ANTIGENIC MODIFICATIONS OF ALTERED TISSUE ANTIGENS

AS TYPIFIED BY VIRUS-TREATED ERYTHROCYTES

DISSERTATION

Presented in Partial Fulfillment of the Requirements

for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State

University

By

NANCY JANE BIGLEY, B. S., M. So.

$$$$>}($

The Ohio State University

1957

Approved by:

'hdrtf-.d. Adviser Department of Bacteriology ACKNOWLEDGEMENTS

The writer wishes to express sincere appreciation to

Dr. M. C. Dodd for his help and giidance throughout the course of this investigatioh.

The writer also wishes to thank Dr. F, W. McCoy and

Dr. W. Zeman for the histologic examinations of tissues so necessary in this work; Dr. M. S. Rheins, B. E, Kirk, M. Sc.,

R, W. Chandler, M. Sc., and W.B. Wacker,M. Sc, for their time and interest in this work as well as their valuable technical TABIE OF CONTENTS

Page

INTRODUCTION...... 1

LITERATURE REVIEW...... ^

MATERIALS AND METHODS...... 29

EXPERIMENTAL RESULTS...... ^

DISCUSSION...... 200

SUMMARY...... 2U5

BIBLIOGRAPHY...... 2l±7

AUTOBIOGRAPHY...... 2$7

iii LIST OF TABIES

Table Page

I Titrations of Anti-Rho(D) Saline Agglutinin with Rho(D) Red Cells Treated with Periodate, Trypsin, and Mumps Virus 50

II Comparison of Titers of Normal and Modified Erythrocytes in Antisera to PR8 and MDV- Treated Human Red Cells 52

III Comparison of Titers of Normal and Modified Erythrocytes in Antisera to Normal and Mumps- Treated Rad Cells 56

IV A Further Comparison of Titers of Normal and Modified Erythrocytes in Antisera to Normal and Mumps-Treated Red Cells 58

V The Effects of Heat on the Stability of Cell Agglutinins in Antiserum to Mumps-Treated Human Erythrocytes 62

VI Titrations of Antiserum to RISC-Treated Human Erythrocytes with Virus-Treated Red Cells 65

VII Titrations of Antiserum to Eluate from RDE- Treated Human (0 Rho) Erythrocytes with Virus-Treated Red Cells 66

VIII Titrations of Antiserum to RDE-Treated Chicken Erythrocytes with Virus-Treated Red Cells 67

IX Titrationsto Eluate from RDE-Treated Chicken Erythrocytes with Virus-Treated Red Cells 68

X Comparative Titers in Antisera to Human and Chicken Erythrocytes Treated with RUE and the Eluates from Such. Cells 69

XI Antibody Response to Shock Doses of Mumps- Treated Red Cells in a Rabbit Passively Sensitized with PR8 Absorbed Antiserum to the Homologous Cells 73

XII Titrations of Rh Antisera with Normal and Treated Red Cells 75

iV. Table

XIII Inhibition of Incomplete Rho(D) Antibody by Eluates from Rho(2) Cells by Mumps Virus

XIV Inhibition of Anti-Kho(D) Albumin Agglutinins by an Eluate from Rh2 (cBE/c— ) Erythrocytes Treated 100 Minutes with Mumps Virus

XV Inhibition of Saline Agglutinins Anti- 1 and Anti-CD by Eluates from Rh^cBE/c— ) Erythro­ cytes Treated 10 0 Minutes with Mumps Virus

XVI Inhibition of Anti-Rho(D) Saline Agglutinins by Eluates from Rh^cBE/c— ) Erythrocytes Treated with Mumps 'Tirus

XVII A Comparison of Inhibition of Anti-CD by Eluates from Mumps-Treated Rho Positive and Negative Stromata,

XVIII Inhibition of Anti-Rho(D) Sera by Eluates from Rh2 Red Cells Treated for 30 Minutes with Periodate

XIX Inhibition of Albumin Agglutinins Anti-Rho(D) by Eluates from Mumps-Treated Rho Stromata

XX Inhibition of Anti-Rho(D) Saline Agglutinins by Eluates from Mumps-Treated Rho Strornata

XXI Inhibition of Anti-Rho (D) Albumin Agglutinins by Eluates from Mumps-Treated Rho Stromata

XXII Inhibition of Anti-Rho(D) Albumin Agglutinins by Eluates from Mujjps-Treated Stromata

XXIII Inhibition of Anti-CD Albumin Agglutinins by Eluates from Mumps-Treated Rho Stromata

XXIV Inhibition of Anti-Rho Sera by Eluates from Rho Stromata Treated 60 Minutes with Periodate

XXV The Effect of Increased Temperatures on Absorp­ tion with cde/cde cells of Eluate from Mumps- Treated Rho Strcmata Table

XXVI The Effects of Storage on the Inhibition of Hyland Anti-Rho(D) Albumin Agglutinins by an Eluate from Rho Stromata Treated for 10$ Minutes with Mumps Virus 93

XXVII The Effects of Storage on the Inhibition of Hyland Anti-Rho(D) Albumin Agglutinins by an Eluate from Rho Stromata Treated for 28-| Hours with Mumps Virus 99

XXVIII The Effects of Storage on the Anti-Rh Inhibition of an Eluate from Rho Stromata Treated for 30 Minutes with Periodate 2.01

XXIX Inhibition Titrations of Anti-Rh Sera with Eluates from Erythrocytes Treated with Other Viruses and Enzymes 1(32

XXX inhibition Tests of Anti-c, Anti-C, and Anti-E with Eluates from Mumps-Treated and Periodate- Treated Rho Stromata

XXXI Precipitin Tests with Mixtures of Eluates From Mumps or Periodate-Treated Erythrocytes and Anti-Rho (D) Sera 2_q6

XXXII Precipitin Titrations of Rho Antisera with Eluates from Mumps-Treated Rho Positive and Negative Stromata 1 08

XXXIII Enhancement of Agglutination of Normal Rho ( I)) Cells in -dilutions of Specific Antiserum by Eluates of Erythrocytes Treated with Periodate or Mumps Virus

xxxrv The Effects of Anti-Rho(D) on Hemagglutination by Mumps Virus -jj_0

XXXV Titrations of Saline Anti-Rho(D) with REE-Treated Red Cells 11)|

XXXVI Titrations of Anti-Rho(D) Serum with Normal and Virus-Treated Rho(E) Positive and Negative Erythrocytes. Table

XXXVII Demonstration of Hemagglutinins to Virus-Treated Rho Positive and Negative Erythrocytes in Anti- Rho Serum after One Absorption with Rho Cells 119

XXXVIII Titrations of Anti-Rho(D) Serum with Rho Positive and Negative Erythrocytes after One Absorption with Normal Hr Erythrocytes 122

XXXIX Titrations of Anti-Rho(L) Serum with Rho and Hr Erythrocytes after One Absorption with Trypsin- Treated Rho(D) Erythrocytes 12li

XL Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with Trypsin- Treated Hr Erythrocytes 127

XLI Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with Mumps- Treated Hr Erythrocytes 132

XLII Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with PE8-Treated Rho(D) Erythrocytes 133

XLIII Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with PR8-Treated Hr Erythrocytes 1314

x u v Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with NEV- Treated Rho(D) Erythrocytes 135

XLV Titrations of Anti-Rho(D) Serumwith Rho and Hr Erythrocytes after One Absorption with NDV- Treated Hr Erythrocytes 139

XLVI Effect of Virus-Treated Cell Antibody on the Agglutination of Rh Positive Cells by Rh Anti­ body lit?

XLVII Titrations of Anti-Rho(D) Albumin Agglutinating Antibody with Normal and Virus-Treated Red Cells 1U9

XLVIII Titrations of Anti-Rho(D) Albumin Agglutinating Antibody after Absorptions with Normal Red Cells 1$2

vil Table Page

XLIX Titrations of Anti-Rho(D) Albumin Agglutinating Antibody after Absorptions with NDV-Treated Erythrocytes

L Inhibition and Enhancement of Agglutination in Anti-Globulin Serum of Red Cells Sensitized by Anti-Rho(D) Serum and Serum to Virus-Modified Cells 160

LI Modified Cell Agglutinins in Normal and Hemolytic Anemia Sera 166

III Titrations of Saline Anti-Rho(D) after Absorptions with Pneumococcal Polysaccharides Types I and III and Their Respective Tronic Acids 170

LHI Latex Fixation Reactions with Serum and Eluates from Red Cells of Rabbits Injected with Uronic Acids 1 7 8

LI7 Attempts to Demonstrate Agglutinins for Normal and Glucurone-treated Rabbit Red Cells in Serum Globulin of Glucurone-injected rabbits 180

LV Altered Reactivity of Erythrocytes from Rabbits Injected with Response of Galacturonic Acid 182

m i l LIST OF FIGURES

Figure

1 Electrophoretic Patterns of Serum 18 Days After Injection 18U

2 Electrophoretic Patterns of Preliminary Serum 18^

3 Electrophoretic Patterns of Sera from a Rabbit Injected with Alpha-D-galacturonic Acid Incorporated in Freund's Adjuvant 186

ix INTRODUCTION

For autoimmunization to occur it is necessary that some normal body component be sufficiently altered so as to assume antigenic properties. This change or changes may be evoked by the action of viruses, bacteria, enzymes, adjuvants, or combination

■with foreign substances or some other modifying factor. Thus, it has been postulated that in rheumatic fever and other of the so- called collagen diseases, hemolytic anemia, allergic encephalo­ myelitis, and certain opthalmic diseases, cells have been altered by microorganisms or abnormal metabolic events. Such a process may play a critical role in initiating an immune phenomenon, resulting in antibody to normal tissue components.

Because of the many alterations produced in red cells by the action of certain hemagglutinating viruses, both in vitro and in vivo, virus-treated erythrocytes have been extensively studied as possible initiators of both naturally-occurring and experimental hemolytic anemias. Among the alterations produced in red cells by viruses of the mumps-Newcastle disease-influenza group are modifications in electrophoretic mobility, receptor activity, antigenic specificity, and serologic reactivity. The latter two modifications are most pertinent to the work presented here.

Burnet et al. (19U6), Wallace (1953), and Evans (1955) have shown that specific antigenic alterations occur in chicken or human red cells treated with either influenza or Newcastle disease viruses.

More recently, Bigley (1955) has shown a similar effect with mumps

1 2

virus and perhaps of greater significance, the fact that antibody to such mumps-treated cells is also an incomplete hemagglutinin for normal red cells. Similar changes also have been demonstrated by Gardner (1955) to occur in vivo in chickens infected with

Newcastle disease virus (NDV). Likewise, the serologic reac­ tivity of virus-treated Rh positive erythrocytes in specific antiserum has been shown to be altered since such cells are frequently agglutinated in saline by the incomplete form of this antibody. The pertinency of this phenomenon to the problems of autoimmunization is suggested by the recently reported fact that autoantibody in some cases is specific for Rh antigens.

In addition, Gottschalk (1953) has reported that the muco- protein of virus inhibitor is altered by virus action, liberating products composed of a carboxypyrrole structure and various hexose residues, such as glucose, galactose and their amines. These are similar to those released from susceptible red cells by comparable action of virus. Considering the wide distribution of such compounds in various tissues of the body and the fact that muco­ polysaccharide depolymers are liberated in the disintegrations of "ground substance" or tissue matrix in many diseases, includ­ ing the collagen diseases, the possibility of such substances as autoimmunogens must also be considered.

The work to be presented consists of three phases: l) the further characterization of the antigenic specificity of virus- 3

treated erythrocytesj 2 ) some aspects of the altered reactions of such cells in anti-Eh sera; and, 3) the effects of injecting combinations of uronic acids and adjuvants into rabbits. Although seemingly diverse, these aims were derived from experimental approaches directed at demonstrating that tissue alterations, typified by virus-treated erythrocytes, do evoke immunologic responses, although these are not usually demonstrable by clas­ sical techniques. Similarly, the injection of uronic acids may be justifiably considered analogous to the situation created by viruses and bacteria which have left altered tissue structures and components in the wake of their pathogenic activities. These may then serve the role of autosensitization. LITERATURE REVIEW

In practically all of the examples of autoimmunization contain­ ed in the literature, an alteration of the antigenic material as a result of virus action, autolysis, bacterial action, combination with foreign substances, or some other means is necessary for this phenomenon to occur. Several workers have attempted to elucidate the mechanism for the initial antigenic alteration of erythrocytes responsible for the subsequent appearance of autoantibodies in the sera of certain acquired hemolytic anemia patients. It has been demonstrated that red blood cells of many acquired hemolytic anemia patients were specifically agglutinated in the presence of antibody to enzyme treated cells (Dodd, Wright, et al., 1953).

This group also demonstrated the presence of trypsin cell antibody in the sera of some of these patients (Bouroncle et al. 1931).

More recently Crowley and Bouroncle (1936) have corroborated the findings of Weiner et al. (1933)> Dacie and Gutbush (193U), and

Hollaender (1933) that some patients with autoimmune hemolytic anemia may form antibodies against specific blood group antigens present in their own erythrocytes. In twelve cases reported, they found anti-e specificity in the sera of two individuals and anti-D as well as anti-E in the serum of a third individual.

Stefani, Magalini, and Patterson (1936) have recently suggested that neoplastic tissue may represent an antigenic

u 5

stimulus for the formation of antibodies against formed blood elements and indicated that antigens common to malignant cells and blood cells are responsible for the development of such antibodies. Two patients with metastatic prostatic carcinoma and one patient with metastatic breast carcinoma, previously splen- ectomized with unsuccessful results, were studied. Tumor tissue was available through surgery, while fresh autopsy tissue served as control. The patients' sera were absorbed with the indivi­ dual's own malignant tissue, malignant tissue of identical histologic type from other donors and normal tissue from indivi­ duals with identical blood groups. The platelet agglutinin titers decreased consistently and markedly after absorption of serum with the patient's own neoplastic tissue and, in some instances, with homologous neoplastic tissue. In contrast, the decrease in such sera of platelet agglutinins was inconsistent and insignificant when absorbed with normal tissue. The auto- and iso- erythrocyte antibodies in a patient with a reticulum cell sarcoma were absorb­ ed by tissue from tumoral nodules and not by the surrounding splenic tissue. On autopsy, extensive involvement of all organs was later shown. In several instances, thrombocytopenia and, in one case, leucopenia were transitorily reproduced in normal individuals receiving intravenously 2j?0 ml. or more of such a patient's plasma.

Stats and Wasserman (1952) suggested the possible virus 6

etiology o.f certain acquired hemolytic anemias and Mooten and

Clark (19^2) isolated Newcastle disease virus (NDV) from the blood of a patient with acute hemolytic anemia. The latter workers subsequently reported the isolation of NDV and uniden­ tified viruses from the blood of patients suffering from various blood dycrasias. Thus, it has been postulated that virus al­ teration of erythrocytes could also function as the antigenic stimulus for the modified cell antibody in certain of the acquired anemias by a process of autoimmunization.

In vitro alterations of erythrocytes resulting from the action of bacterial enzymes, proteolytic enzymes such as trypsin, and viruses have been observed frequently. Thomsen (1926) observed that red cells, previously contaminated by bacteria, were agglutinated by normal sera. Subsequently Friendenrich (1928) showed that this alteration of erythrocytes, which rendered them agglutinable in normal sera, was brought about by the action of bacterial enzymes. The antibody, present in the normal sera of humans and many mammals, which brings about this agglutination has been termed the "T" agglutinin by Burnet and Anderson (19U7)•

Lind and McArthur (19U7) demonstrated that this agglutinin is not an immune antibody. Burnet, Anderson, and McCrae (19U7) have shown that erythrocytes acted upon by Vibrio cholerae enzymes (RDE), Clostridium welchii toxins, and certain hem- agglutinating virus enzymes were also rendered agglutinable in 7

the presence of the ’’T" agglutinin.

In the hemagglutination phenomenon, Hirst (191*2) showed that cells, after absorption and elution of the influenza virus, were no longer agglutinable in the presence of the original virus.

Subsequently, Burnet, McCrae, and Stone (191*6) showed that a receptor gradient existed for the action on red cells of the mumps-influenza-Newcastle disease group of the hemagglutinating viruses. Following absorption and elution of one member of this group, e.g. influenza, the red cells were no longer agglutinable by the same virus or by viruses (NDV and mumps) situated lower in the series. This receptor gradient varies depending upon the species of red cell used and the strains of virus. These same workers also demonstrated that enzymes from Vibrio cholerae and

Clostridium welchii progressively modified red cells in the same linear order as do the viruses of the above mentioned group.

Anderson (191*7) showed that erythrocytes, treated with NDV o at 37 C, were agglutinated by the homologous virus antiserum, indicating residual virus on erythrocytes treated by this virus at body temperature. More recently, Evans (1936) infected chick embryos with both human and chicken erythrocytes that had been treated with NDV and washed several times. His "inelutable'1 virus was not removed from the cell surface with subsequent RDE treatment, nor did previous treatment of the erythrocytes with

RDE prevent the attachment of this form of virus. Moore and 8

Diamond (1956) selected out and cultured a similar form of NDV in tissue cultures of Ehrlich carcinoma cells, Burnet and Lind (1950) found that NDV was hemolytic for chicken erythrocytes above 2cP C and for human cells above 21+°G. When the receptors had been destroyed by RDE or by viruses higher in the receptor gradient, the cells were not susceptible to the hemolytic action of NDV,

In comparing the NDV hemagglutination with that of influenza the following facts are noted: less active clearing of the cell suspension when reading the agglutination by the pattern of cells

(Lush, 191+3) 5 rapid elution of virus and complete subsequent

stabilization of cells (Burnet, 191+2)5 and that treated cells

are insensitive to the agglutinating action of NDV and mumps virus but show almost unaltered susceptibility to agglutination by influenza viruses (Burnet, 191+5). In the preceding comparison,

human erythrocytes are slightly less agglutinable than are fowl

cells, but show all three features as clearly as the latter

(Burnet, 191+7). Evans (1956), using the factor of Donald and

Isaacs, calculated values for chicken cells of 83O and 1660 receptor sites red cell for influenza (PR8 ) and NDV respectively.

Anderson (191+7) reported the existence of two types of NDV particles: one type is able to free itself from red cells by

destroying cell receptorsj the other is unable to dissolve this

union. It was postulated that the stabilization of such cells

by these two types of NDV particles may involve two mechanisms\ 9

namely, destruction of the erythrocyte receptor associated with the elution of the more active particles and the blocking of the receptor by the retained, less active virus particle.

Previously, Barnet and Anderson (19U6) reported that the changed character of the NDV-treated cell was due to the adsorption to the cell surface of an agent other than the virus produced during the growth of NDV in chick embryo cells. Von Magnus (1953) has observed that when dilute inocula of both influenza and

Newcastle disease viruses are employed as seed, the relation between infectivity and hemagglutinating activity is remarkably

constant (one hemagglutinating unit by the Salk method equals

10^ egg infective doses). Under certain experimental conditions all of which involve the use of large inocula, virus particles are produced which are hemagglutinating but apparently non- inf ective. Such viral particles are termed '•incomplete" virus.

Whether incomplete and active virus differ in their actions at

the red cell receptor site is a moot point.

The mumps virus also demonstrates an hemolytic activity.

Gardner and Morgan (1952) demonstrated that the hemolysin of mumps is a labile substance with many of the characteristics of an enzyme. These same workers presented evidence that the mumps

hemagglutinin and hemolysin are separate entities. Inhibition

of the mumps hemolysin occurred without a corresponding decrease in hemagglutinating potency in the presence of urethane and hydroxylamine chloride. Reducing agents markedly enhanced the 10

hemagglutinating capacity of this virus while suppressing hemolytic activity. Lind (19U8) reported that the activities of the mumps virus are the result of the actual mumps particle. Furthermore, the red cell sensitizing agent of either mumps virus or NDV is essentially that fraction of virus particle which is capable of adsorption but not of subsequent elution. Levens and Enders

(19U5) demonstrated that rabbit antisera against viruses of influenza A and B did not significantly inhibit the hemagglutina­ tion of the mumps virus.

Burnet (19H6) and Burnet and Anderson (19U6) showed that NDV and mumps could be irreversibly attached to red cells. This was

established by showing that after treatment with the Tirus at

37°C cells were agglutinable by specific anti-viral immune sera and retained this agglutinability after treatment with receptor destroying enzyme (RDE). Similarly Burnet (1952) demonstrated

the irreversible binding of the influenza virus to the red cell

surface. This was shown by the capacity of the cells to agglutinate normal fowl cells, by agglutination of treated cells with soluble inhibitors, and by agglutination in immune serum previously treated with RDE. This phenomenon is not shown with active virus nor with human red cells.

