepted by the lymphoid tiene surrounding the portal of entry of the antigen (Cartiel by the migrating eosinophilu), ·102 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY or whether it avoids contact with the lymphoid barrier and penetrates the interior of the organism via the blood (p 14) An example of the first possibility is the migration of eosinophils from the cryptal lining of the intestinal mucosa into the interior of the yıllus, where the lymphocytic barrier of the lamina propria arrests the moving eosinophils (for the mechanism of the arrest see p. 35) An example of the second possibility is the migration of the eosinophilised leucoblastic cell from the bone-marrow into the blood When eosinophils with reproduced antigenic protein in their cytoplasm pass again through the intestinal lymphoid barrier (which is a zone containing specific antibodies in high concentration) they again make contact with the specific antibodies; the resulting union of antigen and antibody causes their arrest. A zone of high titre of antibodies surrounds the portal of entry for any antigen (Drinker et al, 1941). Such a local concentration of specific antibodies, undiluted by the body fluids, is sufficient to precipitate local antigen-antibody interaction and to immobilise the moving antigen, this local accumulation of antibodies need not necessarily be reflected in the circulating blood As soon Eosinophils as Source of Antigen. as eosinophils regain their mobility by relieparınisation of their gelated cytoplasmic protein (p. 76) they move away into the interior of the organism via lymphatics or blood-vessels Such cosmophils are now deprived of their antigenicity by the antigen-antibody reaction which occurred in the lymphatic harrier, but they still retain the power of Te-synthesising the antigenic protein by means of the anabolic phase of their newly formed proteinases Such re-synthesis of the antigenic protein by eosinophils is possible. The renewal of antigenicity in eosinophils is limited by the fact that the Posinophils have moved away from the place where the antigenic protein entered the body, a region where they could receivo further supplies of antigen to incorporate into their own cytoplasm, If by chance an eosinophil, having re-synthesised its antigen, should pass a second time through the lymphoid barrier surrounding the portal of entry of its antigen, it would be again arrested and de-antigenised In this way a permanent rycle is established of de-antigenzsation by the lymphoid tissue ·THE ROLE OF EOSINOPHILS IN ANAPHYLAXIS 103 and re-antigenisation by the newly formed proteinases of the proteins of eosinophils Those eosinophils which avoid the barrier of the antigenantibody reaction, or which have re-synthesised the antigenic protein, represent a potential source of the antigen In normal conditions these cells are either repeatedly de-antigenised in the lymphocytic tissues or, if they make contact with sensitised cells, they may precipitate a micro-anaphylactic reaction Eosinophils with retained antigenicity were removed from the peritoneum affected by anaphylactic inflammation in esperrments on guinea-pigs (Godlowskı, 1948 b) Such eosinophils, not having made contact with the lymphoid barrier, possessed a strong antigenic power Eosinophils as Source of Anaphylactic Reaction. Eosinophils carrying antigenic proteins in their cytoplasm are a potential cause of anaphylactic reactions The antigenic protein, however, being incorporated into the structure of the cell, cannot be liberated until the cell disintegrates This was shown in an experiment in which a suspension of specifically sensitised eosinophils could not produce an anaphylactic reaction in the Schultz-Dale test until they were completely destroyed by repeated freezing and thawing (Godłowski, 1948 b) The life-span of eosinophils is not known It is possible that eosinophils, of the same age and carrying the same antigen may disintegrate in the normal ageing process at the same time and in sufficiently high numbers to liberate a toxic dose of antigen, but in normal conditions such possibility is very remote. The minute amount of antigenic protein liberated from a small number of eosinophils represents a sub-toxic dose which can be neutralised by free antibodies in the blood, thus in normal conditions no such micro-anaphylactic reactions take place therefore the antigenic capacity of cosinophils under normal conditions, from the point of riew of pathogenicity, 13 small. If, however, the number of circulating eosinophuls is very high, the liberation of antigen may become sufficient to precipitate various degrees of anaphylactic reaction. Pathogenetic Significance of Eosinophils. Eosinophils rould thus seem to represent a form of cells without specific origin, and with or without antigenic power.

·104 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY The presence of eosinophils in the blood proves that there is in the organism at least one portal of entry for one or more antigens Since eosinophils are at present regarded as normal elements of the blood there are two alternatives to be considered with regard to their physiopathological state 1 Anaphylaxis is a physiological phenomenon at occurs physiologically when an unsplit protein molecule enters the organism by any channel, or originates within the normal organism In such circumstances the pathological manifestations of anaphylaxis begin to appear only when physiological counter-measures normally taken by the organism do not balance the ill-effects caused by the entry of unsplit protein Here the eosinophils, if their number is within normal limits, are normal cells which are unable to proteolyse the invading (physiologically) unsplit protein molecules. This inability of eosinophilised cells results from the wear and tear process of their cellular enzymes in normal or in accelerated metabolism. The failing proteinases are thereupon reorganised and so enabled to reproduce the invading protein, 2 Anaphylaxis is a pathological phenomenon of universal incidence This latent physiopathology is brought to the light only in its more severe forms In its mildest form anaphylaxis may be regarded as a pathological condition appearing in every organism, and subjecting it to strain which, in turn, accelerates the process of ageing of the intracellular enzymes. The eosinophils in such a concept must be regarded as pathological elements, even if their number is within normal limits By their non-specific origin and noxious character they exhibit a primary imperfection in the biochemical constitution at the time of the formation of the organism ·CHAPTER I GENERAL CONCLUSIONS TH HE cells liable to be sensitised are those whose proteolytic enzymes possess insufficient potency to proteolyse the antigenic protein molecules which have invaded their interior. This enzymatic disability may be caused by 1. Primary constitutional abnormalty of the enzymatiu system, this feature may be organ and species specific. this enzymatic pattern is ruled by the Mendelian law in hereditary transmission 2. A normal ageing process of proteolytic intracellular enzymes. 3 The action of cytoplasmic toxins, directed towards cellular enzymes To acquire antigenic potency a protein must fulfil the following conditions 1. Its molecules must be smaller than those of the host-cell. 2 Its physico-chemical structure must enable it to stimulate the nucleus of the host-cell for enzymatic adaptation 3. It must escape proteolytic cleavage by the existing enzymes in the invaded organism The antigenic protein molecules remaining unaltered in the interior of the enzymatically disabled cells strongly stimulate the cytoplasmic proteolytic enzymes to reorganise themselves by the mechanism of enzymatic adaptation The cells which cannot in these circumstances reorganise their proteina es disintegrate completely The cells which are able to reorganise their proteinases become sensitised This intracellular enzymatic transformation constitutes the primary feature of sensitisation. The adaptation or reorganisation of the tryptic enzymes in the sensitised cells is arranged in the following manner, specific proteolytic toxic enzyme system is formed intracellularly by the mechanism of coupled reaction, this system consists of the nuclear organising factor and molecules of the antigenic protein. This enzymatic system cannot start proteolysis until and unless it is augmented by another molecule of the same antigenic protein; thus augmentation occurs in the second (shock) invasion of antigen А 103 ·106 ENZYMATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY ?) In the initial stages of anaphylactic shock the toxic hydrolysis yields various proteoses intracellularly which, by intrinsic