♦ • • The chief implication of the work is that it points to the existence of tyro well defined stages in the union of enzyme (E) to substrate (S). There are hints of more than two stages but for simplicity only the two main 11

ones vri.ll be discussed. The first is common to both active and indicator virus, it takes place rapidly in the cold or at any temperature up to ij.0oC and it demands a considerable ion concentration in the medium. It is essentially reversible and almost cer­ tainly represents a dynamic equilibrium which only develops binding power sufficient to give demonstrable red cell agglutination when virus and red cell (or indicator macromolecule) are united by two or more such unions. Virus in the first stage of union can be liberated from red cells by RDE, by immune serum, and some indicator viruses at least, by a soluble inhibitor. The first stage is regarded as an un­ oriented attraction between enzyme and substrate mediated in accordance with Puck's hypothesis (1951) by cations adsorbed in definite sites on one or other reagent, most probably on the enzyme. The second stage or more correctly the final stage is one proceeding relatively slow at 37°C and much more slowly at lower temperatures and with lower ionic requirements. With active virus it takes the form of receptor or substrate destruction except in the case of NDV and the mumps virus where a proportion of the second stage unions give rise to irreversible union and hemolysis. With indicator virus there is an irreversible binding. The final stage is regarded as the establishment of a correctly oriented apposition of E to S resulting typically in enzyme-substrate union with activation and destruction of substrate with the reliberation of the enzyme. Where E and S are concerned the comple­ tion of chemical union gives an irreversible union and attachment of virus to the substrate carrier that can be broken only by gross physical forces. It seems highly probable that an intermediate stage of relatively firm orientation adsorption exists in which for one reason or other the final chemical union is delayed. This may be characteristic of some types of inhibitor-indicator virus union and is most likely the explanation of the failure of human cells to show reaction.

Burnet et al. (19U8) as well as Hirst (19U8) interpreted the modifying effect of low concentrations of periodate on the cell receptor and on the inhibitory mucoproteins in terms of the 12

well-known oxidation of alpha glycol groups in carbohydrates.

The presence of a sugar as one of the split products of inhibitory mucoprotein (Gottschalk, 19h9 and Gottschalk and Lind, 1951) supported the view that the carbohydrate complex of the muco­ protein was subject to attack by the viral enzyme. Chromatographic and spectroscopic analyses of the carbohydrate complex of the urinary mucoprotein revealed glucosamine, gaiactosamine, galac­ tose, mannose, and fucose as well as an acid-labile substance

(Gottschalk, 1952). This substance was later identified as pyrrole-2-carboxylic acid by comparing its properties with an authentic sample of the acid and by its catalytic reduction to proline (Gottschalk, 1952). This imino acid was found in all mucoproteins, inhibitory or not, and in the glycoproteins that

Gottschalk tested. It is present in the compound released enzy­ matically from inhibitors and is thought to be a constituent of sialic acid. Klenk and Lauenstein (1952) and Klenk and Faillard

(195U) showed that in bovine submaxillary gland mucoprotein, sialic acid contains two acetyl groups while N-acetylneuraminic acid (NANA) contains only one acetyl group. Either compound in the presence of alkali will form a U-hydroxypyrroline unstable derivative which through the stabilizing loss of a water molecule becomes 2-carboxypyrrole. In the same manner the split product of the urine inhibitory mucoprotein was then identified as NANA.

Gottschalk claims (1957) that from this work it has been proved 13

that the neuraminic acid in its mono-or diacetylated form is the genuine product released from inhibitory mucoproteins by the influenza virus enzyme. Gottschalk and Perry (1931) showed that the enzyme associated with the influenza virus is an intrinsic part of the viral particle, in that, 1 ) when the influenza virus was purified by three independent procedures, removing up to 99 per cent of the nitrogen of the crude virus preparation, the ratio of hemagglutinin titer to enzymatic activity was found not to be significantly changed; and 2 ) upon repeated attempts, they were never able to detect any trace of this enzyme in the host cells

(chorio-allantoic membrane) or in the normal allantoic fluid.

Gottschalk (1933 and 1936) demonstrated that concomitant with the enzymatic release of sialic acid from homogeneous bovine sub- maxillary gland mucoprotein, the sialic acid content of the re­ sidual mucoprotein was reduced to half that of the original muco­ protein. Burnet (1931) and Tamm and Horsfall (1932) showed that the same enzyme, now identified as neuraminidase, is present in mumps and Newcastle disease viruses. In a 1937 review, Gottschalk reported that neuraminic acid is present in various amounts in all virus hemagglutinin inhibitory mucoproteins and in a glycoprotein from the lipid-free stroma protein of bovine red cells.

The action of neuraminidase either as a portion of the viral moiety or as the exo-enzyme of RDE is the hydrolytic cleavage of the glycosidic linkage joining the keto group of neuraminic acid to D-galactose or to D-galactosamine or possibly to other sugars.

As to the function of the influenza virus neuraminidase, Ackerman and Francis (195k) have presented data suggesting that the enzyme plays its role in the liberation of the assembled viral particle from the host cell membrane. They were able to show that the liberation mechanism can be inhibited by a sulfonic acid deriv­ ative. This phenomenon of liberation-inhibition, however, is not observed when RDE is added to the virus-host tissue system after the initiation of infection. Similarly, heated-influenza virus though noninfective will adsorb to susceptible red cells but cannot elute from them. Ackerman and Francis state that "the function of the influenza virus enzyme is to facillitate the escape of the progeny from the surface of the defunct cell".

This concept is in agreement with the earlier presentation of Gottschalk (1953):

.,. From the studies on the nature of the virus receptor it was concluded that the first step in cell­ ular infection (by influenza virus) is the adsorption of the virus onto the surface of the host cell. It is fairly certain that the cellular receptors are conjugated proteins, the prosthetic groups being distributed in pattern form over the protein surface. These pros­ thetic groups are heterogenous polysaccharides; the tail of these polysaccarides contains a pyrrole-carboxylic acid joined most probably by an amide link to a hexo- samine residue. On the viral surface are areas with a pattern fitting the loose ends of the polysaccharides3 the chemical groupings building up these patterns are endowed with enzymatic properties. Adsorption of the virus to the host cell is the result of mutual attrac­ tion by electrostatic forces of the complementary areas at the virus surface and at the free end of the pros- 15

thetic groups of the host cell receptors. When both the polysaccharide and the virus enzyme are in their natural state, adsorption is followed by enzyme action on the amide link of the pyrrole derivative. If by periodic acid oxidation some of the alpha glycol groups of the polysaccharide are oxidized, adsorption of the virus to the cellular receptor occurs, though not followed by enzyme action. For the initiation of cellular infection, only adsorption of the living virus at the host cell is necessary. Enzyme action on the pros­ thetic group of the receptors is not a prerequisite for cell infection as demonstrated by infection of host cells the receptors of which were' modified by pretreat­ ment with periodate. Once adsorption has taken place, the virus is ingested into the cell, an act proceeding at a higher rate than the spontaneous elution.

Metalloporphyrins have been observed to inhibit competitively hyaluronidase as well as the hemagglutinating "aggressin" activity of partially depolymerized hyaluronate and also have been reported to inhibit the agglutination of human red cells by type-specific serum or by influenza B, Newcastle disease, mumps and vaccinia viruses. Examinations of the steric configurations of materials found to exhibit this range of activity revealed a common de­ nominator underlying all of them. Some antihistamines, heparin, and glucurone were specifically studied in this respect. (Barnard,

1951+). Barnard (195U) reported that the only chemical concordance among these diverse competitive inhibitors of viral hemagglutinins, iso-agglutinins and hyaluronidase is the resonating heteropen- tacyclic ring in each.

...The pyrroles of chlorophyllins are isoteric with the furanoid form of glucuronolactone, as well as thiophenes, thiazoles and imidazoles with anti-viral properties. It becomes permissible to assign a furan resonance contribution to hexuronate, hexosamine or hexose as these glycoid moieties are linked in ground-substance 16

polymers of either plant or animal. This premise of aromatic pentacyclic resonance configuration is further justified since chelation of transition element metal in a vinyl porphyrin enhances its antihyaluronidase, antiviral, antiaggressin, anti-thrombic and anti- hemagglutinative as ■well as its general heparinic activity, and it is known through magnetometric data that such chelation enhances pyrrole resonance.

On this basis Barnard advocated an hypothesis of viral multiplica­ tion and speculated as well on the manner in which photosynthetic aldol condensation "clips'1 at the hexose stage.

...Once assigned, a resonating furanoid structure for the ribose molecule or the saccharide moiety of intra­ cellular matrix has certain implications. The cell receptor to which hemagglutinating virus or other hemagglutinin or aggressin attaches must be the steric negative of the heteropentacyclic ring. As such it is a potential template for furanoid or pyrrole rings. Such a receptor could then constitute the agency by which any ground substance is elaborated. A similar templating mechanism could be invoked to explain the porphyrin production that occurs in megaloblastic and erythroblastic anemias. Cell receptor template could dynamically mould simple carbon atoms into sterically stable aromatic rings of sufficiently high resonance and consequent reactivity to be incorporated in chain (chitin, cellulose, chondroitin, ets.) or ring (porphyrin) polymers. This templating cell receptor, therefore, exhibits activity that is indistinguishable from that of an enzyme.

Equilibrium studies"(Barnard, 195U) showed that the hem­ agglutination inhibition by metalloporphyrins to be competitive, the inhibitor having the same configuration as "hemagglutinating antibody" thus enabling it to compete with the specific antibody for the cell receptor. Barnard has designated such inhibitors as "antibody configured haptenes" and claims that close scrutiny of the structure of this class of compounds predicates the 17

existence of an apposed class of "antigen configured haptens" which are usually macrocationic. It is feasible that such structures may well be hexose moieties and their amines which are integral constituents of tissues and tissue matrices.

Further studies of the sera of humans as well as sera from commonly used laboratory animals have revealed the presence of both heat-labile and heat stable virus-inhibitors. Hilleman and

Werner (1953) discussed two heat-stable influenza viral inhibitors in human sera designated as alpha and beta inhibitors. The alpha inhibitor is inactivated by the RDE of Vibrio cholerae; is most active against heat-killed viruses$ and presumably is the factor functioning in the Francis phenomenon. They described an alpha- inhibitor active against A, A 1, and B strains of influenza viruses.

The beta-inhibitor is not destroyed by RDE and is most active against unheated A prime influenzas isolated in 195l« Howitt

(1 9 5 0 ) describes a property of unheated human sera which he found to be virucidal for NDV. Heating at 56°C for 30 minutes destroyed this inhibitor. Bang and Foard (1956) studied the inhibition of mumps and NDV by chicken, rabbit, and human sera.

Both chicken and rabbit antisera against mumps virus failed to neutralize NDV. Chicken antisera to NDV failed to neutralize mumps virusj but rabbit antisera prepared against three strains of NDV gave inconsistent results against mumps virus. Several human sera with high titers of neutralizing antibodies against 18

NDV were successfully absorbed with mumps virus. The absorption procedure was most effective when the serum was diluted; one serum which had a low neutralizing titer against mumps virus it­ self was included. Karzon (1956) described a heat-labile NDV- inhibitor present in human and rabbit sera. This inhibitor could also be destroyed by trypsin, streptokinase, ethylenediamine tetraacetate (EDTA), and by a component or components of tissues, especially kidney. Recent evidence indicates that this non­ specific heat-labile NDV-inhibitor (VIS) is the same as the pro­ perdin system. VIS differs from specific NDV antibody in that

VIS-virus unions are totally irreversible. Karzon postulated a system of species or natural resistance to NDV based on the exist­ ence of circulating VIS, an intracellular inhibitor of VIS, and serum proteases which potentially may alter the level of VIS.

In 191+7 Burnet and Anderson reported investigations which established the alteration of the antigenicity of treated red cells. These workers reported the production in rabbits of anti­ body to RDE-treated cells and modified cell antibody by immun­ ization with eluates from enzyme-treated cells.

Chu and Coombs (191+7) studied the effect of treatment with this group of viruses on the agglutination of Rh positive and Rh negative erythrocytes in incomplete anti-Rh sera. They found Rh negative cells were not agglutinated after virus treatment, but that Rh positive cells were agglutinated in saline dilutions of 19

antisera. These same workers reported that NDV produced the most dependable and uniform modification for the detection of incom­ plete Rh antibody by this method. In contrast to this, Makinodan and Maoris (1955) demonstrated the agglutination of trypsin- treated Rh negative (cde/cde) red cells in a pooled RhQ(D) serum.

They further demonstrated that this agglutination was due to a serum factor other than Rh0 (D) antibody.

Electrophoretic mobility studies on treated cells carried out by Hanig (19U8) and Ada and Stone (1950), demonstrated that virus or RDE treatment decreased the mobilities of the erythro­

cytes. Similarly, Ponder (1951) showed that trypsinization produced the same effect. Wallace (1953) demonstrated this same effect with PR8 and NDV-treated erythrocytes.

Burnet and Anderson (191*6) and Evans (1955) reported that red cells treated with NDV develop a new antigenic character, which allows them to be agglutinated to high titers either by experimental NDV immune serum or by serum from most cases of infectious mononucleosis in man. Stewart and Meenan (1951)

concluded that the changes in antigenicity produced by influ­

enza virus and RDE were indistinguishable from each other but different from changes produced by trypsin and periodate, which in turn were different from each other.

Dodd, Wright, et al. (1953)> postulating that subsurface antigen sites exposed by enzymatic alterations of red cells might 20

be responsible for the stimulation of antibody associated with certain hemolytic anemias, were able to show that the cells of many of these patients were agglutinated by specific trypsinized

cell antibody while normal cells were not. Wallace (1953) demonstrated that NDV modification of human or chicken erythro­

cytes produced in vitro encompasses the change produced on these

cells by PR8. When antisera to both NDV and PR8-altered cells were absorbed with homologous and heterologous virus-modified

cells, only Newcastle cell specificity remained. Likewise, when antisera to red cells from chick embryos infected with these viruses, were comparably absorbed with erythrocytes from infected

embryos, this same relationship existed (Bigley, 1955).

Karzon and Bang (1951) detected a viremia followed by the appearance of agglutinins for virus-treated red cells in chickens experimentally infected with Newcastle disease virus, while

Gardner (1955) demonstrated antigenically modified erythrocytes during such infection. "Working on the basis of postulations similar to those of Dodd, Wright, et al. (1953), Smith (1953) was able to produce an experimental hemolytic anemia in rabbits by

the injection of antisera to trypsinized and normal compatible

cells prepared by the transfusion of trypsinized and normal com­ patible cells.

French and Ada (195)-).) demonstrated the in vivo modification

of guinea pig erythrocytes after injection of RDE. The elec­ 21 trophoretic mobilities of the erythrocytes were decreased, followed by a rapid appearance of red cells with normal mobilities. These investigators explained this phenomenon on the basis that the damage caused to the cell membrane by the RDE is repaired in the course of a few days, the virus receptors being regenerated while the cells remain in the circulation. To substantiate their claim, these workers cite the work of Muir et al. (1950) who reported that there is an active turnover of cholesterol in the red cell.

French and Ada, therefore, claim that there is a continuous reconstruction process of the erythrocyte surface going on throughout the lifespan of this non-nucleated cell.

In addition, it has been shown that erythrocytes which have been modified with enzymes or virus, sensitized with antibody, or taken from patients with acquired hemolytic anemias are more susceptible to phagocytosis than normal red cells (Wright, Dodd, et al., 1953).

From reviewing the literature it is evident that viruses, enzymes and certain ions can effect the alteration of erythrocytes in vitro, and that there is reasonable evidence to support the supposition that red cells may be modified in vivo; and so, more strongly substantiates the fact that such in vivo changes may in turn stimulate the initiation of autoantibody production. The cell changes brought about by the mumps-NDV-influenza group of viruses include panagglutinability; direct agglutination by in­ complete anti-Rh sera; decreased electrophoretic mobilities; 22

inability to agglutinate in the presence of homologous virus; and alteration of antigenicity (Briody, 1952). Furthermore* there is present in the literature subtle evidence to incriminate in several types of disease states altered reactivity to simple tissue structures. Such structure^ upon degenerative alterations of cells, have been thought to be innocuous. This is illustrated by various clinical studies such as that reported by Stefani,

Magalini, and Patterson (1956) in which auto- and iso-erythrocyte antibodies were absorbed with isologous tumor tissue but not by the surrounding normal tissue. Furthermore, isoagglutinins dis­ play mainly carbohydrate specificity directed against the hexose configurations present in the receptor structure.

Although the literature contains little work dealing with the antigenicity of uronic acids, per se, this present work calls for some examination of the occurrence and properties of these substances. Hexose uronic acids structurally are nothing more than their respective hexose sugars oxidized at the sixth carbon to contain a carboxyl grouping.

This uronic acid configuration is present in such basic structures as "intercellular cement substance" (polymerized hyaluronic acid), platelets, collagen, cartilage, salivary and gastric mucins and heparin. These same configurational units are present in plant pectins, pneumococcal capsular polysaccharides, and in the capsular portions of some streptococci. In mammalian organisms, glucuronic acid functions in detoxification mechanisms, 23

being conjugated with the toxic molecule and the conjugate removed through the 'urine- Similarly, it has been observed that-sex hormones are eliminated as glucuronides, suggesting the possibility that the conjugation mechanism may function in the regulation of hormones (Kleiner, 1951).

Serologic specificity can be shown to reside in the uronic acid moiety (Heidleberger, 1956). The antibody specificities of pneumococcal polysaccharides II, III, and VIII seem to be directed chiefly toward glucuronic acid. Goebel (1937) showed that anti­ gens prepared from glucuronic acid are precipitated even in high dilutions of antipneumococcus horse sera of these types, while the corresponding glucose compounds exhibited little or no act­ ivity. In the same manner, Goebel and Hotchkiss (1936) demon­ strated the specificity for galacturonic acid which is contained in Type I pneumococcus polysaccharide. Furthermore, Goebel observed that glucuronic acid and galacturonic acid did not cross-react serologically. The same is true for glucose and galactose.

The linkages involved in this specificity are clearly de­ lineated by Heidleberger (1956). The specific capsular sub­ stance of Type III pneumococcus is a polycellobiruonic acid, com­ posed of glucose and glucuronic acid in which the glucuronic acid is linked in the beta position to the four position of glucose.

Each of the cellobiuronic acid units is linked, probably through the beta linkage, to the three position of the glucuronic acid in front of it, so that there are alternate k>3 linkages. The

Type III substance is all polycellobiuronic acid while the Type

VIII substance has only about 50 per cent of this acid. The react­ ivity of the Type VIII substance in Type III antisera or the re­ activity of the Type III substance in Type VIII antisera would be due to the occurrence of multiple cellobiruonic acid units in both substances.

Of the blood group substances, one seems to have a galactose specificity. Kabat (19U9) showed that A substance reacts in

Type XIV antipneumococcus serum. The more fucose present in the

A substance the less its reactivity in Type XIV antiserum. The fucose, probably in the furanose form in the A substance, is readily removed and consequently the reactivity as A substance decreases. On the other hand, the reactivity toward Type XIV serum increases, because the main chain of the blood group sub­ stance seems to be composed of galactose and N-acetylglucosamine groupings just as is much of the Type XIV polysaccharide, and some of their linkages must occur in the same positions. Similar­ ly, Gottschalk (1953) describes the existence of acetylhexosamines, hexoses, and 2-carboxypyrrole as degradation products resulting from the action of influenza viruses on red cell receptors and urinary mucoprotein. Recently, Odell and Anderson (19 ) have further indicated the production of mucopolysaccharides by 25

platelets.

In various rheumatic disease states, acid mucopolysaccharides

of connective tissue ground substance and synovial fluid mucin have been shown to be abnormal. In rheumatic fever the altera­

tions are indicative of an immediate type of hypersensitivity,

characterized edema, vascular defects, and collagen degenera­

tions. Other syndromes in which hypersensitivity may be in­

volved are rheumatoid arthritis, disseminated lupus erythematosus, periarteritis nodosa, glomerulonephritis, and certain pupuric

diseases. Streptococci are implicated in the development of

several of these syndromes. Although the manner of the estab­

lishment of the hypersensitive state has been attributed to the

somatic portions of the streptococci either altering or reacting with host tissue, the fact that many strains of virulent strep­

tococci possess hyaluronic acid capsules themselves should not be overlooked.

Even though there is very little literature which is

directly pertinent to this work, the implications of uronic acids

as components of certain basic cell structures is evident.

Hypersensitivity to abnormal uronic acid conjugates may be shown

to be the common denominator existing in the various rheumatic

disease states. Badin, Schubert, and Vouras (1955) found an

anionic polysaccharide containing hexosamine, glucuronic acid and

sugar in human euglobulin, but not in euglobulin-free plasma. 26

This substance differs from chondroitin and contains protein.

This glucuronate containing polysaccharide is increased in plasma of patients with rheumatoid arthritis, and the authors suggested a relationship to cartilage destruction and to the larger amount of euglobulin found in plasma in this disease.

Quantitative differences in D-glucuronolactone metabolism in arthritic, hepatic and mesenchymal tissue diseases were claimed to have been found by Fishman et al. (1951) and Fretwurst and

Ahlhelm (19$% but the differences were generally not remarkable.

Hartmann and Berg (1952) reported that intravenous injec­ tion of hyaluronidase increases the permeability of the rabbit's blood vessels to protein, as measured by a decrease in plasma albumin and the appearance of albumin in the liver and kidney.

This effect is intensified by deoxy-corticosterone, and inhibited by 1 g. of D-glucuronic acid. McCrea and Duran-Reynolds (1953) have shown that the factor in hydrolyzed hyaluronic acid, which apparently inactivates vaccinia virus is D-glucuronic acid. Many of these phenomena may be related to the inhibition of beta-D- glucuronidase which can be produced by D-glucuronic acid (Spencer and Williams, 1951)* Toxicity studies have shown D-glucuronolac- tone to be relatively innocuous.

Teague (195U) discusses the functional, reactive groupings in such a molecule:

...It is impressive that these same groups not only are responsible for the lipid solubility, surface- 27

tension action, and pH of solutions of the molecule, but are also frequently those primarily responsible for the biological activity of the substance. The bio­ logical activity of a compound, be it specifically physiological or pharmacological, or merely toxic, is an expression of the compound to "fit'or combine in some way with certain enzyme systems. Let us call these systems '‘activity11 enzymes to distinguish them from "metabolic" enzyme systems which bring about the degradation or conjugation of a molecule. We can then state that the functional hydroxyl groups which combine as substrates for metabolic enzymes also frequently combine with activity enzymes. When the functional hydroxyl group is suppressed, it naturally follows that the biological and chemical effects dependent on the integrity of these groups are lessened.

Furthermore, Teague (1951+) advocates that compounds such as ethanol which have no specific biological action still exhibit pharmacological effects which are generally described as toxic.

The toxic effects are suppressed when the functional glucur- onogenic hydroxyls are masked by conjugation or lost by degrad­ ation. Teague (1951+) has postulated that functional groupings such as the hydroxyl may combine as substrates with both meta­ bolic and activity enzymes. The metabolic system would bring about a chemical change in the compounds involved, whereas the activity enzyme systems would cause some biological events on combination with the functional configurations. In addition, it is conceivable that in certain cases the activity system and the metabolic system are the same. As an example of this duality of function, Teague (1951+) cites the monoamine oxidase system which has been shown to be involved simultaneously in the oxidative 28

deamination of epinephrine and in an activity system in the cell receptor initiating the reactions in which epinephrine effects adrenergic cell stimulation. The relationship between the mechan­ isms of mucopolysaccharide synthesis and the D-glucuronic con­ jugating system may be similar.

Glucosamine and galactosamine structures are found as in­ tegral parts of red blood cell receptors, brain, and other tissues.

Kabat (1956) has recently shown that oligosaccharides inhibit a dextran-human 1*••*6 antidextran system and that the dimensions of the complementary areas of some of the antidextran combining sites include areas complementary to tri-, tetra-, and hexa-, saccharides. Thus, antibody of carbohydrate specificity may actually be directed against molecular units which arise in vivo from the liberation of degenerated tissue components. Furthermore, the serologic specificity of molecular units such as the hexuronic acids would reside in the linkages of these compounds either in

the simple conjugated state or in biological materials of which

they are structural components. The present investigation was undertaken for the purpose of further studying the serologic alterations of virus-treated erythrocytes with special emphasis on carbohydrate specificity resulting from such treatments. MATERIALS AND METHODS

Influenza Virus

The Type A influenza virus (PR8 strain) used in this work was initially received as infected mouse lung from the virus col­ lection of the Department of Bacteriology of The Ohio State

University. This mouse lung virus was adapted to the developing chick embryo and subsequently maintained by passage in the allan­ toic cavity of 9-12 day old embryonated eggs. Stock PR8 virus was prepared by inoculating 0.1 ml. of virus into the allantoic cavity of the embryonated eggs. Following a J4.8 hour incubation period at 37°C the eggs were refrigerated for 3-13 hours and the infected allantoic fluid was removed. The virus-containing fluid was then tested for sterility, titrated for hemagglutinin, ampuled, and stored at -30°C.

Newcastle Disease Virus

The strain of Newcastle disease virus (NDV) used in this in­ vestigation was isolated from a fatal case of the disease in a chicken, and was obtained from the Department of Veterinary

Pathology of The Ohio State University. The virus had been pas­ saged many times in fertile hen's eggs and was received as frozen allantoic fluid. Stock NDV was prepared and preserved in the same manner as described for the PR8 .

29 30

Mumps Virus

The Binders strain of mumps virus employed in this study was obtained from the Ohio State Department of Health, Columbus, Ohio.

The virus had been passaged many times in chick embryos and was received as frozen allantoic fluid. Stock mumps virus was . optimally prepared by the method advocated by Weil, Beard, Sharp, and Beard (19U8). Seven day old fertile eggs were inoculated allantoically with 0.1 ml. of the virus suspension. Following a seven day incubation period at 3'7°C the infected eggs were refrigerated for 3-13 hours and the allantoic fluid was tested and preserved in the same manner as previously described for the

NDV and PR8 virus.

Virus Hemagglutination Titrations

Virus hemagglutination titrations were performed in round bottomed tubes measuring 7 X 75 mm. Doubling dilutions of virus

suspensions were prepared in 0.5 ml. quantities of phosphate buffered saline (pH 7*U) and 0.5 ml. of 0.5 per cent human 0 cells

or 0.25 per cent chicken erythrocytes was added to each virus dilution. Titrations with the ER8 virus and NDV were incubated

16 minutes - 2 hours at room temperature. Best results with the

titration of the mumps virus were obtained by incubation at 37°C for JU5 minutes - 2 hours. Titrations were read on the basis of

the pattern of sedimented cells. 31

Receptor Destroying Enzyme (RDE)

A toxogenic strain of Clostridium perfringens (OSU) was incubated in Brewer's thioglycollate medium for 12-18 hours. The bacterial cells were removed by centrifugation and the supernatant material used as the unpurified RDE. Also used as RDE was the crude filtrate from a 16-20 hour culture of Vibrio cholerae strain I4Z incubated at 37°C in heart-infusion broth (Difco).

Preparation of Freund Adjuvant

Two volumes of antigen suspension in physiological saline were added dropwise to one volume of autoclaved and melted Falba.

Freund advocates mixing antigen and Falba with mortar and pestle, but with soluble antigens dropwise addition of the Falba followed by frequent and vigorous shaking was adequate to suspent the saline

solutions into a smooth mixture in the Falba. Two volumes of autoclaved mineral oil (medium grade) containing non-viable

Mycobacterium tuberculosis var. hominis (or Mycobacterium phlei) were added and an emulsion formed upon vigorous shaking. The proportions of ingredients are thus 0.3 ml. antigen in saline

solution to 0.15 ml. Falba to 0.3 ml. parafin oil containing

0.1 mg. of the tubercle bacilli (Freund et al. 191+8).

Uronic Acids and Pneumococcal Polysaccharides

The alpha-D-galacturonic acid and glucurone were obtained 32

from the Eastman-Kodak Company, Rochester, New York.

The pneumococcal polysaccharides used in this work had been obtained from the Bureau of Laboratories, Michigan Department of

Health.

Periodate

A stock solution of 0.1 M KIO^ was prepared with distilled water and stored at U°C. Final dilutions of 0.001M were made in physiological saline (pH 7-U) and used in the treatment of ery­

throcytes and red cell stromata.

Periodate Inhibitor

A stock solution of periodate inhibitor was prepared by mixing

25 g. of d-glucose and 30 g. of trisodium citrate in 250 ml. of

distilled water after which the solution was stored at Ijpc. In

order to prepare an isotonic solution which would destroy excess

periodate, the stock solution was diluted 1 in U with distilled water as needed.

Preparation of Erythrocyte Stromata

For the preparation of red cell stromata, erythrocytes from

a pint of human, group 0, Rh positive or negative, blood were

washed three times in phosphate buffered saline (pH 7«U).

Plasma and leucocytes had been previously removed from the 33

centrifuged samples. The washed erythrocytes were then added to

60-70 volumes of cold distilled water, pH 6 .0-6.1. After vigorous shaking, the particulate matter was allowed to settle out at room

temperature and the supernatant fluid subsequently removed. An equal volume of 0.2 per cent formaldehyde in saline was added to

the sedimented stromata which were then stored at i|PC. Prior to use, the stromata were washed three times in borate buffered

saline, pH 6 .8, and each time sedimented at 3300-Lj.OOO rpm in the

Sorval angle-head centrifuge.

Preparation of Normal Erythrocytes

Normal chicken or human erythrocytes were used for virus

hemagglutination, virus treatments, immunizations, and absorption while normal rabbit red cells were employed in titrations.

Chicken and rabbit erythrocytes were obtained from normal

animals by cardiac puncture. Blood, thus collected, in a 3*0 per

cent citrate solution, was freed of plasma and white cells and the

red cells washed three times in phosphate buffered saline.

Human red cells, group 0 Rh0(D) positive and negative, in

citrated blood were obtained from the Red Cross Blood Bank. Plasma

and leucocytes were removed from centrifuged samples and the red

cells were washed three times in buffered saline. 3U

Preparation of Treated. Erythrocytes and Stromata

Virus-treated erythrocytes were prepared according to a modification of the method used by Ada and Stone (1930). Washed and packed chicken or human 0 red blood cells were added to un­ diluted virus, containing at least 1021). hemagglutinating units, to make a final red cell concentration of ten per cent. These virus- red cell mixtures were incubated in a water bath at 37°C for four hours with agitation at thirty minute intervals. Following in­ cubation, the red cells were separated from the mixture by cen­ trifugation and were washed three times with phosphate buffered saline. In the treatment of red cell stromata with mumps virus the ratio of stromata to virus was varied as was the time of treatment.

Trypsinized erythrocytes were prepared by incubating two volumes of packed red cells with three volumes of 0.1 per cent trypsin (Difco 1:25>0) for 30 minutes at 37°C. The cells were subsequently washed three times in buffered saline.

The method of treatment of erythrocytes with periodate is essentially the same as that described by Coffin and Pickles (1933).

Two volumes of a 10 per cent red cell suspension were mixed with

one volume of a 0.001 M KI0j^ and one volume of physiological

saline for different periods at room temperature. At the end of

this period an equal volume of the periodate inhibitor was added

to the red cell-periodate mixture. The cells were centrifuged

and washed three times in saline. Varying concentrations of red 35

cell stromata were treated in the same manner as that described for erythrocytes.

Human 0 and rabbit erythrocytes were treated for various periods of time with 0.25 per cent glucuronic acid or 0.125 per

cent alpha-D-galacturonic acid at room temperature and at 37°C.

The cells were then washed several times in buffered saline.

RDE-treated red cells were prepared by exposing one part red

cells to two parts crude RDE for 30 minutes - 2 hours at 37°G.

After RDE treatment, the cells were washed several times in buffered saline.

For the purpose of merely altering the charge on rabbit

erythrocytes chromium treatment of the cells was used. One

volume of washed, packed red cells was exposed to nine volumes of

0.001M CrCl^*6H20 made-up in unbuffered saline for 20-30 minutes

at room temperature. The cells were subsequently centrifuged

and were not washed because continued washings removed the altered

charge.

Preparation of Eluates

Eluates were obtained from red cells and from stromata

incubated with either periodate or mumps virus for various spe­

cified periods. The cellular material was centrifuged and the

supernatant material, designated as eluate, was removed. The

eluates consisted of: (l) saline from suspension of periodate-

treated cells and stromata containing eluted cell material with 36

and without the periodate oxidative inhibitor and (2 ) chorioallan­ toic fluid from mumps-treated erythrocytes and stromata containing mumps virus and eluted cell material.

Inhibition Titrations

One-tenth ml. quantities of saline were added to each tube of a series in which the eluate was serially diluted. One-tenth ml. amounts of an agglutinating dilution of anti-Rh0(D) serum were added to each eluate dilution. The contents of each tube in the titration were well mixed and incubated for 10-20 minutes at either room temperature of 37°C. Subsequently, 0.1 ml quantities of a 2 per cent suspension of either untreated Rh D positive or trypsinized Rh positive red cells were added to each tube and the mixtures incubated for an additional 30 minutes after which the tubes were centrifuged (1500 rpm) for one minute and read for agglutination.

Rh antisera of human origin were used in this work.

Commercial preparations are indicated in the various tables.

The plasma used as diluent consisted of pooled human plasma from 0Rho(D) blood.

Preparation of Erythrocyte Antisera

The antisera to normal and in vitro treated human red cells were prepared by giving rabbits five intravenous injections of 1.0 37 ml* of a five per cent suspension of red cells at three day intervals. The rabbits were bled ten days after the last injec­ tion and the sera inactivated at $6°C for thirty minutes, and frozen at -5>0°C.

Because of the weakly reacting antiserum resulting from the first intravenous immunization of rabbits with mumps-treated cells, an antiserum was prepared by injecting rabbits intramuscularly with one injection of 3 .0-5.0 ml. of equal parts of treated-red

cells and Freund adjuvant. The rabbits were bled four weeks after

the injection and the sera were inactivated and stored as indi­

cated above.

Hemagglutination Titrations

Doubling dilutions of erythrocyte or Rh antisera were pre­ pared in 0.1 ml. amounts of buffered saline and 0.1 ml. of a two per cent suspension of red cells was added to each serum dilution.

Tubes if ere allowed to incubate at 37°G for thirty minutes, after which they were centrifuged at about 1000 rpm for one minute and

read for agglutination.

Precipitation Titrations

Antiserum was diluted in doubling dilutions and transferred

to precipitin tubes in 0.1 ml. amounts. One-tenth ml. of the anti­

gen material was added to each tube. The tubes were incubated for

30 minutes - U hours at 37°C and subsequently for k-5 days at 8°c 38

after which they were read for the presence or absence of pre­ cipitated material.

Absorption of Antisera

Ivlodified-cell antisera were absorbed at room temperature with homologous and heterologous cells in the following maimer:

Ratio of Absorption Serum to Cells Time

1st 1:1 10 min. 2nd 1:1 30 min. 3rd 2:1 1 hour iith 2:1 1 hour 3 th 2:1 1 hour 6 th 2:1 1 hour

Rh antisera were absorbed in the following way: 1 part serum to 1 part cells followed by incubation for 30 minutes at room tem­ perature, After each absorption the cells were sedimented by centrifugation and the supernatant serum removed.

Precipitation of Globulins

Gamma and beta globulins were separated from serum by sodium sulfate precipitation. Nine ml. of 20 per cent aqueous Na2S0^ were added slowly to one ml. of serum and the mixture agitated constantly during the addition and periodically over a 3 hour period at 37°C. The mixture was then centrifuged for 20 minutes in a Sorval angle head super centrifuge, the supernatant was removed, and the precipitate restored to a one ml. volume with 39

double-distilled water. Electrophoretic analyses of the recon­ stituted globulins revealed only gamma and beta globulins.

Preparation of Antiglobulin Serum

Anti-rabbit globulin was prepared in chickens injected intra­ muscularly with reconstituted rabbit globulins, which had been precipitated by 20 per cent Na2S0^, incorporated in Freund adju­ vant.

Standardization of Anti-Globulin Serum

The chicken anti-rabbit globulin serum was standardized in the following manner: Rabbit erythrocytes from several normal animals and these same red cells treated with the uronic acids were titrated in this anti-globulin serum (designated in the tables as CARG). The uronic acid-treated erythrocytes agglutin­ ated to higher titers that the untreated red cells. The dilu­ tion of CARG selected for use in developing tests was selected as the next dilution of serum beyond the titer for uronic acid- treated cells. Doubling dilutions were employed.

Injection of Animals with Uronic Acids and Sera

Rabbits were injected with low molecular weight carbohydrates, so as to create, if possible, an excess of these substances in areas of injured or degenerating tissue. For this purpose, doses ko

of 0.23 to 3*0 g. of alpha-D-galacturonic acid or 1 .0-3 g. of glucurone incorporated in as much as 10-20 cc. of adjuvants of the water-in-oil type were injected intramuscularly. The animals were bled by cardiac puncture prior to injection and at weekly intervals thereafter.

At various intervals these animals were skin tested with 0.1 ml. of 0 .3-1.0 per cent of saline solutions of glucurone and ga­ lacturonic acid.

Animals were injected intraveneously' with sera from animals previously injected with uronic acids and with such sera mixed with the homologous uronic acid. These methods are further de­ scribed in the results.

Complement Fixation Tests

Complement fixation tests were performed on occasion with the sera of animals injected with uronic acids. The uronic acids and normal rabbit brain suspension were used as test antigens.

In antigen titrations 0.23 per cent glucurone and 0.1,23 per cent galacturonic acid were found to be suitable. Higher concentrations were anti-complementary. A 1:100 dilution of rabbit brain was suitable (Petker, 1932). The tests were performed using the

Kolmer full method (Kolmer, Spaulding, and Robinson, 1931)• ui

Latex Fixation

Latex fixation reactions were performed with the sera of several animals, previously injected with glucurone and galac­ turonic acid. The polystyrene latex of uniform particle size

(l.l? micron) was obtained from the Dow Chemical Company,

Midland, Michigan. The latex was a suspension which contained

11 per cent solid material and was prepared for serologic use in the following manner: twenty ml. of water was added to 2.0 ml. of the suspension and filtered through Whatman #U0 filter paper.

The resulting stock solution of latex should be sufficiently dense so that when approximately 0.1 ml. of the stock solution is added to 10.0 ml. borate buffer, a light transmission of 7 per cent is obtained when this diluted latex suspension is examined in a spectrophotometer at 6£0 millimicra with a red filter. This stock solution may be kept for several months in the refrigerator.

The borate buffered saline used as diluent in this test was pre­ pared by mixing 5>0 ml. of 0.1 M boric acid and 3.9 ml. 0.1N NaOR and made up to 100 ml. with distilled water. The pH was adjusted

to exactly 8.2jisotonicity was achieved by adding 0.83 g. of

NaCl to each 100 ml. of buffer.

Either uronic acid or globulin was added to the prepared latex suspension and comprised the test antigen. Dilutions of

sera (1:20 - 1:U0) were employed to avoid non-specific reactions

observable in more concentrated sera. The tests were incubated

at both 5>6°C or 37°C for 30 minutes and were read for flocculation U2

after light centrifugation.

The stock gamma globulin solution was prepared from polio­ myelitis immune globulin which contained 150 mg. of globulin per milliliter and was diluted to 5 mg. globulin per ml. with the borate buffer.

The test antigen consisted of 0.1 ml. stock latex suspension,

0.5 ml. of the gamma globulin solution and 9.5 ml. borate buf­ fered saline. The test antigen containing the uronic acids was prepared in the same manner with 0.5 ml. of a 1 per cent uronic acid solution replacing the gamma globulin.

Erythrophagocytic Tests

Phagocytic studies were carried out by using rabbit splenic macrophages in tissue culture according to the technique of Bass

(1953)* Macrophages and test red blood cells were incubated at

37°C for thirty minutes, after which the phaocytic indes (P.I.) was determined by calculating the percentage of macrophages which

contained phagocytized erythrocytes.

Paper Electrophoresis of Sera

Buffer: The buffer consisted of 2.76 g. diethyl barbituric acid

and lS»b g* sodium diethyl barbiturate (0.075 ionic strength) dis­

solved in a total of 1 liter distilled water. The resulting pH was

8.6 and was checked on a Beckman glass electrode pH meter (Model

H2). Dye; Fifty ml. of 95 per cent ethyl alcohol were added to 0.1 g. bromphenol blue and 31 g* zinc sulfate (l. H2O) or U9.7 g* zinc sulfate (7 H2O). This resulting slurry was stirred for several minutes and then dilated to 1 liter in 5 per cent (by volume) acetic acid. This solution, stirred until all crystals were dis­ solved, served as the dye.