action, disrupt the intracellular lipoprotein molecules thereby exposing structural proteins of the cytoplasm to tryptic hydrolysis In the later stages of anaphylactic shock the proteoses, by extrinsic action, inactivate lipoids in the lipoprotein conjugation in the cells not primarily affected by the anaphylactic reaction. This action starts chaotic proteolysis of toxic and non-toxic types and liberates toxic catabolites which are responsible for the manifestations of the anaphylactic reaction Thus the initial and primary stages (in the intrinsic phase) of the anaphylactic reaction take place in the sensitised tissues; in the extrinsic phase of the anaphylactic reaction the whole organism is involved in the “proteolytic storm Antigen-antibody union, per se a non-noxious reaction, is a defensive measure which takes place mainly in the lymphoid tissue and in the body fluids, it has the task, in the first place, of preventing the access of antigen to cells with an inadequate proteolytic system, ze, it prevents sensitisation In the second place, it prevents the access of antigen to sensitised cells and $0 prevents anaphylactic shock Proteolytic antibodies may be regarded as those proteinases which appear in the cells as a result of antigenic stimulation and which can proteolyse only the antigenic molecules to the level of non-toxic catabolites. Toxic proteolytic enzymes are those proteinases which appear in the cells and next in the body fluids as a result of antigenic stimulation, and which can hydrolyse only the antigenic protein (specific) to the level of toxic catabolites. Various toxic intermediate catabolites of protein hydrolysis are set free during the uncontrolled anaphylactic proteolysis and, Uberated into the blood, they exert their extrinsic toxic activities. The chemical stress of this action, in turn, mobilises the pititary-adrenal response which releases a great variety of corticosteroids Released Cil.oxygenated corticosteroids maintain the proteolysis which has been initiated by proteoses kberated intracellularly in the early stages of the anaphylactic The manifestations derived from this endocrine involvement are superimposed upon the symptoms produced originally by the anaphylactic reaction The quałty of the released toxic protein catabolites determines the character of the anaphylactic reaction; the typo reaction ·GENERAL CONCLUSIONS 107 of catabolites released depends primarily on the chenucal composition of the hydrolysed proteins The main toxins released into the body fluds during the anaphylactic reaction are heparin, histamine, and various proteoses Heparin released from protein salts in the early stages of the anaphylactic reaction causes incoagulability of tho blood, in the recovery stage of the anaphylactic reaction a tendency towards an increased coagulability supervenes as & result of the rapid withdrawal of heparin from the blood The de-heparinisation of the blood proteins 19 caused by re-heparinisation of the proteins of the tissues involved in the anaphylactic reaction This increased coagulability favours the formation of intracapıllary clotting which is responsible for the ischemic necrosis so characteristic of the later stages of anaphylactic inflammation Histamine released during the anaphylactic reaction is responsible for most of the dramatic manifestations of anaphylactic reactions by its intrinsic and extrinsic actions. Catabolites of the higher level of protein hydrolysis (proteoses) released during the anaphylactic reaction (leucotaxin, leuco. cytosis promoting factor, leucopenic factors, pyrexin, necrosin, etc.) are responsible for various local and constitutional manifestations. Symptoms identical with those of the anaphylactic reaction can be produced by application of agents capable of initiating chaotic and uncontrolled intracellular proteolysis. These agents are 1. Physical • intravenous injection of fat solvents. 2 Physico-chemical intravenous injection of colloidal solutions, eg, protein, peptone, sulphur, ete, or non-colloidal suspensions, eg, kaolin. 3. Chemical : intravenous injections of oxidising agents such as hydrogen peroxido, iodides Intracellular and extracellular proteolysis can be initiated in viro and in ritro through the disruption of the lipo-protein molecules, Jipods in Lipo-protein molecules protect the protein components against an interaction with the protein of proteolytic enzymes These lipoids may be rendered inactive in the following manner by the same agents as those which precipitate anaphylactic-like syndromes: 1 By removing the lipoid barrier separating the substrato ·108 EXZYBIATIC CONCEPT OF ANAPHYLAXIS AND ALLERGY proteins from proteins of enzymes by fat solrents such as chloroform, acetone, etc. (pbysical action). 2. By altering the dispersion phase of the lipoids in the lipo-protein molecules by surface-acting substances such as colloidal solutions or non-colloidal suspensions (physico-chemical action). 3 By breaking the double bonds in the unsaturated fatty acris of the lipoids, eg , through oxidation by hydrogen peroxide, halogenation byiodides, etc (chemical action). The stress of the anaphylactic reaction releases tlie pituitaryadrenal response · liberated corticosteroids interfere with various phases of peripheral metabolism, those steroids which Rre involsed in protein metabolism initiate proteolysis by the disruption of the lipo-protein conjugation in those tissues which are rich in proteolytic enzymes. Lipo-protein conjugation has the task of protecting the substrate proteins against undue hydrolysis carried out by the proteolytic enzymes normally present in all organs Any action which disrupte the lipo-protein molecules is apt to release proteolysis of the organism's own proteins Rapid disruption of the lipo-protein conjugation leads to partial Jiydrolysis which leaves behind highly toxic protein catabolites. C'1-oxygenated corticosteroids disrupt the lipo-protein conjugation by saturation of the double bonds in the fatty acids of the lipoids ; this action is closely co-ordinated with the tryptic capacity of the proteolytic enzymes available this proteolyais leaves no toxic degradates of protein. The beneficial effects of the C11-oxygenated corticosteroids in allergic conditions are produced by elimination of the secondary histo-chemical changes in the affected tissue, these changes take place in the third stage of anaplıylactic shock, and the steroids remore them by initiating the proteolysis of the accumulated inflammatory exudates this proteolis, however, leates the primary mechanism of the first and second stages of the anaphylactic shock unaffected. The process of formation of eosinophils or eosinophilisation takes place in those cells which do not possess sufficiently active proteinases. These cells, jaraded by unsplit protein molecules, trorganise their intracellular enzyme systems under the stimulating action of foreign protein molecules The nex}y reorganised proteinases in their catabolic phase rrconstitute ·potency are thus mobilised and shifted into the rintott, Here, however, the antigenicity of eosinophils liny w peatustel by the anabolic action of their newly reorganisoil prodottant During the eosinopenia induced by hormones (lumiin eosinophily are destroyed in the blood by the lytir motus al C11-oxygenated corticosteroids which accelerate their inte because of their altered physico-chemical klatr, AIR A By contrast the tin tinitilla, white blood cells are affected in a similar way lry tl Air susceptible to the eosinolytic action of corticosteroika, Allows action of corticosteroids, but the coexisting with all of polymorphonuclear leucocytes masks their destructie Eosinophils are to be regarded as carriers of various antigens. If large numhers of these cells are destrogerl, their att lycenie protein may be liberated in sufficient amounts tus precipitat But & destrnetion of timinophie sufficiently masure to cause an anaphylactic trtir nuet GENERAL COSCLCSIONS the invading foreign protein and incorporate it into the structural proteins of cytoplasm Thus eosinophils my become a source of antigen Eosinophils are cells which are formed plıysiologiantly liye the same mechanism in the bone-marrow, in the niucon the alimentary canal, or in any other non-loinntopolutter tissue in which the conditions for eosinophilisation niny «XH, Those formed in non-hæmatopoietic tissues may be denignato il as pseudo-eosinophils because, according to cuiryompirul classification, they do not belong to the true blood clomania, Antigenic protein u hen absorbed in the intestine any ratore not only eosinophilisation in the cryptal epithelium but also may stimulate the intestinal lymphord tissue bautier to forma specific antibodies These two effects are produced