Fixative: Nine grams of sodium acetate (3H2O) dissolved in 1 liter of 10 per cent (by volume ) acetic acid constituted the fixative.

Procedure; The paper electrophoresis of sera was conducted in a Spinco Model R paper electrophoresis system. The cell was set up using 8-lhatman 3mm. paper strips, S and S lj.70 wicks, and the buffer solution. The wicks were first inserted into the cell.

The rack loaded with the eight paper strips was placed on the

support stand of the cell, folded into position and locked.

Eight-hundred ml. of the buffer were placed in the cell. The

cover slot was sealed with tape, the cell tilted to equalize

fluid levels in the compartments, and 15 minutes were allowed for

the liquid-vapor equilibrium. A 0.010 ml. serum sample was trans­

ferred with a pipette to the striper and then applied to the

appropriate strip. The cover to the cell was sealed with tape

and the cell attached to the duostat. The separations were 14;

conducted either at 3 milliamperes per cell, constant current, for 16 hours or at 13 milliamperes per cell for 6 hours. Upon completion of the separation the strips were dried for 30 minutes at 120-130OC. The strips were dyed for I4I - 6 hours at room temperature with the dye solution, rinsed twice in 3 per cent acetic acid (6 minutes/rinse) and then rinsed for 6 minutes in the fixative solution. The strips were then blotted, transferred to the drying rack, and heated for 13 minutes in an oven preheated to 120-130°C. Ten minutes prior to scanning the strips were exposed for 1 minute to fumes from 1 ml. concentrated ammonium hydroxide. The strips were then scanned in the analytrol. Because the terminology characterizing the various treatments of red cells is involved, the following abbreviations are used throughout this work:

NH - normal, untreated, human 0 Rh0(D) positive erythrocytes N-H - normal, untreated human, 0 Rh0(D) negative erythrocytes

HTryp - trypsinized Rh positive red cells H-Tryp - trypsinized Rh negative red cells

HPR8 - Rh positive red cells treated with PR8 virus H-PR8 - Rh negative red cells treated with PR8 virus CPR8 - chicken erythrocytes treated with PR8 virus

HNDV - Rh positive red cells treated with NDV H-NDV - Rh negative red cells treated with NDV GNDV - chicken erythrocytes treated with NDV

HM - Rh positive red cells treated with mumps virus H-M - Rh negative red cells treated with mumps virus CM - chicken erythrocytes treated with mumps virus

HP - Rh positive red cells treated with periodate H-P - Rh negative red cells treated with periodate

NR - normal, untreated, rabbit erythrocytes (usually from 2-3 donor animals)

GR - glucurone-treated rabbit red cells

ADGR ~ rabbit erythrocytes treated with alpha-D-galacturonic acid

HRDE - Rh0 positive human red cells treated with RDE (Clostri­ dium welchii) H-RDE - Rh0 negative human red cells treated with RDE CRDE - chicken erythrocytes treated with RDE The commercial Anti~Rh0(D) sera used in this work are designated in the tables only by the name of the manufacturer. These sera arei

Anti-Rh0 (D) saline agglutinin Anti-CD saline agglutinin

Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York

Albumin Agglutinin Anti-RhQ(D)

Hyland Laboratories, Los Angeles, California

Saline Anti-Rh0(D)

Knickerbocker Biosales, New York, New York

Anti-Rh0(D) saline agglutinin Anti-Rh (D) albumin agglutinin Anti-Rh0(E) Anti-Rh0(C) Anti-Rh0(c)

Ortho Pharmaceutical Corporation, Raritan, New Jersey EXPERIMENTAL RESULTS

Antigenic alterations of red cells acted upon by the mumps-

Newcastle disease-influenza hemagglutinating group of viruses are demonstrated by the fact that such cells are agglutinated by "T" agglutinins of normal sera, in incomplete anti-Rh sera, and by specific antisera to virus treated cells. These latter two aspects are further studied in this investigation. In an attempt to more fully understand the occurrence of modified-cell anti­ body resulting from in vivo erythrocyte degradation, agglutinins for virus and enzyme-treated red cells were characterized and compared in both anti-Rh sera and in specific sera for the treated

cells.

Wallace (1953) showed that the specificities of human and

chicken erythrocytes treated with influenza (PR8) virus or

Newcastle disease virus (NDV) were primarily concerned with species antigens. Alterations of chicken cells by mumps virus however were relatively minor in comparison to the modifications of human

cells, suggesting an alteration of antigens peculiar to the human red cell (Bigley, 1955)* Since it was known that reducing agents greatly enhance the hemagglutinating potency of mumps virus

(Gardner and Morgan, 1952) while suppressing the hemolytic act­ ivity which results from viral elution at the receptor areas, the

elution-hemolysis was inferred to be an oxidative process. Thus,

the action of mumps virus on erythrocytes was compared with that

U7 1*8

of periodate, which in low concentrations has bean observed to alter oxidatively these same viral receptors (Fazekas de St.

Groth and Grahm, 19U9).

Morgan and Watkins observed that oxidation or red cells destroyed partially or completely, the Rhesus D (Rh0) antigen as evidenced by reduction or absence of agglutination in specific antiserum. Coffin and Pickles (1953) reported that specific agglutination of periodate-treated cells may be restored by trypsin if periodate treatment has not been too vigorous.

Presumably D antigen sites previously unaltered by the periodate are made available by the action of trypsin. Since mumps- treated erythrocytes reacted minimally or not at all in incomplete anti-Rh, mumps-treated cells were trypsinized and titrated in the same anti-Rh serum as seen in Table I.

As is indicated in this table, periodate and mumps-treated red cells failed to agglutinate in an anti-Rho(D) saline agglutinin which also contained incomplete anti~Rh0 as indicated by the trypsin-cell titer. These same cells upon subsequent

trypsinization agglutinated in this serum to essentially the same titer as did trypsinized Rh positive erythrocytes. The reversal of this did not occurj namely, trypsin-treated red

cells upon subsequent treatment with mumps virus did not lose

their agglutinability in anti-Rh0(D) serum but agglutinated h9

to the same titer as did trypsinized red cells. This phenomenon was repeated using several other commercial Rh antisera. Thus, periodate and mumps-treated red cells behave similarly in anti-

Rh0(D) sera.

To determine whether further differences existed between

Rho(D) positive and negative erythrocytes after treatments with viruses several absorptions and titrations were performed with the virus-modified cell and normal cell antisera previously characterized by Wallace (1953) and Bigley (1955)* These results are presented in composite form in Tables II, III, and IV. To reiterate the results previously presented (Wallace, Dodd and

Wright, 1955): Absorptions of the antisera for virus-treated cells with normal human red cells removed the agglutinins for the absorbing cell but left the agglutinating antibodies for the virus-treated cells. Trypsin cell antibody was mark­ edly reduced by the normal cell absorptions. From the data obtained in homologous and heterolobous absorptions of the modified-cell sera (Table II) it is obvious that HPR8 and HNDV cells showed similar specifities while those of mumps-treated red cells were in certain ways similar yet exhibited some differences. Absorption of either Anti-HNDV or Anti HPR8 with

FR8-treated erythrocytes removed agglutinins for the absorbing cell leaving in the sera antibody reactive for the NDV treat- TABLE I

Titrations of Anti-Rh0(D) Saline Agglutinin* with Rh0(D) Red Cells Treated with Periodate, Tripsin and Mumps Virus

Test Dilutions of Serum in Saline Cells 2 4 8 16 32 64 128 256 512 1024 Cell Control

NH 3 3 1---- HTryp 4444321 HM HM-Tryp 444432 2 1 HP HP-Tryp 4433211 HTryp-M

HE Untreated Rh0(D) erythrooytes. HTryp Rh0 erythrooytes treated with trypsin, HM RhQ(D) erythrocytes treated with mumps virus, HP Rh0(D) erythrooytes treated with periodate. HM-Tryp Rh0(D) erythrocytes treated first with mumps virus and then with trypsin, HP-Tryp RhQ(D) erythrocytes treated first with periodate and then with trypsin. HTryp-M Rh0(D) erythrocytes treated first with trypsin and then with mumps virus.

* Knickerbocker

va o 5i

ment. When HNDV cells -vrere used to absorb these two sera, the predominating specificity remaining in the sera was for the NDV-treated red cell. Wallace et al. explained the inability of PR8-treated red cells to remove from homologous serum the HNDV-agglutinin by postulating the creation of

HNDV specificity by the immunized animal during the in vivo degradation of the red cells treated in vitro with PR8.

They also observed that absorption of modified cell anti­ body was enhanced by the removal of the normal cell agglu­ tinin. Basic similarities in the specifities present in antisera to chicken erythrocytes (Wallace, Dodd, and Wright,

1955) and to chick embryo red cells either from the infected chick embryo or from in vitro treatments (Bigley, 1955) with either FR8 or NDV were revealed. In antisera to chicken or

chick embryo erythrocytes modified by either NDV or PR8 virus,

the Newcastle-cell specificity predominated. This observ­ ation further substantiated the in vivo degradation of ery­

throcytes postulated by Wallace, (1953) and thus contri­ buted to the 'understanding of the serologic reactivity of virus-altered red cells. 52 TABLE II

Comparison of Titers of Normal and Modified Erythrocytes in Antisera to PR8 and NDV-treated Human Red Cells

Red Cells U s e d in Test Absorptions Cells Anti-HPR8 Ant i-HNDV

0 NH 512 1024 HPR8 4096 4096 HNDV 4096 4096 HM 2048 16384

2 x NH with HPR8 2 - HPR8 HNDV 64- 256 64 HM 8 - • HP -

2-3 x NH with HPR8 8-16 - HNDV HNDV 32-64 16 HM 2

2 x NH 2 «* with HPR8 512 256 HMHNDV 512 256 HM 8 - H-M 64 HP -

5 x NH with HPR8 2048 2048 NH HNDV 2048 2048 HM 2048 2048 TABLE II (Continued)

Comparison of liters of Normal and Modified Erythrocytes in Antisera to PR8 and NDV-treated Human Red Cells

Red Cells Used in Test Absorptions Cells Anti-HPR8 Anti-HNDV 2x 3x 5 x with NH N-H -- and 2-3 x BPR8 -- with HPR8 HNDV 64 64 HM 4 128 H-M 8 128

5 x with NH N-H tm and 2 x with HPR8 - - HNDV HNDV 2 2 HM 2 zones 1024 2 zones 2048 H-M 2 zones 4096 2 zones 1024

4 x with NH H-H __ and 1 x HM HPR8 512 32 HNDV 512 256 HM 4 - H-M 64 Still further differences were noted in these two sera when mumps-treated red cells were used as absorbing cells (Table II).

Mumps-treated cells absorbed from anti-HNDV-cell serum antibody for normal and HM cells as was the case with anti-HM (Table IV) and anti-HPR8 (Table II). In contrast to the results with anti-

HPR8, normal cells removed some mumps-cell specificity, but not all of it, as found in anti-HM. Thus, the serum contains anti­ body completely removed by HM and partially by normal cells. The part not removed by normal cells is also not absorbed by HPR8 in contrast to anti-HPR8 serum in which all mumps specificity was absorbed by HPR8 cells. Absorption with HPR8 cells removed all agglutinins with this specificity and some antibody for HNDV, while antibody for both HPR8 and HNDV was not removed by mumps- treated cells.

Absorption of both anti-IIPR8 and anti-HNDV with mumps-treated cells removed agglutinins for both normal cells and the absorbing cells, leaving considerable antibody for cells treated with both viruses as noted in Table II. This finding indicates antibody specific for mumps-treated cells was present in these two sera and that such cells also removed all normal cell hemagglutinins.

Both of these phenomena were observed with the homologous mumps­ cell serum (Table III). Whereas all antibody for the three virus- modified cells was removed by mumps-treated cells from homologous serum (Table III), such absorptions of the heterologous sera left $ $ considerable antibody for both PR8 and NDV-treated cells. These two antisera contain antibody not absorbable by mumps-cells, but which can be removed (Table II) by HPR8 or HNDV cells. However, these two sera demonstrate a titratable but Incomplete agglutination of both HM and H-M after absorption with HNDV. After the removal of normal cell agglutinins HNDV cells did not remove from anti-HM all HM agglutinins while HPR8 cells did (Table IV)•

Two absorptions of anti-HM with HPR8 cells not only removed all antibody for the absorbing cells but also all agglutinating antibody for normal cells, and all but a small amount for NDV- treated red cells (Table III), leaving mumps-cell specificity present in high titer.

As evident in Table IV, this mumps-cell specificity was absorbable by normal red cells, and thereby may be considered an incomplete antibody for normal erythrocytes. Practically the same results were obtained by absorption with NDV-treated red cells except for a small amount of agglutinin for normal cells, which was evident in a comparable absorption of antisera to normal human cells (Table III). It was then evident that anti-

HM contained a mixture of antibody specificities, the residual agglutinin being specific for antigens in homologous cells, but not for the heterologous virus-treated cells. This residual mumps­ cell agglutinin was present in normal cell antiserum though less pronounced (Table III) than in the homologous serum. TABLE III

Comparison of Titers of Normal and Modified Erythrocytes in Jntisera to Normal and Mumps-Treated Red Cells

Red Cells Used in Test Absorptions Cells Anti-HM Anti-NH

0 NH 64 2048 N-H 64 2048 HPR8 256 4096 HNDV 256 4096 HM 16384 16384- 65536 H-M 2048 16384

2x 3x 2-3 x with NH - 8 HPR8 N-H 16 4 HPR8 - 2 HNDV 4 4 HM 4096 32 H-M 16384* 8 HP 32 HTryp 2 - CM - CP 2

2 x with HPR8 NH and 1 x with NH N-H - HM - H-M-

2 x with HPR8 N-H _ and 1 x with CM HM - HM** - H-M 16384*

* Non-agglutinating zone. Completely negative at 1:128. ** No agglutination in presenoe of ohicken anti-rabbit globulin. 57

TABLE III (Continued)

Comparison of Titers of Normal and Modified Erythrooytes in Antisera to Normal and Mumps-Treated Red Cells

Red Cells Used in Test Absorptions Cells Anti-HM Anti-NH

2x 3x 2-3 x with NH 32 16 HNDV N-H 32 HPR8 - 64 HNDV _ 2 HM 1024 1024 H-M 256 HP 64 HTryp 16

2 x with HM NH HPR8 - 16 HNDV - 16 HM 4 4 58

TABLE IV

Further Comparison of Titers of Normaland Modified Erythrooytes in Antxsera to Normal and Mumps-Treated Red Cells

K . e d Cells TJ sed in Test -^Absorptions Cells Anti-HM Anti-NH

4 x with N H NH m, N-H -- HPR8 32 8 HNDV 64 4 HM 64 - H-M 32 16

4- x with N — H NH N-H - HM 16 H-M 16

*4 x with N H and HPR8 i x with H P R 8 HNDV 2 HM -

x with N H and HPR8 3. x with H N D V HNDV - HM 16

-4 x with N H and N-H 1 x with H M HPR8 - HNDV 4 HM - H-M 2 59

Periodate-treated Rh0(D) cells and normal Rh0 negative red cells reacted to approximately the same titer with the so-called incomplete antibody present in the mumps-cell serum. Mumps-

treated Rh0 negative red cells agglutinated more extensively than did the homologous Rh0 positive mumps cells in this serum, ex­ hibiting several zones of agglutination (Table III). Both normal human Rh0 positive and negative erythrocytes removed from this

serum the agglutinins for themselves and both types of mumps-cell agglutinins, while mumps-treated chicken erythrocytes removed the

RhQ mumps cell agglutinin and the Rh0 negative normal cell anti­ body but left untouched the Rh0 negative mumps cell hemagglutinin.

Even in the presence of anti-globulin serum the positive mumps­

cell did not agglutinate (Table III). From this, it is evident

that mumps-treated chicken erythrocytes, though not agglutinated in this serum, possess some antigenic similarity with the mumps-

treated Rh0 positive red cell and normal Rh0 negative cell, but not the Rh0 negative mumps cell. Furthermore, in this serum

(anti-HM absorbed twice with HPR8) it is obvious that normal Rh negative and periodate-treated Rh0 positive erythrocytes behave

similarly, while in anti-Rh0(D) sera periodate and mumps-treated

Rh positive red cells react apparently in the same manner (Table I).

These differences may be explained if the mumps-treated Rh0 positive red cell surface is considered to be essentially lacking

in RhQ antigen loci, according to the argument presented by Coffin 60

and. Pickles (1951) for the periodate-treatment of Rh0 erythrocytes.

Thus, in the immunized animal during the in vivo degradation of the positive mumps cell, the first specificity to register may be thought of primarily as an Rh0 negative-mumps alteration which is substantiated by the greater reactivity in this serum displayed for the negative mumps-treated red cell and explains the agglu­ tination in the incomplete antibody fraction of the normal negative and periodate-treated Rh0 positive red cells. Since the specific­ ity of the serum is for a mumps-altered cell, the only specificity in common among the immunizing cell, the periodate-positive cell, and the normal negative cell is the apparent lack or reduction of

Rh0(D) antigen configurations on the cell surface. Further degradation of the HM cell in the immunized animal would uncover

Rh0 antigenic loci which in the rabbit register poorly. However, the specificities shared in common among the three viruses in the alteration of red cells is probably due to the fact that they all

share an affinity for the same receptor sites on the cell surface.

In lieu of these facts and speculations it is not odd that mumps-

treated chicken erythrocytes leave only the Rh negative mumps­

cell agglutinin in the incomplete fraction for they possess not only a similar group of viral receptor areas on their surfaces, but a minor mumps-cell alteration (Bigley, 1955) and thus can remove the HM and N-H cell agglutinins but cannot remove the primary specificity from the serum, i.e., that for the negative 61

mumps-treated cell as depicted in Table III. However, as might be expected from this argument, they could reduce the degree or strength of reaction of the H-M cell which it did in the first

zone (zone in which N-H and HP cells agglutinated) though this is not evident due to the nature of the table. To substantiate further this explanation are the observations presented in the data of Table IV. Normal Rh0 positive cell absorption reduced

all modified-cell agglutinins to essentially the same titer while normal negative red cells reduced the mumps-cell agglutinins

(both positive and negative) to the same extent. In direct absorp­

tion procedures, both types of normal cells were able to remove all normal cell agglutinins. Furthermore, mumps-cell absorption of

the anti-HM serum removed virtually all cell-agglutinins (Table

IV) including the reactivity for the mumps-treated RhQ negative

red cell.

Since the saline anti-RhQ agglutinin is heat-labile while

the incomplete form is not, an attempt was made (Table V) to

determine whether the agglutinins in the antiserum to mumps-

treated Rh0 cells were heat—labile. The unabsorbed serum was

heated at 60, 65, and 70°G for varied periods of time as seen in Table V. Heating of the serum at 60OC for 60 minutes seemed

to increase the agglutinins measured i.e., the normal and trypsin­

cell agglutinins. However, this was attributed to the fact that

as a serum is being heated at such elevated temperatures, TABLE V

The Effects of Heat on the Stability of Cell Agglutinins in Antiserum to Mumps-Treated Hnm*m Erythrocytes

Temp Time Test Cell Dilutions of Serum (uc) (Min) Cells Control 2 4 8 16 32 64 128 256 512 1024 2048 ..... 8192

HE •m S S S 4 4 2 _l j _. 60 60 EH - S S s 4 4 3 2 1 - -- 65 120 NH - s S s 4 2 + -- - - - 70 40 BH - 3 3 2 2 1 ------70 110 NH - 4 4 4 3 ------HTryp - S SS S SS 4 4 4 1 - 60 60 HTryp - S S SS s 4 3 2 2 1 + 65 120 HTryp - 4 4 4 4 3 2 1 + - -- 70 40 HTyyp - 4 4 4 4 3 3 - - --- 70 110 HTryp - S S S s S S 4 1 -- - - - HPR8 - S S S 4 4 3 2 1 -- - 70 110 HPR8 - 4 4 4 4 4 3 ------HNDV - S S S 4 4 3 "2 1 - -- 70 110 HNDV - 4 4 4 4 2 2 1 ------HM - S S S 4 4 4 3 3 3 2 2 ..... 1 70 110 HM 4 s s S 4 4 4 2 2 2 2 .....

O r o 63

structural bonds are being broken so that an apparent increase in antibody is measured although actually there is no increase in antibody content in this denaturing process. This rearrangement of protein configurations is more evident when the titers for the trypsin-cell agglutinin are compared. Heating at 70°C for UO minutes reduced the titer from 1:102U to 1:61). but continued heating at this temperature for 70 additional minutes revealed a titer of 1:256. Generally, the normal and modified cell agglu­ tinins were not greatly affected by heat. The slight decrease in titer evident in each instance was -undoubtedly due to the denaturation of more labile linkages. This serum was not like

Rh antiserum it its heat lability for the saline agglutinins were not greatly diminished by heating at 60~70°C for 60-110 minutes.

Since RDE treatment of human or chicken erythrocytes can render such cells inert to the attachment of any of these three viruses. Antiserum against RDE-treated human and chicken red cells, and the eluates resulting from these treatments, were examined for virus-modified cell agglutinins. These results are presented in Tables VI, VII, VIII, and IX and composited in Table

X. It is obvious from these results that the same phenomena observed in antisera to virus-treated cells are present in these sera. However, there is no reactivity for heterologous normal cells, similarly observed by Wallace (1953) between human cell 6U

and chicken erythrocyte antisera. Moreover, the cross-reactivity

for treated cells of both species which Wallace reported is also

evident in these sera. That is, virus-treated human cells were

agglutinated by antiserum to RDE-treated chicken cells, and vice versa. (Tables VI and VIII). This also occurred in the antisera

to eluates from the RDE treatments (Tables VII and IX).

It is interesting that the antiserum to the eluate from RDE-

treated human erythrocytes contained agglutinins for virus-

treated cells to practically the same titer as did the antiserum

to the RDE-treated human cells, but the normal Rh0 positive cell

agglutinin was present in the eluate antiserum in low titer (1:8)

while the Rh negative cell agglutinin was present in a much higher

titer (1:128) as was the positive normal cell agglutinin (1:512)

in the antiserum to the RDE-treated human cells (Table X).

On the other hand, in the antiserum to the eluate from RDB-

treated chicken cells the virus-treated, chicken cell reactivity

was markedly less than that observable in the antiserum to RDE-

treated chicken erythrocytes. In fact, in one instance, the HNDV-

cell agglutinin was present in higher titer than the CNDV-cell

antibody in the antiserum to the eluate from RDE-treated chicken

cells (Table IX). As was observed in the antiserum to mumps-

treated Rh0 positive erythrocytes,, the H-M cell agglutinin in the

antiserum to the eluate from RDE-treated human 0 Rh0(D) cells was

markedly stronger than even the agglutinins for the other virus- TABLE VI

Titrations of Antiserum to RDE-Treated Human Erythrooytes -with Virus-Treated. Red Cells

Test Dilutions of Serum Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 16384

NH - S SSS SS 4 3 1 -- ---

HFR8 - S S S s S s 4 4 2 1 - - - -

HNDV - s S s s s s S SS 4 2 2 2 2

HM - s S s s s s 4 4 4 2 2 1 - -

NO c*

CPR8 _ 4 3 2 1 - _ - — - -_ — - -

CNDV - 4 3 2 2 2 1 + ------

CM _ 2 —_—_------TABLE VII

Titrations of Antiserum to Eluate from RDE-Treated Human (0 Rh0) Erythrocytes with Virus-Treated Red Cell 8

Test Dilutions of Serum Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 16384 32768 65536

tm NH 4 3 1 ------

N-H - S 4 4 4 3 2 1 - - - a s - - a s - a s

HPR8 - S S 4 4 4 3 3 2 2 1 - a s - - --

HNDV - S S S S S S 4 4 3 2 1 + -- - -

HM - S 4 4 4 3 3 3 2 2 2 1 + - -- -

H-M -SS SS S 4 4 4 4 3 3 2 2 2 2 1

M3 as

CPR8 4 2 + — _

CNDV - 3 + ------

CM _ 1 _ —_- — -- - - -

= 8! TABLE VIII

Titrations of Antiserum to RDE-Treated Chicken Erythrocytes with Virus-Treated Red Cells

Test Dilutions of Serum Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096

HC - S S 4 4 4 4 4 2 - - - -

+ CPR8 - s SSSSSS 4 2 - -

CNDV m s SSS s S 4 2 2 + - -

CM mm s S S s s S 4 4 3 2 2 -

NH ------

HPR8 - s 2 1 ------

HNDV - 4 4 2 1 1 + — — — —

O s -J TABLE IX

Titrations of Antiserum to Eluate from RDE-Treated Chioken Erythrocytes with Virus-Treated Red Cells

Test Dilutions of Serum Cells C 2 4 8 16 32 64 128 25 6 512 1024 2048 4096

HC - s s 4 3 ------

CPR8 - s s S 4 3 2 ------

CNDV - s s 4 4 2 ------

CM - s 4 4 4 1 ------

NH m

HPR8 s S 4 2 1 _ _ _

HNDV s 4 4 3 2 2 2 2 1 - - -

mm EM mm * mm *•

O n CD 69 TABLE X

Comparative Titers in Antisera to Human and Chicken Erythrocytes Treated with RDE and The Eluates from Such Cells

Test Anti- Anti- Anti-HRDE Anti -C RDE Cells HRDE CRDE Eluate Eluate

H-H 128

NH 512 - 8 -

NC - 256 - 16

HPR8 1024 8 1024 32

CPR8 16 512 8 64

HNDV 4096 32 2048 512

GNDV 32-64 512 2 32

H-M 32768

HM 4096 - 2048 -

CM 2 2048 2 32 70

treated cells.

No further work was performed using these sera, but it is obvious that the alterations which occurred at the receptor areas, shared with mumps, influenza (PR8), and Newcastle disease viruses, was degraded in such a manner during the treatment of red cells with RDE and the subsequent in vivo breakdown in the immunized animal, that the specificities shown in the antisera to virus- treated erythrocytes are evident. It is interesting that the mumps-treated and the normal, untreated, Rh negative red cells show greater reactivity than did their Rhc positive counterparts in the one serum (anti-HRDE-eluate) in which they were titrated.

RDE is not a single enzyme, but several (a lecithinase, a neur- amininidase, and other unidentified enzymes). Since it is known that under certain experimental conditions this collection of enzymes can render greater alterations of the erythrocyte surface than any of the three viruses studied, it is feasible that RDE can, perhaps, alter the protein portion of the Rh antigens in such a manner that the Rh0(D) negative red cells, which may have more of this moiety exposed, are more reactive with antisera to portions of the red cell surface from Rh positive cells which were lost upon treatment with RDE.

Because of the presence of the incomplete normal cell anti­ body in the mumps-cell antiserum, passive anaphylaxis was attempted in rabbits. One animal received intravenously 1.0 cc. of 71

unabsorbed. anti-HM. Sixteen and one-half hours later this animal was injected with £.0 cc. of a 10 per cent suspension of normal

human 0 Rh0(D) erythrocytes intravenously. At this time there were no visible signs of distress in the animal. Two hours later

an additional f>.0 cc. of the same cells were given to the animal.

Immediate signs of distress were evident and typified by rapid

intercostal breathing and apparent apprehension. Within two

hours the animalfe head had dropped to one side and paralysis was

apparent and death soon ensued. Blood removed by cardiac punc­

ture from this animal shortly before death was dark and clotted

slowly. The serum from this blood inhibited hapamine precipi­

tation in an antihapamine serum. Thus, it was concluded that

the normal cell antibody present in the anti-HM serum could

induce anaphylaxis in rabbits when normal cells were used as the

shocking dose.

Another animal received 2.1 cc. of anti-HM antiserum pre­

viously absorbed twice with PR8-treated 0 Rh0 human red cells.

Sixteen and one-half hours later 5.0 cc. of a 10 per cent suspension

of normal human 0 Rh0 cells were given intravenously to this

animal. At this time the rabbit showed mild shock symptoms. Two

hours later the same amount of normal cells were again admin­

istered to the animal and four hours after this the animal was

given £.0 cc. of a 10 per cent suspension of mumps-treated 0 RhQ

red cells. Mild symptoms of shock were again evident. Four days 72

later, 10 cc. of the mumps-cell suspension was injected intra­ venously and the animal once more showed signs of distress and within 2-3 hours, the ear veins were markedly injected. Seven days were allowed to elapse so that the animal might produce anti­ body to the cells which had been used as shocking doses. The rabbit was then given two shocking doses, each consisting of 10 cc. of 3 per cent suspension of mumps-treated red cells. After the first intravenous injection the animal went into immediate but mild shock which was still prominent at the time of the second injection four hours later. The second injection merely strength­ ened the shock symptoms. Twenty-four hours later, the animal exhibited heavy intercostal breathing, the head was dropped to one side, and the ears drooped. Paralysis of the hind legs preceded convulsions and death followed. After death, it was observed that the liver was enlarged with hemorrhagic areas prominent. The liver also contained small white areas regularly spaced through­ out the organ and it was thought to be connective tissue involve­ ment. Small hemorrhagic areas were also observed throughout the skin of this animal. Table XI demonstrates the sera reactivity of this animal. Prior to any injections, the serum of this animal exhibited merely a 1:U titer for trypsinized red cells. On the

day prior to death, (11th day) the following reactivity of this animal's serum was observed: 1:256 titer for normal cells, 1 :2014.8 and 1:512 for trypsin and mumps-treated cells respectively. TABLE XI

Antibocty' Response to Shock Doses of Mumps-Treated Red Cells in a Rabbit Passively Sensitized with PR8 Absorbed Antiserum to the Homologous Cells

Serum Test Dilutions of Serum Sample______Cells______C 2 4 8 16 52 64 128 256 512 1024 2048 4096

Initial Bleeding NH ------

HTryp -21-

HM

11 days after NH - S s 4 4 4 3 3 1 + 1st Shocking Dose HTryp - s s s 4 4 3 3 3 2 2 1

HM - S s s 4 4 2 2 2 1 - -

12 days after NH - s s 4 3 1 •• 1st Shocking Dos® (Prior to Death) HTryp - s s S 4 4 4 3 3 2 1 — —

HM - s s 4 4 3 3 2 2 2 1 + - 7k

However, shortly before death (12th day), the normal cell agglu­ tinins were reactive only as high as a 1:32 dilution of serum while the treated-cell agglutinins apparently were left intact.

These results again implicate the normal cell agglutinins as being involved in the anaphylaxis.

Since this obvious difference existed in the serologic reactivity of mumps-treated Rh0 positive and negative red cells as seen in Table III, and because of the striking similarity of the actions of periodate and mumps virus on the Rh positive red cell agglutinability in anti-Rh0(D) sera (Table I), eluates from both kinds of treated cells were tested for the capacity to inhibit the agglutination of Rh positive cells by specific antiserum. It seemed likely that Rh0(D) antigen, either part of or in close proximity to the viral receptor of erythrocytes, might be literally removed from the cell surface during viral elution and not de­ stroyed.

The eluates from mumps and periodate-treated red cells inhibited the agglutination of both normal and trypsinized Rh positive erythrocytes by anti-Rh D sera (Tables XIII through XXI).

The reactivities of normal and trypsinized red cells in these Rh0 antisera are presented in Table XII.

The eluates from mumps-treated Rh D positive erythrocytes have been shown to inhibit both saline and incomplete forms of Rh antibody, anti-D or anti-CD (Tables XIII through XVI). This TABLE XII

Titrations of Rh Airti-sera with Normal and Treated Red Cells*

Test Dilutions of Serum Serum Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048

Ortho Plasma NH -22- Albumin

Agg’n Plasma HTryp -SS443 3 2 2 I Anti- Eh0(D)

Lederle Plasma NH - 1 (85*)

Albumin Saline HTryp - S S S 4 4 2 2 W a m Agg‘n Anti-RhQ(D)

Lederle Plasma NH - 4 1 ------(87$

Albumin Saline HTryp - S S S 4 4 3 2 Agg'n Anti-Rh0(D)

* Rh positive Cells Rhg q DE/o —, Rh negative Cells rr cde/ode. TABLE XII (Continued)

Titrations of Rh Anti-sera with Normal and Treated Red Cells*

Test Dilutions of Serum Serum Diluent Cells C 2 4 8 16 32 64 128 2 56 512 102 4 2048

Hylend Pla sina NH - 1 ------Albumin

Agg'n Saline HTryp -SS44432 1 Anti-Rh0(D)

Abs.l x Saline HTryp -S444321 with 0 ode/ode

Abs. 1 x Sal ine HTryp _ 1------with 0 c DE/o— 1 1 I 1 t 1 Lederle Sal ine NH 0 3 M Anti-CD Sal ine HTryp -SSS321- - - - -

* Rh positive Cells Rhg oDE/c—. Rh negative Cells rr ode/cde. TABLE XIX (Continued)

Titrations of Rh Anti-sera with Normal and Treated Red Cells*

Test Dilutions of Serum Serum Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048

Ortho Saline NH . 4 3 3 1 . Anti-D Saline HTryp - SS 4 3 3 3 2 2 - - mm

Saline HM- SS 4 3 2 - - - - - mm

Saline HP ------mm

Lederle Plasma NH 3 •> — _ _ Anti-D (H*o) Plasma H-M ------Saline HM------

Plasma HM - 4 3 3 3 2 - -- - mm

Saline HPR8 - 1 1 ------

Plasma HPR8 - 3. 1'; ------Saline HNDV- 4 2 ------Plasm HNDV - 4 4 4 4 3 3 3 3 3 --

* Rh positive Cells Rhg oDE/c — ", Rh negative Cells rr ode/ode . TABLE XIII

Inhibition of Incomplete Rh0 (D) Antibody by Eluates from Rh0 (D) Cells by Mumps Virus

Treatment of Eluate Dilutions of Eluate after Serum Test* Removal Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

1 j 2 Pla sma NH _ _ _ - a i 2 2 2 2 2 24 hr. 1:16 Plasma NH - - - - - 2 @ 4°C. 2 2 2 2 18 hr. Plasma NH @ 4°C. - S 4 4 4 4 4 4 4 4 4 30 Hr. 1:2 Plasma NH m 4°c. and 40 min. @ 58°C.

* Rhg (c DE/o — ). Eluate from cells treated 100 minutes with mumps virus. TABLE XIV

Inhibition of Anti-Kh0 (D) Albumin Agglutinins* by an Eluate from Rhg (g DE/o —) Ecythrocytes Treated 100 Minutes with Mumps Virus

Serum Test Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

1*2 Plasma NH + 1 2 2 2 2 2

* Ortho. TABLE XV

Inhibition of Saline Agglutinins* Anti-D and Anti-CD by Eluates from Rhg( oDE/o— >) Erythrooytes Treated 100 minutes with Mumps Virus

Treatment of Eluate Serum after and Test Dilutions of Eluate Remom 1 Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

Anti-D Saline NH S 4 4 4 4 4

24 hr. Anti-D Saline NH 1 1 4 4 4 5 5 5 © 4°C.

30 hr. Anti-D Saline NH - S 4 4 4 4 G 4°C. and 40 min. @ 58 °C.

Anti-CD Saline NH

* Lederle, 81

inhibitory activity was retained for at least twenty-four hours on storage at U°C (Tables XIII and XV), but was lost upon storage for 30 hours followed by heating at 58°C for forty minutes (Tables

XIII and XV). The data in Table XVI in which eluates prepared from mumps-treated Rh2 red cells inhibited slightly the saline agglutinins of Ortho anti-D, illustrate that the degree of in­ hibition exhibited by the eluate depends, in part, upon the time of treatment of the cells with virus as well as the presence of active virus in the eluate. When an eluate, prepared by exposing

Rh2 red cells to mumps virus for 180 minutes was first examined for inhibition of Ortho anti-Rh0(D) saline agglutinin partial inhibition was observed in eluate dilutions as high as 1:32.

However, as seen in Table XII, this serum did not possess normal

cell agglutinins in the dilution of serum used in inhibition, though it did contain a strong trypsin-cell agglutinin at the same dilution. This observation was noted especially in this sera and occassionally in other Rh0 antisera and was thought to be due

to the union of other cell structures present in the eluate

combining with antibody for them and subsequent absorption to the red cell. This will be discussed further with subsequent data.

However, when this eluate was stored in the refrigerator for UU

hours and then reincubated for 30 minutes at 37°C complete inhibi­

tion of the Rh serum was observed by eluate dilutions as high as

1:6U with incomplete inhibition being exhibited in eluate TABLE XVI

Inhibition of Anti-Rh0(D) Saline Agglutinins* by Eluates from Rh2 (cDE/o— ) Erythrocytes Treated with Mumps Virus

Time of Treatment in Eluate Prep, and Serum Test Dilutions of Eluate Subsequent Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

Treatment 100 min. 1:2 Saline NH - 1 2 4 4 4 S SSSS

18 hr. @ 4°C. 1:8 Saline NH - 1 1 3 4 4 4 4 4 4 S

£0 hr. @ 4°C. 1:16 Sal ine NH 1 1 1 1 2 2 2 3 4

180 min. 1:32 Saline NH - + 1 1 2 4 4 4 4 4 4

72 hr. @ 4°C. 1:32 Saline NH - 1 2 2 3 3 3 3 3 4 4

96 hr. @ 4°C. 1:16 Saline NH 3 3 3 3 3 3 3 3 3 3

* Ortho. TABLE XVI (Continued)

Inhibition of Anti-RhQ (D) Saline Agglutinins* by Eluates from RhgCcDE/o— ) Erythrocytes Treated with Mumps Virus

Time of Treatment in Eluate Prep. and Serum Test Dilutions of Eluate Subsequent Dilution Diluent Cells 2 4 8 16 32 64 128 256 512 1024

180 min. 1:32 Sal ine NH treatment followed by 44 hr. @ 4°C and |r hr. incubation at 37 °C

Above 1:32 Saline NH material stored sin additional 24 hr. m 4°C.

* Ortho, TABLE XVII

A Comparison of Inhibition of Anti-CD by Eluates from Mumps-Treated Rh Positive and Negative Stromata

Test Dilutions of Eluate in Saline Eluate Cells C 2 4 8 16 32 64 128 256 512

HM NH - 1 1

H-M* NH - 333333 3 3 3

* CdE/cd- Erythrocyte Stromata. 85

dilutions up to 1:512. Thus, it became evident that the mumps- virus left in the eluate may be attached to cell structures lost in the elution process and that upon elevating the temperature, virus is liberated once more with a resultant increase in inhibi­ tory activity displayed by the eluate.

From the observations reported for the eluate material resulting from RDE-treatment of erythrocytes, it is evident that red cell structural configurations other than Rh0 antigen are readily removed from the cell surface when this particular set of viral-hemagglutination receptors are being degraded. Further evidence to support the contention that antibodies other than anti-Rh0(D) are contained in Rh antisera is the observation by

Makinodan and Macris (1955)* that an antiserum to Rh0 possessed agglutinins for cde/cde trypsinized red cells. It is quite logical to assume that in the immunization of an individual with

RhQ(D) antigen that other stromal structures present in the immunizing cell may also register. Such antigens may be the same as or similar to structures present deeper in the stromata net­ work of the immunized individual's own erythrocytes.

Table XVII demonstrates that this inhibition of anti-Rh sera by eluates is a result of the treatment of Rh0 positive red cells and not of negative cells. An eluate from mumps-treated Rh negative red cells did not exhibit any inhibition of the agglu­ tination of normal Rh D positive red cells in anti-CD saline 86

agglutinin, while the eluate from mumps-treated. Rh0 cells inhi­ bited completely the Rh0 cell agglutination by this serum in dilutions as high as 1:128 and partially inhibited this agglu­ tination up to and including an eluate dilution of l:102lj..

Eluates prepared from Rh2 cells treated with periodate were found to exert a similar inhibitory action on the agglutination of red cells containing the RhQ(D) antigen (Table XVIII). This inhibitor was still present after 2k hours storage in the re­ frigerator, but the activity was diminished after an additional

2k hour storage of this eluate. After 72 hours at U°C this periodate anti-RhQ inhibitory activity was lost.

Red cell stromata were also treated with mumps virus and periodate in an attempt of rid the eluates of hemoglobin and to obtain a more potent inhibition of anti-Rh0(D) reactivity by these eluates. Tables XIX through XXIII demonstrate the inhibi­ tion of anti-Rh sera by eluates from mumps-treated Rh0 stromata.