by diferent parts of the same antigenic protein Thus eosinophllasurutabrdun antigen in their cytoplasm, while moving the onsete la lymphocytic barrier, meet a high titre of spoilie Huntlantida, As the result of the antigen-antibody reaction (outlye il cu eosinophils and antibodies of lymphoid tissut) coniunphilesin temporarily arrested there and temporarily deportail lil then antigenicity The arrested tissue eosinophils can be michalina when the arresting mechanism of the antigen-antilmny w dorson becomes exhausted Eosinophils deprived of their unity cellular proteolys19 an anaphylactic reaction & rare occurrence # Eosinophil-arexamples ofzsolated cellsnbieter ·110 ENZYMATIC CONCEPT BP XYAPIYLAXIS AND ALLERGY & nec function that of synthesising foreign protein ; this actirīty of eosinophils is maintained in physiological as Tell as in pathological conditions The basic difference between these two conditions lies (50 far as the function of cosinophils is condemned) in the type of the synthesised protein A Fuller study of eosinophilisation (a phenomenon of minor significance in itself) may sked Isght on the enzymatic synthesis of protein in generl and neoplastic protein in particular. The presence of anaplıylactic phenomena, whether histochemical of constitutional, can be traced in all living organistus. Eosinophils may be found in the very young embryo, a finung which should be looked upon as evidence of anaphylaxis. The difference betreen the physiological and pathological Anaphylactic response lies in the type of antigen which induces this response and in the degree of the response The presence of anaphylactic symptoms in normal individuals may be regarded, therefore, as the physiological response of a normal organism to stimuli inflicted upon it by the environment. This physiological anapkylaxis may be aggravated if the immunological counter-measure's (in anaphylactic reaction) taken by the organism against these stimul are inadequate Thus eosinophilisation is regarded a3 & result of the inadequacy of the intracellular proteolysis which also forms the basis of anaphylactic reaction; hence eosinopbils may be looked upon as a histo-chemical evidence of anaphylactic mesponse Anaphylactic response, in turn, is an expression of either normal or pathological exhaustion of the proteolytic capacity of the enzymatic system So long as the ager enzymes are replaced by normal adapnee enzymes, the whole process may be regarded as beneficial because it provides the cell with » the cellular enzymea. A new potent enzyms. jt " rejuvenates When the regenerated adaptire entymes acquire a toxic charseter, this process involves the cell in a yacious circle which constitutes the basis of sensitisation The pathology which results from this involrement is shaped by (I} the character of the antigens which has induced sensitisation, (2) the breakdown products which are liberated during the toxic proteolysis, and (3) the predisposing factors of the inrolred organ (eg., anatomical structure, superadded infection, etc.). In other words, clínical allergy 18 an aggravated response of physiological anaphylaxis, whics as conditioned by locally coexisting predisposing factors ·REFERENCES ABDERAALDEN, E, and Percussohn. L (1909) Hoppe-Seyl 2. 62, 243 ABDERHALDEX, E, and Processown, L (1910 a) Hoppe Šey! 2, 64, 100, 433 ADDERHALDEN. E, and PECUSSORN, L (19106) Lloppe-Seyl 2, 68, 88, 277 ABDERHALDEN, E, and PENCUSSOIX, L (1911) Hopp- Seyi 2, 71, i to ADLERSBERG, D, SCHAEFFER, L, and DRACHMAN, S R (1950) j Amer med A88., 144, 909 ALL.IXOX, J B, and ANDERSON, J A (1945) J Vutr, 29, 413 ALSTYNE, E, and GRANT, P (1911) melab Res , 25, 400 ANDERSON, O E, WIESEL, L L , HELMAN, Rw, and STUMPE, W D (1951) Proc Soc erp Bunl, N Y 76, 825 BERGMANN, 31. (1936) Harvey Lect, 31, 57 BERGMANX, MI (1912) Adranc Enzymol, 2, 49 BERTELLI, J. H, FALTA, W. and SCHWEENGER, O (1910) Z Hin Med, 71, 23 Best, W, and SAMTER, M (1951) Blood, 6, 61 Biedl, A , and KRAUS, R (1909) Il'ten Ilın 1schr, 22, 363 BIGOART, J H (1932) Path Bact , 23, 799 BLASCIXO, H (1045) Advanc Enzymol, 5, 67 BLOOM, B, and PIERCE, F T (1952) Velabohem, 1, 155 BORNE, G H (1931) Cytology and Cell Physiolngy." 2nd ed (Oxford Clarendon Press) BRACHET, J (1950) Ann Soc Zool BHg, 81, 185 BRUNNER, M, and WALZER, MI (1928) Arch vnt 11ed, 42, 172 BURNET, FM, and FENNER, F (1919) The Production of Antibodies, Melbourne." (London Macmilan & Co Ltd CALVARY, M (1911) Alunch med Wschr , 58, 670 Cannon, T. P, and Burt, K L (1931) Immunol, 27, 173 CANNON, PR, and SCLLIVAN, FL (1932) Proc Soc erp Bil, N , 29, 517. CHARGAFF, E , ZIFF, M, and NOORE, D H (1911) Bsol Chem, 139, 383 CHARLES, A P. and Srort, D A (1933) Jbid Chem, 102, 431 CLASE, M (1952) Amer J Med, 3, 352 CLAUDE, A. (1943) Science, 97, 451 COCA, A F, and Geove, E F (1925) Immunol, 10, 145 CODE, C F Conx, (1939) Amer J Physiol, 127, 78 JW Conx, J W. Luts, L 1, and JOAXSOX, 21(1919) J Lab chin Med, 34., 255 COON, H A CoWDRY, E V. LEDUC, I E, and KAPLAN, M II (1951) J exp lled. 93, 173 (1932) "Special Cytology," 2nd ed, vol 1 (New York PB CRAMPTON, C. F., and HarrowITZ, F (1930) Science, 112, 300 DALE, H , and LAIDLAW, P P (1910 11) J Physsol, 41, 318 DALE, H H (1929) Lancel, 1, 1179, 1295 DEVEL, II J (1931) "The Lipids," vol i (New Yorh and London Interscienco Fed. Dixon, F J, BUKANTZ, SC, MAMMEN, G J, and Telmage, D W. (1951) Proc, 10, 553 DOMSRT, A (1931) Disch Arch. Ilın Yed, 69, 499 DOMENICE н (1909) Fol Homatd, 8, 99 DOTCHERTY, T F CRASE, J H, and 'WAITE, A (194ļa) Proc. Soc erp Biola DOTCHERTY, T F and Arte, A (19415) Endocrinology, 35, 1 DOTCHERTY, T F. CRASE,J H7, and 11 HITE, A (1945) Pror. Scorp Bid. T. DOCGHERTY, TF (1951) Lancet, 2, 733 Long's "Symposium on Indsence of Hypophysis and Adrenal Cortex on Biological Reactions." DRAGSTEDT, C A (1941) Phyed Pet, 21. 563 DRINKER, C. K. and YoFrey, J M (1991) Lymphatica, Lymph, and Lymphoid Tiesue" (Cambridge, Massacbusetts. Har: ard University 'ress) 111 Hoeber ·11: EXZYMATIC COXCEPT OF ANAPRYLAXIS AXD ALLERCI DCRABOKB, F (1943) Lancet, 2. 186. DXURFT2B), , CODE, C. F., and irgis, & M. (1970) Sy', 75, X) Prre. Si err, Biod. EXLE, H. (1933) J from endl, 29, 41 Enric", WE,, and HERREY, TX (1949) Jesp. Jled, 7, 333 FARLOCK, P (1875-79) Arth Andi Phyusof, Ly:. 571 EXGIL, F L (1949Endendogy45, 370 E GEL F, L. (1953) Amer. J Jed 10,5.56 EGPI. F L (1931 by "Proceedings of the Serond Clinical ACTH Confereise,"br JR Hote tidladelphia and Toronto Blakston Co FSO FROM, IV (1949) Yale y Bind Wed, 21,1 ERDSSTIN, S, AGL EPI), 3 G, and CATALANO, A (1945) de Cal raf. Tubere. 8 deres, 7, 113 Fip R. LF 39.39) Symposum on Steroid Hormares, by Ed Garden {conun Lissersity Press FER, A (1933) Brochen 2, 240, 364 FIER, &, and HFRYS, H (1837) Erzywdlogsa, 3, 187 ForestAN, P. BAPTEA, PE, ALARICHT, F, DESPE), E, CARROL, E. Amet LEXANDER, 3 (1990) der mest. 29, 1462 For, J. 1 (1936) Immunol. 31, 233 Foxy-1) TYSING, A (1953) SubmicroscopNS Morpbolngs of Protoplasm (New York, Amsterdans, and London Elsevier Publishing Ca lac } FXTON, JS (1946) J bood Chem, 166, 721 Ferro, JS HRING, & », and BERGU4X4, (1942Swall Chen, 238 3 FR, G (1939) Proceedings of the Eosinophd Conference, Rar Harbor" (IN pront) GABRILE, J V (1970) Jc Endocrin, 10, 617 Ginot Y, J 1 (1918) Brus meil J. 1, 867 ADVEX JH (1931) Brut med J. 2,387 GAI THIER VILLES, (1951) Jo the Ervdes clusques, experimentales et therapeutiques sur l'Allergre," by Pasteur l'allery-Radiot And A Trench (I'aris L'Expansion Scientifique Française) GERXHBERG, X, FR, EG, BADECA, JR, and Loxo. CNR (1950) Lale J. Bid Wed, 23, 32 Goolong, Z 2 (1946) Brw und J., 737 GODLOWSKI, Z. 2 (1948) brut med 1, 48 Gonowski, Z Z (1948) Bnler Palk , 29, SII Goprotekl. 2 Z. (2010) thn Park, 9, 19 Gootits, % Z (1931) Bril, med vi GutowsX1, Z. 2. 1932) Endorill, 8, 10% Godtowski, Z Z. (1952) Arch in Pharmacodyn, 01, 103 GONDON, A S (1912) Proceedings of the Eostrophe Conference, Ear Hartvor. CREEN, J. (1950) Brut med J. 2, 1165 GREEX, RN and GHAXDIALLY, FX (1951 Bnt med W.1, 496 GREEX, I. X., and SATIGEAR, (1831) Brit med 3.2.499 GREEX, J D, and HARRIS, G. WY (1917) J Endocrine, S, 136 Groot, J. DE, BU HARRIS, G W (1950) J Payant. Ili, 33.5 GPOS TENY (1892) Arch, D. 12 381 Qunted by Bal (1917) Gclick, S. (1941) Adirne, Erzymad. 41. HALBERO, F. (1933) Journal. Loxtel, 73, HARRIS, T. (1944). There for VD Degree, Cambridge Unnersity. HARRIS, II (1948) Plynol, An , 28, +39 HARRIS, W. (1951) Brat, med. J. 2827 HARRIS, W. And JACOBSONY, D (19311 Qunted by Harry (1931). HART, D'A, and RER, RJ1. (1930) Lanzul, 2, 391 FL4L ROWITZ, F, ERAMPIOS, C F, and Sarisxs, R. (1931). Fed. Pror, 10, JIP VA. HEIDELBERGER, 31., KENDALL, F. E, and Av Hro, C. 2. 1933) HEIDE RAIY, R (1998). Arek. ges. Physiol. (P), 48. 2, 834 دي ۱۳۹۶ ) 59, 337.