Table XIX demonstrates that eluates from stromata treated with mumps virus for either 180 or 270 minutes inhibited albumin-type agglutinins, the eluate from the 180 minute treatment exceeding that of the 270 in inhibitory capacity. The stroma control, which consisted of eluate from RhQ stromata, incubated for 180 minutes with saline, did not exert any inhibition of the Rh0 agglutinins

(Tables XIX, XX, and XXI). Thus, the inhibitor of Rh0 activity was not a substance solubilized and released in suspension but TABLE XVIII

Inhibition of Anti-Rh0(D) Sera by Eluates* from Rhg Red Cells Treated for 30 Minutes with Periodate

Test Dilutions of Eluate Serum Eluate Diluent Cells C 2 4 8 16 32 64 128 256 512~ 1024

Incomplete 1:2 - Plasma NH 2 2 2

- 18 hr. @ Plasma NH 1 2 2 4°C.

1:16 24 hr. B Plasma NH - - - - 1 1 3 4°C.

Saline Agg’n 1:2 24 hr. @ Saline NH - 1 3 S S 4 (Lederle) 4°C

Saline Agg'n 1:16 Saline NH 1 1 2 3 3 3 3 4 4 4 (Ortho) 1:32 Saline NH + 2 2 2 2 2 3

1:32 48 hr. © Saline NH - - _ + 2 2 2 2 2 2 2 4°C.

1:16 72 hr. © Saline NH - 3 3 3 3 3 3 3 3 3 3 4°C .

* No Periodate inhibitor added. TABLE XIX

Inhabition of Albumin Agglutinins* Anti-Rho (D) by Eluates from Mumps-Treated RhQ Stromata

Time Eluate Serum Test Removed (Min) Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048.... + 1 + 1 + 1 180 1*2 Plasma NH - - ■■ i i + 1 1 1 270 1:2 Plasma NH - - - f 2 2 2 3 1 + 1

Stromal 1:2 Pla sma NH - 3 Control (180)

* Ortho.

as Co 89

resulted, from the action of mumps virus on the Rh0 stromata.

Greater inhibition of saline agglutinins (Table XX) was evidenced with the stromata eluates than -with the cell eluates (Table XVI).

When the stromata-mumps mixture was allowed to incubate for 100 minutes, the eluate from the suspension was capable of inhibiting completely the one plus agglutination (recorded in Table XII) of normal Rh0 cells. When the treatment was allowed to proceed an additional 80 minutes, the resulting eluate partially inhibited a three plus agglutination of normal Rh0 cells. However, there was an obvious "zone" effect in the measurement of the inhibition of the trypsin-cell agglutinin by eluates produced by treatment of cells for 120, 225, and 330 minutes. As noted in Table XX, only partial inhibition of anti-D occurred in the lower dilutions of these eluates while inhibition was marked in higher dilutions

(1:8-1:512). Because these same eluates inhibited other anti-

Rh0(D) sera completely without any such zones, this irregularity was attributed to factors present in this particular serum (Ortho) and their reactivity with the eluate. Greater inhibition by eluates from mumps-treated Rh0 stromata of the trypsin cell agglutinin in this Ortho saline anti-Rh0 occurred with an eluate removed after 225 minutes from the reacting mixture (Table XX).

The variance in time of treatment of the stromata as well as the differences in inhibitory capacity of the mumps-stromata eluates is best characterized in a comparison of the results 90

in Tables XXII and XXIII® Mumps-Rh0 stromata eluates obtained at

100 minutes of treatment completely inhibited the Ortho anti-CD albumin agglutinins in a dilution of 1:5>12 (Table XXIII), while the Lederle anti-D albumin agglutinins were completely inhibited in a dilution of 1:2, but partially up to and including an eluate dilution of 1:128 (Table XXII). Treatment of Rh0 stromata with mumps virus for 180 minutes yielded eluates which inhibited the anti-CD completely up to a dilution of 1:8 and partially up to

1:128 (Table XXIII) but which inhibited the Lederle anti-D completely up to a 1:256 eluate dilution and partially up to

1:201*8. Treatments of Rh0 stromata with mumps virus for 120, 225, and 330 minutes did not produce eluates which were as potent as that produced after 180 minutes treatment in the inhibition of the

Lederle anti-D (Table XXII). However, here again it is observable that when the eluates were only partially inhibitory, zones of varying strengths of agglutinability were evidenced. Thus, it is obvious that the sera used to detect this inhibition of Rh0 activity also varied greatly in the types of reactive antibodies for normal cell structures. These variables are just as pronounced in the eluates prepared from periodate-treated Rh0 stromata (Table XXIV).

The effects of increased temperature on the inhibitory capacity of eluates from mumps-treated RhQ stromata are shown in Tables XXV and XXVI. If eluates from cells treated for 100-

105 minutes were raised either slowly or immediately to 53-5U°C TABLE XX

Inhibition of Anti-Rh.Q(D) Saline Agglutinins* by Eluates from Mumps-Treated RhQ Stromata,

Time Eluate Serum Test Dilutions of Eluate Removed (min) Dilution Diluent Cells C 2 4 8 32 64 128 266 612 1024 2048 4096

180 1:16 Plasma NH

270 1:8 Plasma NH - 1 1112 3 3 4 4

Stromal Control 1:8 Plasma NH - 3

120 1:128 Saline HTryp 1 - 1 2

225 1:128 Saline HTryp - 2 1 « _ _ _ + +

330 1:128 Saline HTryp - 1 - - - 2 3 3 2 1

* Ortho. TABLE X L l x

Inhibition of Anti-Rh0 (D) Albumin Agglutinins by Eluates from Mumps-Treated RhQ Stromata

Time Eluate Serum Test Removed (min) Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 102'

100 1*2 Plasma "NH 1

180 1*2 Plasma NH 1

180 and 20 hr. © 4°C. ls328 Saline HTryp -- - -- 1 2 2 2 4 4

Stromal Control 1:128 Saline HTryp - 3

120 1:128 Saline HTryp ------

225 1:128 Saline HTryp - 3 3 3 33 3 3 3 3

330 1:128 Saline HTryp -- - - + 2 2 3 3 3

60 1:128 Saline HTryp - + 1 2 3 3 3 3 3 3 3

60 (Absorbed with ode/cde 1:128 Saline HTryp _ + + 3 3 3 3 3 3 3 3 Cells)

* Hyland. TABLE XXII

Inhibition of Anti-Rh0 (D) Albumin Agglutinins* by Eluates from Mumps-Treated Stromata

Time Eluate Serum Test Dilutions of Bluate Removed (min) Dilution Diluent Cells C 2 4 8 16 52 64 128 256 512 1024

100 1:2 Plasma NH --11122 2 3 3 3

180 1*2 Plasm NH ------l 1 1

120 1:64 Saline HTryp -122222 2 2 3 4

225 1:64 Saline HTryp - 233342 2 2 2

330 1:64 Saline HTryp -1111112 1 1 1

* Lederle. TABLE XXIII

Inhibition of Anti-CD Albumin Agglutinins* by Eluates from Mumps-Treated RhQ Stromata

Time Eluate Serum Test Dilutions cf Eluate Removed (min) Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

100 1:2 Plasma NH - - - - 1

180 1:2 Plasma NH - - - 1 1 1 1 2 2 2 2

* Ortho, TABLE XXIV

Inhibition of Anti-Rho Sera by Eluates* from Rh0 Stromata Treated 60 Minutes wiih Periodates

Test Dilutions of Eluate Serum______Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048 409

Anti-D Incomplete Plasma NH ----

Ortho Albumin Agg’n ls2 Plasma NH -1334444 4 S S

Ortho Saline Agg’n Saline NH -2233333 3 3 3

" (1:128) Saline HTryp ------2

Lederle Albumin Agg’n (1:64) Saline HTryp ------1 1 1 1 2

Hyland ALbumin Agg’n (1:128) Saline HTryp ------1

* No Periodate Inhibitor Added. 96

the inhibitory capacity for Rh0(D) reactivity was not lost as shown by inhibition titrations of such eluates with Hyland albumin anti-D, Lederle incomplete anti-D, and Ortho saline anti-D. It is interesting that the zones of inhibitory action so charac­ teristically depicted in this Ortho saline type anti-D, when measured for inhibition of the incomplete anti-Rh0 agglutination of trypsinized cells, were abolished when such eluates were heated at i|[)..5>0 C for 80 minutes. Thus, it may be inferred that these eluates contain other soluble stromata antigens which are more heat-labile than the Rh0-like material that was removed from the cell during treatment or that the elevated temperature served to speed up the elution process and simultaneously denatured the combining areas of the viral particles so that the inhibitory property of the eluate was no longer masked. Heating the mumps- stromata eluate at 100°C for three minutes completely destroyed anti-Rhc inhibitory capacity of the eluate (Table XXV). Further­ more, absorption of these eluates with Rh negative (cde/cde) erythrocytes at IjPC to remove free viral particles did not diminish the inhibition of Rh0 antisera by the eluate material

(Table XXV).

The data in Tables XXVI and XXVII show that heating the mumps-

RhQ stromata eluate at 5>U°C for 60 minutes to destroy or partially denature the mumps virus present, stabilized the Rh0 inhibitory activity of the eluate for at least two days in contrast to the TABLE XXV

The Effects of Increased Temperatures on and Absorption with cde/cde Cells of Eluate from Mumps-Treated Rh0 Stromata

Anti-D Test Dilutions of Eluate in Saline Eluate Serum* Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096 8192 16384

From 100 min. Hyland HTryp Treatment Lederle HTryp ------+ + + 3 3 Ortho HTryp - 1 - - 2 2 3 3 3 1 1

+80 min. @ 44.5°C Hyland HTryp + Lederle HTryp 2 2 Ortho HTryp + 1 +30 min. @ 53°C Hyland HTryp + 1 1

Lederle HTryp 2 Ortho HTryp 1 1

+ Abs. @ 4°C with Hyland HTryp + 2 2 2 2 cde/cde oells Lederle HTryp 2 7 2 2 Ortho HTryp 1 2 2

Eluate from 100 min. Hyland HTryp - 3 3 3 3 3 3 3 3 3 3 heated for 3 min. at 100°C

* Ijl28 Byland albumin agglutinin in saline, 1:64 Lederle albumin agglutinin in saline. 1:128 Ortho saline agglutinin in saline. TABLE XXVI

The Effects of Storage on the Inhibition of Hyland Anti-RhQ(D) Albumin Agglutinins by an Eluate from Rh0 Stromata Treated for 105 Minutes with Mumps Virus

Tin© of Storage © 4°C. Serum Test Dilutions of Eluate Eluate (days) Dil'n. Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096

-- 1:2 Plasma NH + + 1 1

+60 min. © 54°C. - 1*2 Plasma NH + + 1 1 tt - 1:128 Saline HTryp + 1

ii 1 1:128 Saline HTryp + +

n 2 1:128 Saline HTryp + 1 1 1 2

n 6 1:128 Saline HTryp + - + + + 1 2 2 2 2

fi 28 1:128 Saline HTryp 2

vo oo TABLE XXVII

The Effects of Storage on the Inhibition of Hyland Anti-Rh0(D) Albumin Agglutinins by an Eluate from Rh0 Stromata Treated for 28-g- Hours with Mumps Virus

Time of Storage © 4°C. Serum Test Dilutions of Eluate Eluate______(days) Dil»n Diluent Cells C 2 4 8 16 52 64 128 256 512 1024 2048 4096

m t - 1*2 Plasma NH 1111111 1 1 1 1 1

60 min. 54°C1 - 1:128 Saline HTryp - 1 1

tt 1 1:128 Saline HTryp - + 1

n 2 1:128 Saline HTryp - + 1

it 6 1:128 Saline HTryp - 1111111 1 1 1

it 28 1:128 Saline HTryp - 2 100

unheated red-cell eluates (Tables XIII through XVI). However, by six days storage at U°C the Rho(D) inhibitory activity of

such eluates was greatly diminished and was completely absent

after a month of storage in the refrigerator.

A similar comparison of the storage stability of the

periodate-Rho stromata eluate containing the oxidative periodate

inhibitor to stop any further action of eluted cell material by

periodate ions is presented in Table XXVIII. In Hyland albumin

anti-D the periodate Rh0(D) inhibitor exhibited the same inhibi­

tion (1:20^8 eluate) after 2h hours storage in the refrigerator

as it did when freshly prepared. After H8 hours storage, the

inhibitory capacity of such an eluate was greatly diminished. On

the sixth day of storage the inhibitory capacity of periodate-

Rho stromata eluates further diminished and the 28th day revealed

only partial inhibition of the anti-D serum. This was thought to

be the result of the presence of the glucose-citrate oxidative

inhibitor added to the eluate. This material, per se, exhibited

slight inhibition of anti-Rh0(D) reactivity (Table XXIX).

Eluates of Rh D positive and negative cells and stromata

treated with influenza (PR8) virus, NDV, trypsin and RDE did not

inhibit the reactivities of the various anti-Rh D antibodies.

Some of these inhibition tests are presented in Table XXIX.

Furthermore, chorioallantoic fluid with (Table XXIX) and without

mumps virus did not exhibit any inhibitory activity in similar TABLE XXVIH

The Effeots of Storage on the Ahti Eh Inhibition* of an Eluate from Eho Stromata Treated for 30 Minutes with Periodate

Time of Storage e 4°c. Serum Test Dilutions of Eluate (days) Dilution Diluent Cells C 2 4 8 16 32 64 128 256 512 1024 2048 4096

- 1:2 Plasma NH +

- 1:128 Saline HTryp + +

1 1:128 Saline HTryp _+ 1

2 1:128 Sal ine HTryp - - + 1 1 1 1 + + 2 - +

6 1:128 Saline HTryp - - + 1 + 1 1 2 2 2 2

28 1:128 Saline HTryp - 1

* Byland, TABLE XXIX

Inhibition Titrations of Anti-Rh Sera with Eluates from Erythrocytes Treated with Other Viruses and Enzymes

Test Dilutons of Eluate Eluate Antiserum Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

H-M Anti-D Incomplete Plasma NH 1 2 2 2 2 1*16

Lederle Anti-D 1*2 Sal ine NH 3 S 4 4 4

HPR8 Anti-D Incomplete Plasma NH 2 2 3 3 3 3 3 3 3 3 1*16

Lederle Anti-D 1:2 Saline NH 2 2 2 2 2 2 2 2 2 2

HNDV Anti-D Incomplete Plasma NH 2 2 2 2 2 2 2 2 2 2 1*16

Lederle Anti-D 1*2 Saline NH 4 4 4 4 4 4 4 4 4 4

HTryp Anti-D Incomplete 1*2 Plasma NH 2 2 2 2 2 2 2 2 2 2

Ortho Anti-D 1*8 Saline NH 4 4 4 4 4 4 4 4 4 4

HRDE Anti-D* 1*2 Saline NH 4 4

H-RDE Anti-D* 1*2 Saline NH 3 2 102

* Knickerbocker's Saline Anti-Rho(D). TABLE XXIX (Continued)

Inhibition Titrations of Anti-Rh Sera with Eluates from Erythrocytes Treated with Other Viruses and Enzymes

Test Dilutions of Eluate Eluate Antiserum Diluent Cells C 2 4 8 16 32 64 128 256 512 1024

CM Ortho Anti-D Saline HTryp - 3 1*128

Hyland Albumin Saline HTryp - 2 Agg’n 1:128

Lederle (85%) Plasma HTryp - 3 Anti-D 1:64

HTryp-M Ortho Anti-D 1:8 Saline NH 2 2 2 2 2 2 2 2 2 2

Ortho Anti-D 1:2 Plasma NH 2 2 2 2 2 2 2 2 2

Chorioallantoic Ant i-D 1:2 Plasma NH 2 2 2 2 2 2 2 2 2 2 Fluid Containing Mumps Virus Ortho Anti-D 1:16 Saline NH 4 4 4 4 4 4 4 4 4 4

Periodate Byland 1:128 Saline HTryp - _ 1 2 2 Inhibitor Anti-D

Ortho 1:128 Saline HTryp - 2 3 3 Anti-D ioU

titrations. However, there were variations in the amount of agglutination in these titrations with the eluates from the treated cells as compared with the control sera titrations (Table XXII).

As has been mentioned previously, these treatments are quite cap­ able of removing from the cell or stromata configurations, materials which may be antigenic and thus reactive with common cell antibody contained in anti-Rh0 sera. The modified-cell agglutinins present in anti-Rh sera will be reported later in this work.

Erythrocytes which contained only C and E Rh antigens were treated with periodate or mumps virus. These eluates did not inhibit agglutination of Rh positive I) cells by anti-Rh sera

(Table XVII). Conversely, eluates from Rh D treated cells did not inhibit agglutination of other Rh and Hr antigens with their specific antisera (Table XXX). In Table XXX, it may be noted that periodate-Rho stromata eluate partially inhibited the agglutin­ ation of anti-E. For the lack of a better explanation, this anti-

E inhibition may also be due to the added oxidative periodate inhibitor.

A further demonstration of the specificity of reaction of eluates from mumps-treated and periodate-treated stromata with

Rh antibody was the visible precipitates in rings tests with anti-

Rh0(D) sera containing either saline agglutinin or the incomplete form plus eluate. More precipitate was observed with eluates from mumps-treated cells and antiserum (Table XXXI). Also, 10$ TABLE XXX

Inhibition Tests of Anti-o, Anti-C, and Anti-E with Eluates from Mumps-Treated and Periodate-Treated Rh0 Stromata

Ortho Sera Rh Type Extent of Eluate Diluent Undiluted Test Cells Agglutination

Saline Anti-C CDE/o — 3-4 Plasma Anti-c cde/cde 2 Saline Anti-E CDE/o— 4 Saline Anti-C CDE/c— 3-4 PM Plasma Anti-o cde/cde 2 Saline Anti-E CDE/c— 4 Saline Anti-C CDE/o— 3-4 M28i Plasma Anti-c c de/ode 2 Saline Anti-E CDE/c— 4 Saline Anti-C CDE/c— 3-4 Periodate Plasma Anti-c cde/cde 2 Saline Anti-E oDE/c— +1

PM Eluate from 1 volume of packed stromata j 9 volumes virus material incubated at 37°C for 100 minutes, Subse

M28-g- Only variation from above eluate was initial 28^- hours of treatement.

Periodate 1 volume packed stromata: 1 volume 0.01M KIO and diluted to 5.0 cc. Treatement for 30 minutes at room temperature after which 5.0 cc of Periodate oxidative inhibitor were added and the mixture incubated an additional 30 minutes. The eluate was stored for 48 hours at 4°C. 106

TABLE XXXI

Precipitin Tests with. Mixtures of Eluates Prom Mumps- or Periodate- Treated Erythrocytes and Anti-Eh0(D) Sera

Serum______Eluate______Precipitation

Anti-CD HM Erythrocytes +

HM Stromata +

H-M Stromata

Anti-D HM Stromata +

H-M Stromata

Chorioallantoic Fluid containing Mumps Virus

Anti-D (1j2) HM Stromata +

H-M Stromata

Chorioallantoic Fluid containing Mumps Virus 107

precipitation occurred in mixtures of eluates and dilutions of anti-D serum as high as l s 6 U and 1:102U (Table XXXII). An eluate from mumps-treated Rho negative stromata in precipitin titration with Lederle anti-D saline agglutinin did not exhibit any pre­ cipitation (Table XXXII). The fact that no precipitate was formed in ring tests performed with Rh0 antisera and mumps virus material (Table XXXI) lessened the possibility that the virus in the RhQ inhibitory eluates was precipitating with anti-viral antibody in the various human sera. Furthermore, sensitization of normal Rh0 positive erythrocytes with non-agglutinating di­ lutions of anti-Rh0 sera did not inhibit these cells from being agglutinated by mumps virus (Table (XXXIV).

The Ortho saline anti-Rh0(D) was titrated in dilutions from

1:8 through 1:128 with various dilutions of eluates from mumps and periodate-treated RhQ stromata (Table XXXIII). The eluates had been stored for 2l± hours in the refrigerator prior to use.

The same enhancement of agglutination noted previously, with regard to Table XVI, occurred with the dilutions of eluates in serum dilutions beyond 1:8. Beyond serum dilutions of 1:6U, this enhancement was minimal. Storage for an additional 2i| hours at U°C markedly decreased this agglutination enhancement as seen in Table XXXIII.

Thus, it seemed that some other factor in these eluates which was labile upon storage enhanced the agglutination of normal red TABLE XXXII

Precipitin Titrations of RhQ Antisera with Eluates from Mumps-Treated Rh0 Positive and Negative Stromata

Dilutions of Serum Serum______Eluate______C 2 k 8 16 32 6It 128 2g6 $12 102k

Lederle Anti-D HM - + + . + + + + + + +

Lederle Anti-D H-M ------

Inland Anti-D HM - + + + + + + - TABLE XXXIII

Enhancement of Agglutination of Normal RhQ(D) Cells in Dilutions of Specific Antiserum** by Eluates of Erythrocytes Treated with Periodate or Mumps Virus

Serum NH Cell Test Dilutions of Eluate in Saline Dilution Eluate Serum Cells C 2 4 8 16 32 64 128 256 512 1024

1:8 HM 3 NH . 3 3 3 3 3 3 3 3 3 3 1:8 HP 3 NH - 3 3 3 3 3 3 3 3 3 3 1:16 HM 1 NH - 4 4 4 2 2 2 2 2 2 2 1:16 HP 1 NH - 4 3 3 1 3 1 2 2 3 1 1:32 HM - NH - 3 3 2 2 2 2 2 2 2 2 1:32 HM* - NH - 3 3 2 ------1:32 HP - NH - 4 4 2 1 ------1:32 HP* - NH - 2 1 ------1:64 HM - NH - 4 3 2 2 2 2 2 2 2 2 1:64 HM* - NH - 3 1 1 ------1:64 HP - NH - 2 2 1 1 2 2 2 2 2 2 1:64 HP* - NH - 2 ------1:128 HM - NH - 2 1 1 ------1:128 HM NH +

" ''''

HM Eluate stored 24 hours at 4°C. HM* Eluate stored 48 hours at 4°C. HP Eluate stored 24 hours at 4°C. HP* Eluate stored 48 hours at 4°C.

** Ortho Saline Agglutinin. 110

TABLE X X H V

The Effects of Anti-Rh0(D) on Hemagglutination by Mumps Virus

Serum Used as Sensitizer Viral Hemagglutinin Titer (Mumps Virus) 1 vol. cells: $ vol. serum Rh0 Positive Cells RhQ Negative Cells

- 1:32 - l:6ii 1:32

1:128 Albumin Agg«n Anti-Rh0(D) Hyland 1:32 1:32

1:128 Anti-D Ortho 1:32 1:16 - 1:32

1:32 Anti-HM abs. with NH 1:32 - 1:6U

1:32 Anti-HM abs. 2x with HPR8 1:6^ - 1:128 1:61* Ill

cells in dilutions of anti-Rh serum. This undefined activity of these eluates was not the mumps virus for this property was evidenced also in the periodate eluate. The titer of mumps virus used in the preparation of this eluate was 1:$12 both prior to and after the treatment of the stromata. If this agglutination enhancement was due to the viral-red cell agglutination, the control system, devoid of serum, containing eluate and cells also should have been agglutinated. It was not.

The data in Table XXXIV was an attempt to discern whether or not Rh0 positive and negative erythrocytes upon sensitization with anti-Rh0(D) sera or the incomplete normal cell antibody in antiserum to mumps-treated Rh0 cells would interfere with the attachment of mumps virus. Obviously, they did not, or if they did, the experimental conditions were such that the interference was not detected.

In general, in the preparation of RhQ inhibitor from mumps- treated Rh0 stromata, maximum inhibition appeared with eluates from stromata treated for 1^, 3j and $ hours. However, as pre­ viously mentioned, this measure depends, in part, on the serum used in the inhibition titration. Eluates obtained after 2 and k hours treatment exhibited relatively poor inhibitory capacity.

It appeared that the viral action on the material eluted early may continue and that these apparent "bursts" of inhibitory act­ ivity imparted to the eluate may be nothing more than new 112

additions to the supernatant of the anti-D inhibitor as it is released from its stromal orientation. This could, in part, account for the instability of the inhibitor.

Preliminary attempts were made to stabilize this inhibitory property of the mumps type eluate. Such attempts were aimed at the removal of viral activity: heating the inhibitory eluate at

53-35°C for 30-80 minutes to destroy the enzyme-like action of the virus and adsorption at U°C of such eluates with 0 Rh negative erythrocytes. Nevertheless, the inhibitory property was stable only for a period of 2-3 days at U°C and thereafter its activity rapidly declined. Likewise, periodate-cell eluates exhibited little inhibition of anti-Rh D sera after 2-3 days. It, there­ fore, seemed likely that the instability of these anti-Rh D inhibitors was a property of the inhibitor material itself. Re­ gardless of their instability, these eluates, when freshly pre­ pared, have repeatedly inhibited the agglutination of the saline agglutinin, the trypsin-cell antibody, and plasma agglutinins in anti Rh0(D) sera.

One common feature possessed by viruses of the mumps-

Newcastle disease-influenza hemagglutinating group is their adsorption to the same receptor areas on the erythrocyte surface.

According to the work of Burnet, Gottschalk and others (19!?3)> this is apparently an adsorption of complementary configurations.

In investigations concerning the nature of the virus receptor of 113

susceptible red cells they have demonstrated that as a result of the enzyme-like action of the viral particles during the elution process, certain chemical entities are removed from the cell to

the supernatant. Such substances were found to be various hexoses, hexosamines, and a material identified as 2~carboxypyrrole.

Various reports by others and the results previously presented indicate that the action of influenza and Newcastle disease viruses on the receptor material increases the agglutinability of

Rh positive cells in anti-Rh D sera. To the contrary, mumps virus acts as does periodate to decrease agglutinability. Unlike most

oxidizing agents, periodate is relatively selective in its action,

splitting the 1,2 alpha glycol groupings so abundant in carbo­

hydrate material. Thus, it may be inferred that mumps virus at

the time of elution acts specifically on a polysaccharide material, which, when liberated from Rh positive cells, inhibits anti-Rh D

agglutinins. Substantiating the carbohydrate nature of the Rh D

inhibitor material is the observation that the periodate oxidative

inhibitor, the d-glucose-citrate mixture, also exhibits slight

inhibition of the Rh D antisera. This material is similar in

structure to the hexose configurations that Gottschalk describes.

Mumps virus selectively removes anti-Rh D inhibitory material

from its stromal position. Treatment of erythrocytes with the

receptor destroying enzyme (RDE) of Clostridium welchii in which

presumably all viral receptor material had been destroyed, T&BLE XXXV

Titrations of Saline Anti-RhQ(D) with RDE-Treated Red Cells

Test Dilutions of Serum in Saline Cells C 2 4 8 16 32 64 128 256 512 1024

HRDE - 4 4 4 1 - - - - - t m

HRDE* - 3 2 1 + ------

H-RDE - 4 2 2 ------m t

H-RDE* ------

RH - 3 3 1 ------

N-H ------t m - - --

HRDE and H-RDE 1 part oelbs: 2 parts crude RDE (Cl.welohii) for 40 min. at room temperature.

HRDE*and H-RDE* 1 part cellss 2 parts RDE (V. cholerae) for 90 min. at room temperature.

* Knickerbocker. 115

rendered both Rh D positive and negative erythrocytes agglutinable in anti-Rh D serum (Table XXXV). However, RDE from Vibrio cholerae did not affect Rh negative cells in this manner nor did it much alter the reactivity of Rh0(D) cells in this serum, (Table XXXV).

Eluates from RDE (Clostridium welchii) treated Rho positive and negative red cells formed visible precipitates in the same anti-

D serum.

Such evidence suggests the presence in Rh sera of anti­ bodies to normal cell constituents in addition to the Rho antigen.

Thus, it appeared that the selectivity of the action of the mumps virus on the red cell surface is the specific removal of Rh D haptenic material and that the action of RDE, which is known to encompass and even surpass the action of any of these viruses on erythrocytes, involves the ultimate in proteolysis of the red cell stromal configurations. In order to study this further, agglutination-absorption procedures involving virus and enzyme- treated erythrocytes in several anti-Rh0(D) sera were performed.

Since it was suggested previously that anti-Rh sera possess anti-erythrocyte antibody other than anti-Rh0(D) and that such antibody may represent a response to antigenically modified red cells, anti-Rh sera were examined for antibodies to trypsinized and virus-treated Rh-(Hr), as well as Rh/(Rh0) erythrocytes.

The data presented in Table XXXVI show that a commercial anti-Rho

(D) serum (Knickerbocker) possessed demonstrable saline agglutinins 116

for both normal and treated Rho cells as well as for Rhc- negative erythrocytes treated with the RDE obtained from Clo­ stridium welchii. As shown previously the RDE of Vibrio cholerae did not render Rh negative erythrocytes agglutinable in this same serum. The order of reactivity in this unabsorbed anti-Rho serum for Rho cells was HNDV, HTryp, HRDE, HFR8, and NC. The Rh negative normal and PR8 and trypsin-treated negative cells did not agglutinate in this serum either in saline or in the plasma diluent. Mumps and periodate-treated Rho erythrocytes did not agglutinate in saline dilutions of this antiserum, but did in plasma dilutions as did the Rh0-negative NDV-treated red cells.

It is interesting to note that the agglutinability of H-NDV cells in plasma occurred in serum dilutions beyond those in which the normal Rh0 cells agglutinated and this agglutination of negative

NDV red cells was stronger in dilutions beyond the Rho-trypsin cell titer.

Thus, it was evident that this Rh-antiserum contained agglu­ tinins for red cell antigens other than Rh0(D) as evidenced by the reactivity of Rh-negative erythrocytes treated with either NDV or

RDE. Although NDV-treated chicken red cells did not agglutinate in saline dilutions of this anti-Rho, they did in plasma dilutions and peculiarly in a 1:128 dilution of serum in albumin. This incomplete type of agglutination of the NDV cell, further in­ dicates the presence in this serum of antibody for cellular TABLE XXXVI

Titrations of Anti-RhoOD) Serum with Normal and Virus Treated Rho(I3) Positive and Negative Erythrocytes

Test Dilutions of Serum Cells C 2 h 8 16 32 61; 128 256 512 1021; m> NH 3 3 1 mm m m NH* - 2 1 1 - - — - -- N-H HI H i t mm mm HTryp mm h h k 3 3 1 a s HTryp* mm h h h h 3 2 aa -- ~ H-Tryp ------~ H-Tryp* cHP r 4. HP* 1 _ HM m m + ------HM* - 2 2 2 1 - - - mm - mm TT ■»/ n-H mm __ _ HPR8 mm u k 1 H-PR8 mm ------HNBV - h 3 3 3 3 2 2 2 1 1 H-NBV ------H-MDV* - - -- - + 2 3 S k 1 CNDV ------CNDV* - 3 U h 3 3 3 3 3 3 2 CNDV** ------1 -- - HRIE - s k h 2 1 - -- -- H-RBE mm h 2 2 ------* Plasma diluent. ■shs- Albumin (Bovine) 1:10 diluent. (Knickerbocker Anti-Rho(D)). 118

structures other than Rh0 and because of this CNDV agglutinin it would seem that such structures are probably those of the viral

receptor. The peculiar reactivity of NDV-treated chicken cells

in albumin dilutions of this anti-Rho may be explained in several ways. Firstly, the albumin was not as viscous a suspending men-

strum as was the plasma. Secondly, the pooled human 0 plasma,

though possessing no demonstrable agglutinins for any of the normal

or treated Rho and Hr cells, could have contributed to these

agglutinating systems undefined factors which enhanced agglutina­

tion. Nevertheless, the one tube agglutination of CNDV cells in

a 1:128 albumin dilution of serum occurred in the next dilution

beyond the trypsin Rh0 cell titer and further indicates a possible

interference in the demonstration of anti-Rho antibody with anti­

body for other cell antigens as was evident in the H-NDV cell

reactivity in this serum. Thirdly, the demonstration of such

antibody in Rh-antiserum by a heterologous species cell should

be more difficult since the only defined similarity between

antigen configurations on chicken and human erythrocytes is the

viral receptor, and thereby, antibody concentration would be of

great importance in the detection of agglutinins.

When this serum was absorbed with normal Rho red cells, the

saline and plasma agglutinins were removed for all red cells

exception those treated with NDV (Table XXXVII). The HNDV titer

was practically unaffected by this absorption further indicating TABLE XXXVII

Demonstration of Hemagglutinins to Virus-Treated Rh^ Positive and Negative Erythrocytes in Anti- RhQ Serum after Che Absorption with RhQ Cells

Test Dilutions of Serum Cells C 2 k 8 16 32 6U 128 256 512 102U

NH mm NH* ------N-H ------mm HTryp mm ------H-Tryp ------•• --- - HP ------HP* ------HM ------mm HM* --- - m m - mm - -- mm H-M _ _ »• am _ mm _ __ mm HPR8 —_ __ - — mm _ • mm mm H-PR8 — -• - -— - - —— mm HNDV — h h 3 2 2 2 1 1 + mm H-NDV* - h 3 3 2 2 2 1 1 + + H-NDV 3 3 2 ------HRIE - - - - -— - - -- - H-RIE — —— • mm —

* Plasma diluent. 120

that this agglutinin in anti-Rho serum for NDV-treated cells has little to do with Rh0 reactivity. Furthermore, this absorbed serum agglutinated negative NDV-treated red cells in saline dilutions as high as 1:8 and in plasma dilutions as high as the saline agglutinin titer for the positive NDV-treated cell (1:256). Thus, once Rho reactivity was removed from this Rho antiserum, the form­ erly incomplete agglutinin for the H-NDV cell was demonstrable to a certain extent as a saline agglutinin.

In contrast to this NDV-reactivity in the Rh antiserum after absorption with Rh0 cells, absorption of the same serum with Hr

(Rh negative) red cells, removed all agglutinability for the H-NDV cell in both saline and plasma, as well as removing a considerable portion of the HNDV cell agglutinins. Since normal Rh negative red cells were used as the absorbing cell, they shoulb be expected to remove any extraneous antibody for red cell structures other than Rh0(D) antigen, and thereby render the serum specific in its reactivity for the Rh^D) structures. According to the data presented in Table XXXVIII, this occurred. The reactivity now demonstrable for Rh0 erythrocytes treated with NDV in this serum was that of Rho(D). There was no reactivity evident for Rh negative cells after this absorption. However, the titer for normal Rh0 cells was slightly diminished; the trypsin-Rho cell titer remained essentially the same as in the unabsorbed serum; the PR8-Rho cell titer increased to parallel that of trypsin cell 121

reactivity} and the most marked finding was the demonstration of mumps-Rh0 cell agglutinability to the same titer as seen for trypsin and PR8-treated Rh positive red cells. This observation affords an explanation for the incomplete agglutination of HM cells in the unabsorbed serum. Obviously, the mumps-treated Rho positive cell, reacted with anti-Rh serum as did trypsini2ed erythrocytes according to the data in this table (XXXVIII), but only after the removal by normal Rh negative red cells of some unknown antibody. Since this HM blocking antibody did not agglutinate HM cells nor Rh negative normal cells, it must be incomplete and the same as the NDV-cell antibody demonstrable in unabsorbed serum and in serum absorbed with normal Rh positive red cells.

The HNDV agglutinins observable in the unabsorbed serum must be composed mainly of antibody molecules for Rh negative normal cells and a little Rh0(D) antibody. This latter antibody is evident in Table XXXVIII, and the data also indicates that HNDV cells did not react as well as the other three treated Rh^ cells did with Rh antibody. This HNDV cell agglutinin was absorbed by normal Rh negative red cells (Table XXXVIII) but not by normal Rh positive red cells (Table XXXVII)j it is incomplete for negative normal erythrocytes and to some extent incomplete for NDV-treated Rh negative cells (Table XXXVII). It must be the previously mentioned HM blocking antibody which also was TABLE XXXVIII

Titrations of Anti-Rho(D) Serum with RhQ Positive and Negative Erythrocytes after ONE Absorption with Normal Hr Erythrocytes

Test Dilutions of Serum Cells C 2 it 8 16 32 6it 128 256 512 102lt

m _. NH 3 mm NH* mm 3 ------N-H ------N-H* mm — - - - — - - - _ _ HTryp - it it it 2 2 + -- - - HTryp* - h it it 3 2 + ---- H-Tryp ------

H-Tryp* ------HM- 3 2 2 2 2 + + + + + HM* - it 3 3 1 -- mm -- - H-M ------H-M* — _ — — _ _ — _ _ - HPR8 • it 2 2 2 2 + - — - - HPR8* - it 2 2 2 2 T - - -- H-PR8 — —------_ H-PR8* - —-—------HNDV - it 3 3 ------HNDV* * it 3 3 1 ------H-NDV ------H-NDV* - + ------HRIE H 3 1 1 + * ——

* Plasma diluent. 123

incomplete for normal Rh0 cells since it was removed by themj and interferes in some manner with the HPR8 agglutination in anti-

Rh as seen by the increased agglutination of PR8-treated Rho cells in Table XXXVIII after negative cell absorption. Thus, it appeared that NDV treatment did not make the Rho positive cells as reac­ tive with incomplete anti-Rh, as did treatment with trypsin.

This anti-Rho(D) serum contained antibody which was incomplete for normal Rh negative cells and partially so for NDV-treated Rh negative cells, incomplete and blocking for HM cells and partially blocking for the PR8-treated Rh positive cells.

Since trypsinized Rho cells have been used extensively to detect specific incomplete anti-Rho(D) and since normal Rho cells remove the trypsin cell reactivity from Rh antiserum, absorption of anti-Rh serum with trypsinized erythrocytes should remove all

Rh0 reactivity. This is verified in Table XXXIX. Absorption of

the anti-D serum, removed all saline agglutinins and all plasma agglutinins excepting those for the NDV treatments. According

to the previous reasoning, these incomplete agglutinins for NDV- treated Rh positive and negative erythrocytes measurable to approximately the same titer (1 :512-1 :10214) are manifestations of reactivity contained in the Rh antiserum for cell antigens other

than RhQ. Absorption of this serum with Rh negative trypsinized red cells (Table XL) removed all antibody except that for tryp­

sinized Rh0 cells, although slight agglutinin titers are TABLE XXXIX

Titrations of Anti-RhoCD) Serum with Rho and Hr Erythrocytes after ONE Absorption with Trypsin- Treated Rho(D) Erythrocytes

Test Dilutions of Serum Cells______C 2 It 8 16 32 6k 128 256______512______102ii

NH ------N-E ------HTryp ------H-Tryp ------HP ------HM - H-M - HPR8 ------H-PR8 ------HNEV ------HNDV* - 31322 22 2 2 1 H-HDV ------H-NDV* - 23332 22 2 1 + HRIE ------

* Plasma diluent. 12£

evidenced for NH (1*4), HM (1*4), and HNDV (1*8). RDE-treated

Rh0 red cells acted as did trypsinized cells, agglutination to the

same titer (1 *3 2 ) though exhibiting a stronger agglutination than

the corresponding trypsin cells. The plasma diluent test was positive only for the negative NDV-treated cells with a non­

agglutinating zone evident in the lower dilutions through a 1*8

serum dilution.

In comparison of the absorptions of this anti-Rho serum with trypsinized Rh positive and negative cells, the following

observations are pertinent. Trypsinized Rh positive (HT) cells

differ by absorption from trypsinized Rh negative (H-T) cells

essentially by the fact that H-T cells did not remove the antibody

for the trypsin-treated Rho cells. After absorption with tryp­

sinized Rh0 cells the titer of the plasma diluent antibody was

the same for both negative and positive NDV-treated cells, but

when H-T cells were used for absorption, only the H-NDV plasma

diluent test was positive. The difference is more readily

explained when the absorptions with normal Rh negative cells

and Rh negative trypsin-treated cells are considered. The normal

Rh negative (N-H) cell was able to remove from this Rh antiserum

the agglutinins for NDV-treated red cells, both Rh0 and Hr.

Trypsinized Rh negative (Hr) red cells, when used to absorb this

Rh antiserum removed the agglutinin for the RIiq-NDV cell, but

were unable to remove all antibody for the Hr-NDV cell (Table XL) 126

as evidenced by the reactivity of the NDV-Rh negative cells in the serum diluted in plasma* However, the trypsin-treated negative (Hr) cell is not, by virtue of the enzyme treatment, the same as a normal Hr red cell and should not be expected to behave exactly as does the untreated cell in absorptions. If the H-NDV cell is, for the purposes of explanation, considered to be the most altered of all the cells used with respect to antigenic modifications other than Rho(D), then its agglutinin should be

the most difficult to remove from this serum except with homologously-treated cells, untreated negative cells or cells in which the treatment does not involve extensive alterations beyond

the normal, such as the M cell. This will be further discussed.

The data in Table XL indicate that it is also likely that the

HNDV agglutinin previously described is also incomplete and absorbable by both kinds of trypsinized erythrocytes.