·PEFERENCES 11 JORLIYC, J. W., and PETERSES, WSGOSTEL AA (19156) Jerp Ved , 22, 141 TER PD. and KRCSE, H (1951) Fed Pror, 10, 564 113 HEIDENHAIN, R. (1891) Arch. grs. Physıdl, 49, 2014 HiGcx, G 11. (1932)' " Proceedings of the Eosinophil Coaference, Bar Harbor" (In print) HILLS, A. 8, FORSK 4X, P H, and Fisch, C A (1918) Bind, 3, 733 HOBEBWAY, H D (1950) Yale J Bin Aed, 22, 341 HOLVAX, R. H, WHITE, A., and FRETON, JS (1947) Prox Sor Btol, X Y., 65, 196. HOLTZ, P. (1937). Kort Ischr, 16, 1361 HOWELL, K. (1928) "The Xeser Knowledge of Ba tenology and Immunology (Chicago University Press) HOME, DH, and WITTENSTEIN. G J (1950) Proceedings of the First Clinxal ACTH Conference," by J R Mote (Philadelphia and Toronto Blabiston Co ) JACOBSEN, K. P, and Prrors, G (1951) Amer Jod, 10, 531 Jarobsox, W. (1939) J. Path Bact, 49, 1 JAQTEY, LB, and WATERS, E T (1940) Amer J Phyuol. 129, 389 JOBLisa, J. W., and PETERSEN, IF (1914) epMed 19, 459 (1915) Johns Hopk Horp Bull, 26, 358 JOBLING, J W., PETERSES, W, and JOBLINO, J W. Petersex, w, and EGGSTERY, A A (1915 c) verp 11ed, 22 401. Joblina, J W PETERSEN, W, and EcasTEIX, A A (1915e) y erp Med 22, 701 JOBLINO, J W., PETERSEY, W, and EGGSTEE A A (1915) Jerp Ved, 22,5911 JOSET, A J, and LAWRENCE, J S (1932 a) Folia Hæmatdl, 48, 303 JOSEY, A.J, and LAWRENCE, J (19326) Polu Hamal, 48, 323 KARSTRÖN, ÉL. (1938) Ergeb Enzymfach, 7, 351 K 199, E H. (1945) Stopnce, 101, 337 Wur Brk lar Hap, 1, 17 KENDALI, E C. (1949) Ann NY Aend Sc , 50, 50". KIRCHHEIM, L (1911) Arch erp Path Pharmak, 68, 372 KIRCHHELM, L (1912-13) Arch erp Path Pharmak. 73, 1 KIRCHHEZ, L. (1913a) drck exp Path Pharmak, 73, 139, 374 KIRCHHEIM, L (1913) Arck erp Path Pharmak 74,374 KOBERNIK, D, and 'MOORE, R H (1950) Proc Sor exp But, 1 1, 74, 602 KUCZYNSKI, M. H. (1922) I trchmus Arch, 239 185 KrLTSCHITZKY, * 1897) Arch makt Anat, 49, 7 LANDSTEINER, K, and SCHEER, J LANDSTEINER, K (1920) Brochen 2, 101, 230 DER (1927) Jorp Wed, 45, 1045 LANDSTEINER, K, and SCREER, J V DER (1929) Jerp Mend, 50, 4117 LANDSTEINER, K, and SCHEER, J V J V DER (1932) exp Jed, 55, 791 LANDYTELNER, K., and SCHEER, V DER (1931) J exp Med., 59, 769 LANDSTETNER, K, and SHEERJ V DER (1939) Jerp el, 69, 705 LAPAOH, J. M. LWRENCE, J. S, and JOSET, A 3 1932 Folu hamater, 48, 330 LEGER, J., Lee, and Rose B (1948) Proc Soc erp Bid, 87, 69, 463 LAISELETR, J (1930) Ann Inst Pasteur, 78, 151 LOISELETR, J, and LEY, M (1947) ana Ina Pasteur , 75, 116 Losa, C X. 1, and Frr, E (1945) Pror Soc exp Bud, F1,53, 67 MICDERMOTT, W R. Fry, E G, BRODECK, J R, and Losa, C. & F. (1930). NCHLASTER, P. D. and HEDACK, S (1935) Jezp yod, 61, 783 "Enzyme and Immunology: The Enzymes," by J. B Sumner and K Jijtbărb, vol 1, part 1, p 343 (New York Academic Press Inc. Publishers) VAXINOW, A (1927) "Handb d mikr dpat d. Mensch" JATIVOW, A, and Blooy, W. (1948). "Textbook of Histologs," Gth ed. (Philadelphia and London Saunders Co ) Jexkrx, ! (1950). Yener Concepts of Inflammation " (Springfield, II, L'S A: C. c. Thomas) V . J.R'A (1950) ·314 ENZYSTATIC CONCEPT OF ANSPRYLAXIS AND ALLERGY MEYER, E, and EXXEBICH, E. (1909). Arch Ilon Urd, 96, 297 MICHAEL J1, CUMMINGS, M, and Beqov, IT. L. (1330). Pror. Sor. 15p, Bas W.E., 75, 613. MONNĖ, L. (1918) ddrnc. Enzynid., &, 1. MEDD, &, HCCUTEREOX, N., and LreKt R, (1031) Płyciol. Rev., 14.210. MCEINKE, R. C. (1932). "Proceedings of the Eosinophil Conference, Bar Harbor." (Ir print) Muerer?, R. C, Lewis, J L, Kark, R. M (1932) Setener, 115, 377. NETYIL, E., and ParTRIEL, X (1946) CR SOC Brol, 141, 186, 198 OAKLEY, L, IARRAK, G. ft., and BATTY, J. (1919) J. Path, Bact. 81. 179 OFTE, K (1904) Amer J med, 86, 27, 999 PADAWER, J, and Gordox, A S (1952) Endorindony, 51, 52 PARLING, L, PRESextax, Đ, CATRPLE, D I. and IKEDA, C (1942) J. Amer. chem, Soc., 64, 3003. PARLING, L, PRESSMAX, D, and GROSSBERG, A L (191) J. Amtr theme for, 68,284 TEARSON, O, H., EIJEL, L. P., RAT80x, R. W , DROBINEA, K., and Roids, C, P, (1949) Carrer, 2, 913. PYNDXPGRASS, E I., CHAMBERLIN, G. 1, GODFRE, E , and BERDEE, E D. (1942). Ater, J Roentgen., 48, 74! PETERxAA, 3. (1943) J phyº Chen , 48, 183 L'ETERS, R A. (1932). I'roc roy Sa, 139, 143 PATERS, I A. (1995)Urt med J.2 1165 PFEIFFER, H 1916) 2. Immun Forech., 23, 515. PFEIFFER, H. and trta, 8 (1915) 2 Immunforec, 23, 408. PCXFORD, M1, and VOOT, A (1951). J. Physiol, 112, 133 PRATEXITZ, C, and Krstver, II (1991), Central Bact Origa. 88, 160 Quick, A J 11936). Amer. J Physsol, 116, 535 Ramsay, G.S. (1953) Brit mrd. , 1, 439 RANDOLPH, Tn. (1941) Allergy, 15, 89 RANDOLPA, T's. 1. (0850) "Procedings of the First Cinscal ACT# Conferenca," by J. R. Mote London : Churchill Ltd. RATNER, B, and GXTEL, L. HI (1935) Anny Na Chad, 49, 287 RATNER, B (233) Allergy, Asephylaxis, and Imaunotherapy." (Baltimorg: Willaras & W9 Co ) RECANT, L, ICE, D 3. FORSA', PI, and Taon, G W (1990). Jc. Endocrin, 10, 187. Roseon, A R (1923). Falua llamada, 27, 14 RixCEOX, A R. (193) "Jardhook of Hæmatology," vol. 2, p. 1B1, by H. Downes. (Now York. P. B. Hoeber Ine) RICK, A. 2! (1935). 17 She, erp Brod, NY, 32, 1J19 RICK, A. R 1914). "Pathogenesis of Tuberculosis" (Springfield, IN., USA C.C Thorus) Ric, A. R , LEW25, D R.And WINTPOE, Y II (1939) Jahns Blopk forp Bull.. 65, 311. RICH, A R, COCKRAN, T H and McGoos, D C (1991). Johns flopk Harr, Bull. 88, 101. Roxas B SILVA, M (1916) " Hustamina è anafilaxia" (Sao Pacto Edigral| ROLA SILTA, , and ANDRADE, S O. (1949) Sesence, 10, 670. Rous, F. P. (1998). J. ezp fed, 10, 337, Res, F. (1997). Ada Poyra, J'eurd. (Suppl. 40. Sarrx, F. R. (1923) Jahn Hopk. Hoep Bull., 24, 277. SABIN, F. R (1939). J, tup: Int., 70, 67 SAXTEL, 31. (1919). Blond, 1,217 SAYERS, G. 19500) Physil, Rer, 30, 241 SAYERS, G. (13386). "Actrenal Cortes Trausation, First Conference, November 1949." (New York J. Macy, jun , Foundation SATERS, G, and Sarens, 3f. A. (1947). Endocrinolong, 40, 261, S&TEAS, G, SATERS, M, A., LIANG, T V, and Loxo, C. X. H.(1945) Endocrinology 37, 06.

·VE H, and SORGE, FL (1916) No. 97, i. REFERENCES 115 SAYERS, G, Burn, T. IV, TYLER, F. H, JAGER, B F, SCHWARTZ, T B, STA, E. L, SAMUELA, L T., and DAVENPORT, H 1 (1949) Jdın Endocrin, 9, 593 SCHILD, H. O (1939) J. Phyeol , 95, 393. SCHWARTZ, O (1909) Fren. Wir 'Il schr, 22, 1151 SELYF, H (1946) 3 con Endorfin, 6. 117 SEVAG, M G. (1946) Aduync Enzymol. 6, 33. SILDER, R H., How , E E, PORTER, C C, and MESAETT, W (1949) Autr, 37, 429 Simox, L. G (1905). C.R Soc Asdl, 59, 649 SIMOXDY, J P (1923) SMITE, E. L. (1948 a) J bed Chen , 173, 571 Amer J Physiol, 65, 512 SMITH, E L (1918) J bord Chem. 176, 21 SPEIRS, R, 8 (1952) "Proceedings of the Eorinopbu Conference, Bar Harbor" (In print) SPEIRO, R. S, and MEYER, R K (1949) Endocrinongy 45, 403 SPIEGELMAN, S (1950) ** Modern Aspect of Enzymatic Adaptation. The Enzymes," vol 1, by J B Sumner and K Myrback, 1950 (New York. Academic Press Inc, Pablishers) SPIEGELMAN, S, and KAVEN, M (1916) Science, 104, 591. SPRAGUE, R G. (1951) Amer Net, 10, 587 SPRACUE, R G, POWER, MH, Lasos, Ý L, ALBERT, A, METHIESON, DR. HENCH, PS, KENDALL, E C. LLOCUME, C. and FOLLEY, IF 1950). Arch Int Med, 85, 429 SrãoB, C (1910) Ergeb u Med Adhesik. 6, 192 SCGDIOTO, T (1913) Arch erp Path Pharmalol, 61, 14 BUSSMANN, H. Davidson, A, and WALZER, M (1928) Arch int Jed, 42, 469 TKORX, G.W , Forges, P H., PROXTY, F T, and HALLS, A G (1948) Amer. med 138, 137, 1005 TOPLEY, W WC, and WILSON, G S (1936) "The Principles of Bacterology and Immunity, "P137, (Londoa E Arnold VAQUES-LOPEZ, E VAUGHAN, v. c, archan ve, jun, and Fatouak, J, W:(1913) - Protein Split Products in Relation to Immunity and Disease” (Philadelphia, NY VERCANTEREN, R. (1951) Enzymologaa, 12, 311, 389 VERZAR, " Absorption from the Intestine; Monographs on Phasiology" (London Longmans, Green & Co) VRIES, J DE. (1950) J Immunol, 65, 1 Voor, M (1950) Bret med 1.2 1942 Voar, M (1951) Phynod, 113, 123 WALSH, T E, and Canson, P R (1934) Arch Oto-laryngol, 20, 820 s, and Drxox, FJ (1948) dmer med Sc, 216, 136. \\'E143, C., And CZARNETZEY, E Arch Path , 20, 233 WELL, B B, and KEYPALL, E C 1940) Proc Mayo Clin, 15, 859, WESTWATER U PA11910) June 7ep. Ficop Bull, 24, 357 F. (1916), Ann Acad Sco, 40, 859 ABRUMI, P. BRISAUD, E. (1921) Pr med, 29, 181 and W'A, BEIERWALTES, W,'H, 1 D. Dort, F., Jorgs, J.R. KNORPT, C'T,, and Eva, 31. (1950) J. Lad dit. Bed, 36, in.. ZAPPERT, J (1893) 2 1/x Ved, 23, 197 2cxz, E (1921). CR Soe Bul. 91, 121 Zrxz, E, and GYÖRGY, P (1915). 2. immunForech, 23, 402. WARREN (1935) WETTE WIDAL ·INDEX Adaptatem efnymes, 11, 13, 39. 25, $41, 72, fulphularis, adapine enzy 34 3, 3), 15, 1%. %. 1, 282105, 10 Allenohy ph 38H, 17. 4. 49 24, 223, 101 135, 114 Adrspathy la allrtzy and ass laws, alimentary (18113 solice of, 82, #3,79 Adrenaline as cher strepto, 19 allergy and, 1 as exwtnogenic factor 4, 8, XU SI 93 94 annikules He is, II, 11, 3.J yraienna Infras o Joan, 8+ *Du-termeat potenty of serum in, 21 nbeutaneoria injectius in dux, 88 chronic form of, 34 Arnals, s 19, *3 clinirl friss of, 33, 34 Ad-cort ankerelds nee tort rosteroids en matie eeept tengt vitepecorilt rozir horside, 47, 49, W, 51, 77, , rektion concept in, it antigen sbody 81, 81, 94, enzymes in, esterase in hood in, 37 10 58 Aftergle renelan e Alytartie Teatriz protease in blood $, . 27. 31. 37. 65, 106 107 Ankle hrklige, 18 įvuteinase ra blood in, 27, 31, 37, 105-107 Amino acid, del, 13 toxic enzymes role is, 3, 9, 17, 21, 27. 12. $7. PANCntial, 63 113-107 Ammium claridt s eusinupor Caetor is CONTUDs role ia, 201 Anveld marts of a sophis, 35 79.71, 05, 203 słty afds role 0 3 3 62 W 105 Axxy photo fraction of adsenectical extracto, 88 foseigts protein role in, 24, 11, 12 Anamnestic reactinn 17 formula of, 25 Anasty Laetle Osbe is scrones, 3, " #1, 197 heredity of 1.9, 1905 4 720p lactle reactkerts Heuves OS promoting factor ist, $ 301 hee All Anapho la tas, attes antitats leucopene fattors 39, 407 restbrenteissati ietintasin 939, 107 hk Altre fint, 15 lipoprotero conjugation relation to, 22. 29, 3.4 #31 Le fon13), 32, 40 11, 45 prind fartler is Intestsbal 1981 role in. 33. ota-antibuy reaction roositron with, 12. 13, 18, 30, 11, +$ 48 102 103 61, 73 73, , 08.97, 101 102 109 w presiLre in 31 1$ uphold tinkte relation t«s, 24, 3d 383 De zovin in 99 100 carrilary, writeREEN, J1 11e D, zat buzzic, t3 707 Paronital 31.35 de pathlopreal phenoramaoni, w, 114 *****istentely Taxe on, 54, 54, 57, 58, I{, 209 e fornin, 17 phesuspeiderhemicat in 5 tutti one aretate alim i», 33, 54, 59 as phylokal phenonenwa wi 110 rating of brod 1, I, 35 pły ek pathological formula of impunity 19 chronie form of, 3+ Diturtari Rdrerii se faith in relation to, 15 16 definition ot. 35 12,419, 50), ectoplo forms of 33 portal of entry of Antigens, 4, 01 42, 105 {xreren protein role #, 4, 10, 11, 12, *), 172, 72, Pretein metabolum teen in winterucaplat 73. 105, 107 structure of Ep11 in.) 99 reynxin action in $1, 33, #0, ##. $4 proteolis in nee Protebe! hlatarin e fa 305 Previn 22, 39, 107 DiaDe wet1093 359, 31, , BU **, 30, 107 werissacion period in 1, 51, 4, 700 hypergly cami state of equilibrium to 13 > proteinpulss, 31 strsy me'dasians of,1 15, 33. *7 , 204 ituorenia 30 31 *ympathietky adrenal discharge to , 03 prala 11, 37.44% thropshots arridents to 13 111M-posteli molecudes mie fra, 14, 17, 19, Anor), Acute, as esimernie raator, 41, 42, 53, 50 37 Anti anaphylaxis, 18 lymphateswin. 33 antzindy Mee a Vezz-it der, Protein Implosd t-sue role in, #, R$ 26, 14 3135, 103 allergie and Anuph Lartit rreton. 31 36.3.3 "Dani of, 24, 41, 35, 37, J, 55, 57, 205 atopic reagir, 17 2UTU shork as, , 101 ortreteroids relate to 17,1, 43 PXin Now , 33, J7, JY letinition of +5 $0$ proje 19,34 e mes relatos to, , , +3.30 plass solame in, 31 seal artumotation w. + pney chrotic format, 13 31 tation of 7 proteses šo body mada 12, 11, 22, 33, 32 Inca) Ingunit me to in tein metabolisat in *nastie phe, 25 43, lymphoid lixpdge relation tod, 7, 14, 16, 39 tahe »3 SA 74 in catabok plane, 30, 45, 35, 58 Dieelarstsas or action %, 14,5 Proteinases is oily Husets in, N 30, 33, 42, 43, 1309) Trofeok 1h 4 34,73 plssstopathological fara u inanity" 19 proteiris 13 Impf in acute farses I relation to 12 giroteolysis fil, 10, 11, 1273, 29, 32, 34 jutultasj-adrenal remise related to, 15, IN, 31, 40, 41, 42, 55, 56, 34, 307 Biruteoses to body Maids 1st, 27, 32, , +2, 35. pmalarlun er 2,5 7 7,1 1: 3, 41, 43 $6, 204-107 sorteristic. $. # $ 3193 Te 1988 Selle ta y up! . 31 ures of, udio Bowlars chronne (EUX Of, 32, 33 top Deation, 3 28, 3 opwonin, 3 prepitin 3,62 teraperature of the meritus vf, I intesty the SM, 12 thlefto pepfa 18, 31 ee 20873 1211c seducioni jokle, 9 17,9,11.48 Antigenerala 'rufen Antigent of protein, 21.01 Daphs texto see a 4 Bagh factic resition, reactin, * Boles slumical untuk of, 2, 233, 105 dronition of 2 }18 stage of 32, 11, 1115 A Rulrones slouz to, 107 (Darby facts shock and stock antiges Amt) sert ·acting in pustential 53 DextTune Bolupain antigens, NDEX 117 Antigen, derlratives, 4 Chemical saturating of fatty acids, 53, 53 57 double marking of, 3, 4 Chemo-receptors role in pituitary-adrenal respons, dye marked, 3, 4 47, 49 Este in the body, 3, 101 Chloroform intravenous Tajectton as shockhaptens 2, + Ake actions, 3, 42-41 107 precipitating factor 43, 55 107 108 Cholesterol 49 51 lymphold tissue response to, 67, 10, 11 Clotting time. 83 Thectarlist of action 12, 14 46, 101, 105 Coinchy pain as shock precipitating factor 78 metabolism of 4, 74, 101 Collodal su)phur 24 shock precipitating factur 99, mode of interaction with antibodt 4.6 43. 55 56 tode of spreading 10 the body bs blood capillanes, solutions as shock.precipitating factor, 29, 13 10, 14, 74, 102 53. 58 107 by lymphatics, 9 101 neutralisation of, 4.8,7 45 46 Suspensions as shock-precipitating factor, 29. 43, 55, 56, 107 physio-pathologiral formuls of immunits. the Complement fratron, 5 role of 11 12 Cortinateroids 49 51 portals of entry of, 2 74 101 anti-enabolic action, 54 50 preudo-antigens, 3, 40-44, 107 antibodies released by, 8 11 15, 41 radio-artive antigens, 3 Berinitisation, the role of, in 2, 3, 9, 12, 14, 33 46, la allergy mechanism of action, 54 56 58 109 catabolic action 50 56, 59 59 98 97, 99 100, 10% 203-204 Cu orygenated, ensinady tır action 50 51, 84, brusiting dose of 3, 10, 14 28, 33 99 abord-done of, 3, 10, 15, 16, 23, 33, 37, 39, 46 103 fatty acids relation to, 55 5A 57, 105 specificity of, 106 furtuation in body fulds 49 xicity of, 2, 45 Irucolytic action 7, 50, 53, 98-200 transportation of 3, 10, 74, 07, 101 lympholytic action, 7, 13 49 50 51, 55, 95 100 htigen-antibody reaction See aLeo Anaphylactie Inactivation by Jecathin, 54 reaction, Anaphy latis, Antigen Antibody Lipo protein conjugation atfected by 51 59 53 in allray and anaphylaxis, 9, 12, 38 41, 41 45, 57 73 75, 108 Oxurlution potential, 53 anaphylactic shock, 12 NPN in plood increase 50 defrnsive barrier of, 9, 10, 22, 24, 45, 48 2012 poly morphonuclear leucocy tony Woning. 5 oubophit artent in tissue by 74 75 10 88 98-100 Laulure of, 1,10 12 13, 45108 reduction potential, 63 mechanism of, 5, 10, 15 45,73 106 Coxygenated, reduction potential 53 nou Doxious character of, 33, 4 101 Cortisone aretate See Clotygenated corti. Tetorulo endothelial system, 3, 10 costeroids 52 80 role in sensitisation. 10 41, 43-43 106 as eosinopenic hormone, 54, 99 100 site of action, 10 15, 17, 41, 14, 106 protein nietabolism mediated by, 53 6X. 59 task of, 10 96-97 94 In eoslaopbits 73, 75, 109 "Couple reaction mechanism 10 seuntisation prei ention of antigua spreading, 10, 12 15 16 23 105 48, 106 (rtopinam structure in sub-microscupital analysuy types of See Antibody types 19 Versus enzymatic coarPpt of allergy 44 Cytoplasmic genes role in adaptation of enzytoes, 25 urgentative cells a source of eosinophils 13 intestinal tract, 72 85, 88 Nenerbic acid in adrenals 49 Atopie reanu, 17 De-antigenisation of proteins, 62, 61 66 67 Augmentation Depression of antihody production, 10, 13 of toxic enzymatiesystent 10 De sensituation, 10 16 Autonomic nervous system role in eosinopedia 95 anophylactie reartion, 25, 28 32 105 DeAcrycholic ald, 53 centres in the brain 48-49 Desoxy corticosterone acetate as exinopeale hot Azn DORIN staining 81 85 mode 91 DR metabolisin, 53 A70-proteins as aptigen, 3 4 redurtron Determinant Partenia agglutination 6 81, 84 Diet atfertung eosinophilia, 62 63 antig air, 62 63 "Bindmg planes thenry of enzyuttir action, 6. de antigenised 62, 63 Blochemical phenotype 9, 29 protein free 61 Biopsy, 63, 84 81 83 81 raw meat 63, 64 Blood-elotting method, 83 ten essential amino acije 63 vitainin supplement, 63 coagulation in anaphs lactie shoh, 31, 75 Dilution foreigla, 82 Dog. adrenalectomised 40 51, 93 fransfusion as shock-precipitating factor in dog. blood eosinophilia 62-63, BI jejunal perfusion 78 82 83, KX-91 Bone marrow eosinnphil count x0,4 100 tissue cosinophilia, 63 84, 79 fosinophil formation In 80 61, 68, 74 17. ins Bridge formatmathenry of enzymatic Action, Broocholat constriction In guinea pig. Ectopic desings ia anapluyla 37 mational trauria as winnpeble factor, 74, 91 Lachtlema cellotar, 18 Catalolic phase of prutein Dietabolum stimulated Fodogenous coridopenia 94 p3 by corticosteronis, 50 58 58-5y, vs 97, Enzymatic capcept of anarhFlasla and alergy. 7. 44 Fozy See proteolytic enzym and adaphants Cava iliac pochet ar method of Assessment of Londolseis hormonal, 54,89 9 100 miectunin 54, 97.0X100 corticosteroids action on a bite blood celle | Cellular cortical fellestra. 62, 63, 66, 67 ·118 WOEY Eosinopols, clreutating, 7, 94, 95, 96 dlurnal ook nopedir cycle, 01-93 Edalnophil, origin in intestinal tract, 70, 103 tosinopeale tekt, 49, 78, B3 pathogenetic signul cane af, 98 87. 103-104, 105 factori leading to a CTH, 49, 37, 78, BO, B4, 91-98 110 ifrenaito. 49, 78, i sympathetien adrenal discharge effect on, 4,9 Tennis chloride, 18 transstionat form 7, 8, BX 72-73, S4, ** 86, 8= Cosyrested cost eceteroids, 45, 50, 85, Eosinoph& Reseat of testo, 83-84,79 , 1049 diets ettect on blond, 62-84 Chiricals. 78 on tegne, 287 Opicky pala, 78 heparin efect on blood 69, 70, 78, 98 notional trapia, 73 og tissue, 60, 78 98 97, 98 fednite conditions, 78 latestiral sinophalla, effects of dlete, 61, 63, faretan protein parenteral injection, 73 $1,63 homolyans intravascular. 79 pRects of bepatan, 35, 69, 76 Porthago, arute, 79 etects of heparin plus edatnopeaic Harone, bypertensive crisis 78 68, 76, 98-97 fotos, scute, 78 machapiam of evainophilsatlon, 61, es, as Etusulin typoglycemia, 77, 78, RO fource of loss.nopatsientides, Argent Cartoe nella 75 Ndum bluarlopate lajection, 74 85 stars stog, 78 izvezlan Oy blood erinopil, 71 turpestine Injection, 78 local foration 60 83, 109 mechanism of, 49, 50, 78, 80, 88 sersetory action of intestinal epithelium, 72 octurnal Onitopenic cycle, 92-95 83, 109 Dout-adrenaline ia adreuatectomised and bypo. sue, assessment of, 79, 06 97 phymectomized namata, 95 seeytiat 9.9ino acids, 03 Preventious of, buy heparla, 87, 75-78. 89-92, Facer brllge, 19 99-100 Estrage in serum la anaphylactic reactioa, S7, 40,54 Eovinopenic hormones, action on eosinophals, in they bridge, 19 ritro, 80, 82, 03-84, 87, 90-260 kucoston, osigolytic action, 83, DO un tird, 60 70, 80, 81, 80, 91-94, 97, 99-100 Extrftssic action of hostamine. 36. 107 Action on white hood cells in euro. 50, 8, 89, phase of anaphylactic shock. 33 100 50, 91-98, 97, 98-110 No, 50, 70, 0, 0, 02-84, 87, 99-100 effect og liseu fusapplus, 75, 90, 91, 95-97 Estasenta u abord-precipitating factor, 3.6 Inactivation by heparin, Tó 307, 108 moediation in proceso metabolism, 58, 87-88, 199 Fatty acids halogenatron 55, 105 108 test, 49, 78, 89 hydrogenation, 63 65, 107 108 Eoloogh, Abeokute count. 70 Ox}dation, 53, 55, 102-104 fo alimentary tracta dos 60-63, 0, 71, 78, 89-97 unsaturate tole 1» protein Detshoes, 30, 37, iu mice, 60, 74 52, 50, 10 * Rrebold motit, 5, 72, 73, 86, 97, 101 Febrile coucions as coopenic factor, 78 Anatolio phase . 81, s. 101 Por of inflammation as wounce of antiga, 60 Anapas lactic reaction, source of 303, 309-110 Foreign protein, 6 22tgen asbody reaction sole in, 10. 73, 75, 109 A4 antigen, mechanenu, 4, 10, 1963, 62, 72, 73 202, 207 34 antigen carriers, 74, 77, 101-103, 109 Arrest ta boze-KRATTOW 81, 8+ corporation to protes of invaded selle * latestinal tract, 35, 73, 75, 77, 84, 83, 69-97, 109 61, 73, 163 liver, 80, 8+ June, 80, 84 lymphold barrier, 80, $4, 98-37, 203 famma globulo reaction after atrassons tafection skeletal muscles, 80, B4 40, +2, 55 akin, 80, 84 Globulaz leucorytes 83 Spleen, 60, 84 Guinea-pix, anaphylactic shock, 29, 38 stomach, 85, 86 Desitonitis, 10.1 etbeutaneowe tissue, BB, 8+ tasauc eoninops, 63 catabolic phase in, 62, 97, 99-100, 101 circulating eisinophul count, B4, 78, 79, 8.5 Classdca tipa ok," puto Pora, 61, 62, 74, 77, Rapters, 109 Hæmatoge sous spread of soligra "true" fora, 01, 74, 75, 103 HPXoconcentration ta shoes, SI corticosteroids effecta on. 49 Hæmorrage rouldopeale factor, 78 in mlrd, OL 94, 97, 99-104 Hemo-transmitter, 49 in 100, 90-94 Homologous penteia as antigen, 37. 43 Otranting methods, 79 Homomolar cohesive bonus, 19 dormigenisation-antige Alsation ycie 1 Kelly'a modification of Brative, 81 102-104, 109 Lepasis to anaphylactic shock, 35, 10.8, 107 dieta relation 1, 63-64 utseoagulant dosage etecting cheulating could dilution forma in eaglaophit unta, 82 phila 15, 76, 82, 09 carly forms of, 61, 62, 63, 68, 71, 72, 84, 85 biorking action of biood triptic enzynors, 44100 Lactors opditioning cosloophulaatios, Bi cary cotitze due to debuit of 35 Stuctisation of, 44 de-paristos of proteins, 35, 75, 101, 107 Posinopenio test asteeted by, 89, 75, 79,99 formation of, adaptive enzymes role in, 61, 73, 109- 209 masele dosagt effect or coinopsil tr dog, 78. 82, 83,98 g 100 boodstuping for formatiat of, 01, 201 ID #3, 64, 76, 94-100 Baratogosette, 60, 61, 77 mohilang efect of these bosinoptils. 35, 764 non-twematopatetie, 60, 63, 63, 64, 72, 77, 88 94 100 Isoch of, 98, 99-97. 101-105. 105 Immobitiostoa ofIn tissue, 35, 70, 80, $4, 97, In peptose shek, 35, 45 presration of colopenia hy. BR, 25 16 8109 Bereday or stiergy tud asphy kuk, , Jos Neterologous protein, 16, 17, 37, 13 lympiad hamler relation 1. 35,80, 98 97, 101, 107 Metropolet como loads, 19 Dobiustit by beparja, 79, 70, 9, 97, 109 fe spador, 103 ·INDEX 119 Hutaminase, 86 Histamine role in allergy and anaphylaxis, 38, 40, 44, 56, 107 Histidine decarboxylase, 36 Histochemical structure of cell, 18, 101 Humoral pathways, 49 Hydrolysis See Proteolysis in adaphylaxis Hypercalcæmia 31 Byperheparinamla in anaphy lactic shock 31 Hyperfustam Dæmia in anaphylactle shock, 31 Hypertensive crisis As eosinopenic factor, 78 Hypoglycæmia as eosinopenic factor, 78 Hypophysretomy in dog, 83 Hypothalamus as relying organ, 49-49 Lymphoid tusspe around portal of entry of antigen, 10, 35, 95 97 101 barrier against antigen invasion, 6,7 10, 66, 64, 96-97, 101, 100 involution of, 50, 54 role in intestinal tract, 35, 66, 73-75, 80, 96 97, 101, 109 Lymphopena, 49, 79 Immobilisation of coenophuls in lymphoid trseve, 35, 75 100 103 Immunity, active 11, 33 phyllopathological formula of, 12 passive, 17 Infections as eollipopenic factor, 78 Inttarination, difference between allergic and non-allergic, 39, 40 focl a source of antigen, B0 proteolytic enzymes in exudates, 39, 57, 58, 100 Ischemic necrobus in allergy, 35 Insuha hypoglycemia as Bosinopenic factor, 78 technique, 84 Internal haemorrbage as compopenic factor, 78 Intestinal tract, absorption of proteins from, 62 63, 73 biopsy, 63, 64,80 crypta, 65, 74. 85, 89, 109 digestion of proteina, 62 109 epithelial cells eosinophilisation, 62, 68, 70, 75, 87,88 lymphoid barner of, 35, 68, 73-75, 80, 96-97. 101-102, 109 un place of eosinophils formation, 61, 70, 77, 84 88, 709 perfusion in dok 78, 82, 83, 88-91 portal of entry of antigena, 61, 62, 96-97 worus treatment in dog. 80 Intrinsic action of histamine, 38 107 phase of anaphylactie shock, 31, 106 107 Ion metal na bridge in specific proteolys, 18, 24 Ischemic DeCrOBIS, 35 Mammillary bodies, 49 Meat-raw dret. 63-64 Median eminence 48 Mue, antigenic diet effects on 66 bone marrow eosinophiha, 68, 68 de-antigenased diet 68, 88 intestinal eosinophilia, 66, 67, 72 spleen eosinophula 66 67 68 Micro-anaphylactic shock, 31 Microsomes, 3 Bruk as murce of antigen 68. 69 Mitochondria, 3 Mitosis effected by corticosteroids 58 Mohilisation of tissue eosinophils, 73 76, 91, 97, 109 Molecules of Spoids rod-shape in optil membrane, 18 of protein, size rule in antigenicity, 21 Abrillar form of cellular structure, 19 Monocytes, 89-90, 91 Necrosid, 39, 107 Nervous pathways in potwtary adrenal responue, 48 Neuro receptor role in pituitary-adrenal response, 18 Non speciale protein therapy, 41 NPN ID anaphylactir shock, 31, 37, 38 in blood following corticosteroids administration, 50 ID peptone shock, 31, 37 Nacirus destruction in leucocytes, 50, 72 90-91 geDPS s precursors of enzymes 25, 105 organising factor of enzymes, 17, 25, 28, 105 mitosis in, 57 68 primary upset ag source of sensitisation, 29 Jacobson's method of staining, 8 Kaolin suspension administered intravenously 28 shock preripitating factor, 107 Karyolyne, 60, 90 kuitchitzky celle," 63 Operatind surgical sy source of endogenous antigen, 2, 89, W, 100 Opeonin, 5 Ovolecithin, 54 Orycorticosteroids See Corticosteroids Oxygenation of fatty acids 53, 55, 107, 109 Papeth cells B3, 66, 68, 71 Pepein gastric optual pH. 23 Peptone chemical structure 20 metabolism of 31, 55 shoek difference between anaphylactic and, 30, 40,55 done or, 30 32, 39, 41 mechanism of, 30 40, 4135, 197 Perfusion, intestinal in dog 76. 82, 89, 99 Periodical fenestration or otuar membrane, 19-22 Permeability of capitarles, in shock, 31 factor, 39 106 Phagocytosis of szo proteiza, 3, Phenotype biochemical 125 Phoepholipolds in blond 61 Pieremeal type of protenlysis 27 Pituitary adrenal response 47 terky and supbylasia, 15, 16, 18, 40, 47. mechanism 13, 17 velomty, 16, 94 PlaamA volume in sprek 31 Polypeptide chains, diagrammaste structure. 19 eod and wide-ground or, 19 moser rate of in cell, 19 21 Dov toxic atabolites, 8, 97 Post-operative Labolk phase, 63 Praise ketare phenomenon, 17 PrecinciD 5L, 62 Protein beorption from alimentary cadal, 6, 73 fod of lipmation, 59, 60 Lamina propda mucose of intestine, 70-72, 74, 84, 85 88, 96 Laparotomy in dog, 63, 80 Lecithin affecting corticosteroids actioo, 54 Leucocytes involution caused by corticosteroids, 60 59 Leurocytosis polymorphonurlear, B8, 70, 90, 100 following cortisode injections 5. 100 in perfuslon, 68, 70, 88 91, 94-100 promoting factor, 39, 100, 107 Leucopenia 81, 89 Leucopenic factors 39, 100, 107 Leucotaxin, 99 100 1017 Lipolds in blood, 51, 58 fatty acids unsaturated, 97 103 Lipoprotein conjugation, z2, 29, 37, 39, 41, 42, 51, 108 disruption of, 29, 32, 33 39, 41, 42, 55, 67, 103 Local Buation of antibodies 6-7 lomualty mechanism, 8 7 Lytophocytes 49, 50, 75, 89-91, 97 Lymphold tlakue, entibodies release from, 6, 7, 10 16, 18, 33 109 ant Igen la relation to, 9, 10, 101, 109 50 97 ·1 ! RO INDEX Protelu altered as antigen, 9. 29, 20, 73, 103 Salt formation by protein, 29.24 as antiboxiles, Ralts la dog diet, 63 aptigen city of. 21..? B), 73 break-down products 11. 36. 105 Schultz-Iale test 103 Sensitisation in ce) strurture, 9, 18-22 28, 33, 61 see also anaphylactic ren de-antigentuation of, 68, 72, 75 Anaphsla tl adaptive enzymes, rote in 11, 19, 25, 26 10 of entfmes, 0, 21 furtign, 48 antigen, 9, 21, 61, 62, 72, 73, 101, 107 in alergy and anaphylaxis 1, 10, 33, 103 10 incorporation in host-cell, 61, 73, 101 antibody. Tote in, 1018, 106 heterologous, as antigen, 16, 37, 43 Aptigen, role in, 10, 33, 46, 106 homologous, 19 antigen, 37 13 antigen-antihody reaction, role In, 10, 12, 1 100 in lipoprotein conjugatlon, 22, 32, 10% * couple reaction" mechanism, 27, 33, 105 marked with azo-dye, 5, definition of, 2 radioacthie 11 cle-sensitisation, 16, 33 orolecoles as coiting agent 21, 33, 61 Tenetrating rell Interior 19, 31, 61, 62, 101 foreign protein, role in, 10, 11, 12 formonla of, 2, 28 prokluct or antigen antihody reaction, 5, 10 "high sensitivity." of experimental aplms. replica of antigen, 26, 61, 73, 101 ** Low nepatity" of experimeptal animals Apecificity of, 6 siructure S kuhinterperopea) ce morphology, lymphoid tissue, role in, 11, 14 mechasm, 7, 9, 11, 22, 16 18-22 by rapid spread of antigen, 10, 14 #3Dthes) intracellular, 28, 61 73, 102 hj slow'spread of antigen, 9, 10 Proteolysis Accelerated by anaphylactic reaction, 29, molecular size of protein in relation to 21 32, 97, 55, 56, 589 DRUTE 17 corticosteroids, 50 51, 62, 53, 54, 58, 96 37, 106 disrupting or ilpo-protesa coafugation, 20, 20, period of, 12, 40, 2, 106 39, 53, 55, 5 **phs slopathological formula of Imanthit." production of specific antibodies in, 2 3 0. fat solvents, 57, +3, 56 12 14, 15, 18, 33, 43 Ramnia globulin Intravenous Injection, 40, 42, Bensiting dose of antigen in, 2, 3, 12, 24, 101 55 shock-dose of antiken in, 3 12,2 32 IOLIO Deptone niolecules, 30-32, 41 specific toxic enzyme systery in 2,0 28 32 11 surrace-acting forces 22, 1, 4, 5, 6, 109 state or equibrium" in, 12, 13 trypsia shock, 40 42, 44, 55 Serimi sickness, 10 "all or none" t3 pe 27 Shock bee Also Anaphylactic reaction in anaphylaxis and allerg), 7, 10, 11, 22-23. 29, 32, anaphylactic, proteolytic power of seruni 5 38, 40-42 55-36, 58, 107 32, 34, 40, 52 chaotic, 29 32, 37, 41, 46, 51, 59, 107-108 in eostnnphils, 61. 96-97. 99-00 109 comparison between enophylactic and pep extracellular, 28. 29, 32, 37, 41 46, 51, 59, 31, 55 107-108 dose of antigen, 32, 33 46 homopal 50. 51 52, 57 heparin in anaphylactic, 31 34, 44 initiated by chemical agents, 30, 37, 51, 55, 58, 107 lustamine in anaphylactic, 31, 38, +4 by physical agents, 30, 31, 32 55, 58 107 Decro-aparlactic, 34, 103 by physico-chemiral agents, 30, 32, 37 organs, 9, 10 42, 48 55, 58, 107 peptone, 30, 99, 44 intracellular, 25, 28 32, 34, 37, 41, 48, 51, 58, Saclum thlopentone anpathenia In der, 81 81, 96-97, 107-209 Speclic enzy wies definition % 22, 308 Tlecement type, 27 Spleen aspiration biopsy 80 24 of specto antigen. 23, 37, 38, 108 eosinophilia in mice, 67 toxic, 3, 28, 32, 41 42, 46, 56, 57, 100 "Suction force" role in nenaltration, 21 fo white blood cells, 50, 51, 53 9 100 phu hoidae in protein walts, 19 Proteolytic enzymes, adaptation of, 11, 20, 61, 73, Surface-acting forces, disrupting lipo protein mol 105 cules, 22, 297 42 Ageing of, 101, 103, 110 & whock precipitating factors, 20, 37 12 5 fa apaphs lactic shock, 26, 32, 37 58, 103 As antibodies, 8, 106 ympathetiro-adrenal discharge, 44, 04 As Cathepsins, , 10, 23 classiteation of, %, 22, 108 definition of. X, 106 Thrombosis Mechanism in aanphinctie reaction, 3 distribution in the body. 7 Torir antibodies, 9, 17, 28, 44. 16 formation of toxic enzyne system, 0, 17, 22, enzymes & i7, 22, 28, 32, 4, 4410.6 23, 25, 46, 106 106-107 sinas às25, 28 Try tejo nepana complex, 95.160 of granulation tissue, 57 Try prin, intestinal difference between Intracellula intracellular, 25, 29, 98-97 enzymes, 23 of lymphoid twie 11, 14, 50 Tuberculosis dissemination ander armunstration o nucleat organising factor, 17, 33, 23 cortimeterolis, 68 proteinases, 8, 10, 11, 14, 27, 33, 33, 13 Turpentine injecto colopenic factor, is external, 20 internal' 20 in senzitted cell 10, 28. 105 speriticity of, 7, 8, 23 27 28, 103 toga, 11 11, 23, 27, 29, 105, 106 of shite hood cells, 50, 51, 5A, 61 Dk1068, 50, 90 Pyrexin, 39 107 Vinis as antigens, 13, 20 tama minture sur plebent in dogs liet, 03 1 Witte peptone, 39 Rabbit Anaphylactie sex h, 33, 51 hypophy 819, 13 Retieulo-edkythelial oystem role in transportation of artiged, 3.0 Zenters flsatire, 1 7 aragon grupules in epihlal relle et intestinal tract. 70 Prated Great Brave at THE DAILY PRESS Lro, Edinburgh

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