Normal Rho and trypsin Rh0 cells differed in that NH cells did not remove anti-HNDV agglutinin whereas N-H, HT, and H-T

cells all removed it. Thus, it seems that trypsinization of

Rh0 cells allowed them to act as negative normal cells with this

antibody. Thus, all treated Rho cells reacted optimally after

absorption of anti-Rh with normal negative red cells, with HNDV

cells reacting the least. It appears that HT cells can react with both Rh antibody and the HNDV agglutinin. Since trypsinized

Rh negative cells removed most antibody for virus-treated cells TABLE XL

Titrations of Anti-Rh^(D) Serum -with RIiq and Hr Erythrocytes after ONE Absorption with Trypsin- Treated Hr Erythrocytes

Test Dilutions of Serum Cells C 2 k 8 16 32 6it 128 256 512 102li

NH _ 2 1 + m m «• .. NH* ------N-H ------N-H* -- - - - — - -- — _ HTryp - k 3 2 2 1 -- - - - HTryp* - k 3 3 2 1 - -- mm - H-Tryp ------H-Tryp* ------

HM - 2 1 + + ------HM* - — ------H-M _ ------— _ H-M* ___ _—_ _ HPR8 __ — _ — -- - —_ HPR8* — — ------— _ H-PR8 -- _ ------H-FR8* ------HNOT 2 1 ------HNW* - 2 ------H-NOT ------H-NW* _ - + 2 2 2 2 1 -- HREE — h k E 3 3 —

* Plasma diluent. 128

from this anti-Rh serum, it seemed plausible to consider the possibility that HT antibody is the only specific Rh antibody and antibody for HM, HPR8, and HNDV (Table XXXVIII) may not be Rh anti­ body, On the other hand, the alterations produced on Rho cells by these viruses may be considered to exhibit primarily modified-eell antibody and, in this case, in the presence of anti-Rho (Table

XXXVIII) to display secondarily'Rh specificity. Furthermore, in the presence of the never-demonstrated dispecific antibody whose determinant groups would be directed toward both modified cell structures and the normal, unaffected, cell antigens (here typified by the Rho(D) configuration) reactivity such as that just described would be expected.

Moreover, when this serum (Knickerbocker anti-D) was ab­ sorbed with mumps-treated Rh0 cells, all agglutinins for Rho and

Hr normal and treated cells in saline or plasma were removed. In light of the observations previously mentioned in which mumps treatment removed Rho activity from the surface of the erythrocyte, and the observations in modified-cell serum to HM cells, that the mumps-treated Rho cell upon primary in vivo degradation registers as an Rh negative virus treated cell, predicates this absorption behavior of the HM cells in anti-Rh0 serum. The most sensible

explanation for this complete removal of Rh0 antibody as well as modified-cell agglutinins in only one absorption of this Rh- antiserum, is that of dispecificity. The absorbing HM cell 129

possesses a surface devoid for the most part of Rho configurations as shown previously in this investigation, by the inagglutin- ability of HM cells in saline dilutions of Rho sera (Table I).

That Rh^ activity is removed from the cell is substantiated by the fact that eluates from such treated cells inhibit anti-D antibody. That more Rho groupings are left masked or deeper into the stromata framework of the cell is obvious upon the restoration of the Rho activity when such cells are subsequently trypsinized.

Thus, the mumps-RhQ serum has several reactive possibilities* firstly, the virus alteration may be such that the altered anti­ gens adsorb both virus-modified cell antibody and antibody to the protein carrier of the RIIq Cd ) hapten simultaneously. However, if

this were the case, a preferential absorption of Rh antibody

should be expected and virus-cell antibody left in low, but de­

tectable amounts. Secondly, the possibility exists that Rh anti­

serum contains antibody mainly for the Rho hapten-protein carrier

combination and that the Rho mumps cell surface bares a pre­ ponderance of the carrier antigen to this antibody and thereby removes all agglutinins from the serum. This concept demands that

modified-cell antibody possess a primary specificity for the Rh0

protein carrier and would be one example of a dispecific antibody.

And lastly, the antibody contained in Rh antiserum could be

of the nature of a mixture containing antibodies for the RhQ

specificity, the protein carrier, for the cell structures bared 130

only upon extensive red cell degration, and combinations of these.

The mumps cell according to its postulated surface could possibly adsorb to it, antibody specific for the Rho carrier and at the same time for antigens exposed during viral elution or to the

Rho antigen, itself, though masked in some manner and to either the masking groups or some viral-altered antigen. This again predicates an antibody of dispecificity. To strengthen this argument are the experimental observations that in this unab­ sorbed serum, HTryp cells agglutinated directly, while mumps- treated Rho cells did not. This difference between the HM and

HTryp cells is that incomplete antibody for normal negative cells

(designated also as the HNDV agglutinin) which, when present, blocked the Rho antibody from the HM cells but not from the trypsinized Rho cells. Since mumps-treated Rho cells removed all agglutinins from this serum as previously stated, including the

HTryp cell agglutinin, while the HTryp cells removed all anti­ bodies excepting an incomplete antibody for both types of NDV cells, it is obvious that the enzyme treatment does not involve the same antigenic alterations of the red cell as do the viral treatments. Wallace (1953) formerly had demonstrated this in antisera to specific virus and enzyme red cell treatments. The significance of this observation in anti-Rh0 serum of the sim­ ilarities and differences between mumps virus and trypsin treat­ ments of RhQ red cells further illustrates the potency of this 131

mumps-treated cell to absorb incomplete normal cell agglutinins.

The greater, yet in a sense more subtle, alterations resulting from mumps virus-normal red cell interaction is further evident in Table XLI in which the reactivity of this anti-Rh serum is shown after absorption with mumps-treated Rho negative erythro­ cytes. Some antibody was left for all treated Rh0 positive erythrocytes, while none was demonstrable for normal Rho cells nor for any of the treated negative cells. Comparatively, more antibody was removed for treated Rh positive cells by H-M cells than by normal Rh negative cells (Table XXXVIII). Also, more antibody for Rho trypsinized cells was removed than was absorbed by H-T cells (Table XL). Plasma diluent tests indicate an en­ hanced agglutination of normal Rh0 and PR8-treated Rho red cells.

A zonal type agglutination of H-NDV cells in plasma appeared as was evident after absorption with H-Tryp cells (Table XL) and with H-PR8 (Table XLIII) as well as with HFR8 cells (Table XLII).

This reactivity for H-NDV cells alone remoined in the serum absorbed with treated negative erythrocytes, while reactivity for both of the NDV cells in plasma remained in serum absorbed with

Rho cells treated with trypsin (Table XXXIX) and with PR8 (Table

XLII). HPR8 absorption of this serum, like the absorptions with

HT and HM cells removed all antibody. Thus absorption with either

PR8 or trypsin-treated Rho cells of this anti-Rh0(D) serum are identical. After absorption with PR8-treated Rh negative cells, TABLE X U

Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with Mumps Treated Hr Erythrocytes

Test Dilutions of Serum Cells C 2 h 8 16 32 6b 128 256 512 102k

NH + + NH* - h 2 2 T - - 2 3 3 2 N-H ------N-H* _ - _—_ — —_— — HTryp - h 3 1 + ------HTryp* - S k 3 2 1 - - - - - H-Tryp «w ------H-Tryp* ------HM - 3 2 2 1 ------HM* - 2 1 1 + ------H-M ------H-M* ___ - — _ _ _ — _ HPR8 _ 3 2 - - ———- - _ HPR8* h 1* 3 2 + + + _ H-PR8 — + - _ 1 T — - - H-FR8* ------HNDV - 3 1 1 ------HNDV* - k 3 2 + ------H-NDV ------H-NDV* + + 1 1 2 2 2 2 2 3

* Plasma diluent. TABLE XLII

Titrations of Anti-Rho (D) Serum with Rho and Hr Erythrocytes after One Absorption with PR8-Treated Rh0(B) Erythrocytes

Test Dilutions of Serum Cells C 2 u 8 16 32 6h 128 256 512 102k

NH _ «» NH* ------N-H —- - _-__- — -_ HTryp - 1 ------HTryp* - 1 ------H-Tryp mm ------

H-Tryp* ------

HP - — ------HP* ------HM ------mm HM* ------H-M .. - — -«• — _ —__- HPR8 __ — - -_ _ ___ • HPR8 _ — . — —— — - — _ * ■ H-PR8 mm - - - — _ - ---- HNDV - ——-— ------HNW* - + 2 3 k + + + -- - H-NOT ------H-NW* - + 2 2 3 2 1 1 1 1 —

* Plasma diluent. TABLE XLIII

Titrations of Anti-Bh0(D) Serum with Rh0 and Hr Erythrocytes after One Absorption with PR8-Treated Hr Erythrocytes H 1 «H Is Test Dilutions o Cells C 2 k 8 16 32 128 256 512 1021; n > NH 1 mm . NH* - 1 ------N-H ------N-H* _ — - _ — — - - - - — HTryp - k 3 2 1 + -- - mm - HTryp* - h 3 2 1 ------H-Tryp ------H-Tryp* ------HM - h 2 2 2 1 - - --- HM* mm 3 ------«M H-M mm ------H-M* -_ —— — — _ «• HPR8 — _——- —— __ — HPR8* - 3 2 1 1 ------H-PR8 MB —__ -- - -_- H-PR8* - ~ ------_ - HNDV - 3 2 1 ------HNDV* - 3 2 2 ------H-NDV ------mm - - - mm + + H-NDV* 1 + 1 2 3 1 •

* Plasma diluent. TABLE X H V

Titrations of Anti-Rho(D) Serum with Rho and Hr Erythrocytes after One Absorption with NDV-treated BhQ Erythrocytes

Test Dilutions of Serum Cells C 2 k 8 16 32 6U 128 256 512 102i|

NH 2 2 1 __ . NH* - 2 2 1 - - — - -- N-H - 2 1 ------— - HTryp - k h 3 3 2 1 --- - HTryp* - k b 3 3 2 1 - - - - H-Tryp - 2 2 2 2 ------HP - + mm -- - - — -- - HP* — 7 - _ — — _ mm HM - 2 1 - - - -• --

HM* - 2 1 ------H-MT T ? u{ HPR8 mm HPR8* - - -—------* H-ER8 ------•- HNDV ------HNDV* -- - - - _ - - -- - H-NDV ------mm HRDE — — —— —— — — — — **

* Plasma diluent. 136

the antibody designated as anti-Rh0 in this serum appeared in usual anounts (Table XLIII), It16 titer for HT; 1:32, for HM; and Is8, for HNDV. Titers for other types of cells, including

HPR8, were essentially negative. Plasma diluent antibody was present, however, for the HPR8 and H-NDV cells with the latter exhibiting a questionable agglutination up to a plasma dilution of 1:6U«

Absorption of this anti-D serum with NDV-treated Rho cells

(Table XLIV), left antibody primarily for the Rho-trypsinized cell and some for normal Rh0 cells (1;8) and for mumps-treated Rho cells (1:U). However, saline agglutinins were now demonstrable for normal negative red cell antigens as exemplified by the 1:U titer for normal Rh negative cells and the 1:16 titer for tryp­ sinized Rh negative erythrocytes. These reactions were not as strong as those observed with the Rho counterparts of these cells.

No plasma diluent antibody was present after HNDV cell absorption.

Furthermore, absorption of the serum with NDV-treated Rh negative red cells (Table XLV) yielded the following reactivities: 1:32 saline titer for normal Rho Cells which was not demonstrable in plasma; 1:32 saline titer for normal Rh negative cells with a zone of a partial agglutinin up to a dilution of 1:8, and which agglutination was abolished upon addition of plasma; titers of

1:32 for both Rh positive and negative trypsinized red cells with the H-T agglutination of lesser intensity; a 1:512 saline titer for mumps-treated Rh0 cells which in plasma diluent only reacted in a dilution of 1:8* In addition, while the HER8 titer was almost negligible (1:2), and HNDV cell titer both in saline and in plasma was 1:8$ RDE-treated Rho cells agglutinated to a dilu­ tion of serum in saline of 1:32 with partial agglutination evident in the 1:2 serum dilution. Negative mumps-treated cells agglu­ tinated to a titer of 1:61* in saline with partial aggregation up to 1:16. This agglutination was absent in the plasma diluent test. Rh negative FR8 and NDV-treated cells showed no reactivity in either saline or plasma. This lessening or loss of reactivity of cells in absorbed serum in the plasma diluent test was unex­ plained, Two possibilities exist. The first is that the agglu­ tinins being measured were more stable in the presence of the ions in the phosphate buffered saline, which were perhaps less available or absent in the plasma. The ionic strength of the saline may have supplied the necessary electrostatic charges prerequisite for the fitting of complementary configurations. Coincident with this, is the fact that plasma becomes more alkaline upon storage.

However, the Rh antiserum (containing preservative) was stored a comparable length of time. On the other hand, the plasma may have contained substances which interferred with the red cell- antibody binding by absorbing both from the serum and serum ab­ sorbed to red cells the antibody reactive with the erythrocytes.

There is no reason why plasma should not contain these modified- 138

cell agglutinins according to the arguments advanced in this investigation. However, it is obvious from the data in Table XLV that this diminuition of reactivity did not occur with the RItq- trypsin cell nor with the slight agglutinability of FR8 or NDV- treated Rho cells.

As can be seen in Table XXXVI, unabsorbed anti-Rh serum contained saline agglutinins for NDV-treated Rh0 cells at a higher titer than either the classical incomplete Rh antibody for tryp­ sinized Rho cells, or the saline agglutinins for untreated Rho positive cells. Rho positive cells treated with FR8 virus behaved about as normal cells, while Rh positive cells treated with mumps virus did not react, i.e., behaved as though Rh negative. Absorp­ tion with Rh0 cells (Table XXXVII) not only confirmed the antibody specificity for the NDV cell agglutinins which were not affected at all, but showed some reactivity for Rh negative cells as well.

As might be expected, Rh positive NDV-treated cells absorbed all this antibody for homologous as well as for Rh negative NDV- treated cells (Table XI*IV), but it is also obvious that these cells did not readily remove the typical Rh positive trypsinized- cell antibody. These facts suggest that perhaps the specificity of the agglutinins for the RIiq-NDV cell is toward the virus

(residual virus on cells) and that the virus, in turn, blocks the combination of Rh antigen and the incomplete trypsinized- cell Rh antibody. An alternative suggestion was the possibility TABIE XLV

Titrations of Anti-Rh^D) Serum with RLq and Hr Erythrocytes after One Absorption with NDV-Treated Hr Erythrocytes

Test Dilutions of Serum Cells C 2 h 8 16 32 62* 128 256 512 102k

NH - h 3 3 2 1 + - - - - TJTT.M.JNlTJr N-H - 2 3 h h 2 + - - - - n—nw*7 t T v HTryp h h h 3 1 + mm — HTryp* - h h h 3 1 - - -- - H-Tryp - 3 3 2 1 1 ---- - H-Tryp* ------+ HM. h k 3 2 2 1 1 1 1 - -- -- HM* k 2 1 - -

H-M - 2 3 3 b 2 1 ---- H-M* _ — —— — _ — _ — mm HFR8 _ 3 + __ •— _ — mm HPR8* - 3 T - — - mm -—- — H-FR8 — — — _ —- mm - — — — H-PR8* - —-- — — mm -- ~ • HNDV - 3 2 2 - - mm - - -- HNDV* mm 3 2 2 ------H-NDV mm ------mm - H-NDV* mm ------HRDE mm 3 h 3 2 1 ** '

* Plasma diluent. that the Rh positive NDV-cell agglutinins are preferentially absorbed from a mixture of the two antibodies. However, these differences also reinforce the separate specificities of the NDV-

Rh negative cell agglutinin and the trypsinized cell agglutinin*

However, the data in Table XXXVIII show that the Rh positive

NDV cell agglutinins were removed for the most part by absorption with normal negative cells. These same cells also removed agglu­ tinins for negative-NDV cells. Again, the Rho trypsin cell agglutinins remained. These facts mitigate the possibility that the specificity of the Rh0-NDV agglutinins is against either virus or Rh antigen alone. Rather, this antibody, is directed against an antigen in untreated Rh negative cells, which was made avail­ able in Rh D, positive cells after treatment with NDV. Whereas untreated Rh positive cells were neither agglutinated by it, nor absorbed it (Tables XXXVI and XXXVTI). Rh positive NDV-treated cells did both. These latter cells possibly can remove Rh anti­ body also. However, as noted before in Table XLIV, Rho-trypsin cell antibody was not removed, and in addition, it should be noted that after one absorption with Rho-NDV cells, antibody for Rh negative trypsinized cells was evident, (Table XLIV). Thus, the essential differences are that absorption with untreated Rh negative cells: Removed all antibody for negative cells, treated or untreatedj increased agglutinability of PR8-treated cells; and that such serum then agglutinated mumps-treated Rho erythrocytes (Table XXXVIII) which unabsorbed serum did not. However, absorp­

tion with Rh positive NDV-treated cells seemed to: reduce all antibody for virus-treated cellss did not remove Rho trypsin cell antibody as rapidly as untreated Rho cellsj and was followed by

the appearance of antibody for Rh negative trypsinized cells.

Absorption with Rh negative cells treated with NDV, pro­

duced results different yet from sera absorbed with untreated

Rh negative or Rh0-NDV cells. In Table XLV, it can be seen again

that antibody for Rh negative NDV-treated cells was all removed,

and only a little remained for Rh positive, NDV cells as with the

above cells (Tables XXXVIII and XLIV), but in addition, antibody

for PR8 negative cells was removed and greatly reduced for Rh

positive, PR8 cells. In addition, normal untreated Rh positive

and Rh negative cells as well as trypsinized Rh positive and

negative cells all agglutinated in saline to the same titer, 1:32,

practically the equivalent of the Rh positive trypsinized cell

titer in unabsorbed serum, with the exception that the untreated

negative cells showed a partial zone effect. The titer for Rh

positive cells treated with mumps virus was increased to 1:512

and negative-mumps cells then agglutinated to a titer of 1:61+ in

comparison to serum after absorption with untreated negative cells

(Table XXXVIII).

These data seem to indicate that when Rh positive red cells

were treated with NDV, they were agglutinated in saline by anti­ body in anti-Rh serum which was absorbed by these same cells ; by untreated Rh negative cells and by Rh negative NDV-treated cells,

although the antibody was incomplete, i.e., did not agglutinate untreated cells at all in saline and the NDV-Rh negative cells

very little. Thus, the antigen is common to Rh positive and

negative cells, although it is not available serologically in the

former until treatment with NDV. This antibody completely inhi­

bited the agglutination of mumps-treated Rh positive cells and

partially inhibited the agglutination of PR8-Rh0 cells, although

they both must have the antigen available since they absorb it

(Table XLIII). When anti-Rh serum was absorbed with NDV-treated

Rh negative cells (Table XLV), this antibody was also removed and

the only other reduction observed was the reduction of agglutinin

for PR8-Rho cells to almost nothing (lower than after absorption

with untreated Rh negative cells (Table XXXVIII) and in unabsorbed

serum (Table XXXVI)). The removal of this PR8-Rh0 specificity

was accompanied by the appearance of saline agglutinins for un­

treated Rh positive and negative cells; Rh positive and Rh

negative trypsinized cells; Rh negative mumps-treated cells; and

a 16-fold increase in titer for Rh positive mumps-treated cells.

Obviously, this Rh positive, NDV cell specific antibody was

not removed by untreated normal Rh positive cells (Table XXXVIII)

nor was there any evidence that it combined with such cells. After

such absorption, Rh negative, NDV-treated cells showed a low titer Ui3-

of 1:8 also. The assumption that what was removed, was Rh antibody supposedly can be confirmed by noting that antibody left after absorption with untreated Rh negative cells (Table XXXVIII), was to Rho-trypsin, Rho-mumps cells, with the additional assumption that some of the Rho-NDV cell antibody was also anti Rh. However, data in other tables cast doubt on these assumptions. Thus, the

Rh positive PR8 specificity, most of the mumps cell specificity and all but a trace of the Rhg-NDV cell antibody were removed by Rh negative, trypsinized cells (Table XL), and Rh negative mumps cells reduced the titer of antibody for trypsinized cells appreciably (Table XLII).

Furthermore, absorption with untreated Rh positive cells (Table

XXXVII) removed saline agglutinins for Rh positive cells, Rh positive trypsinized cell antibody and the small amount of anti­ body for Rh positive, PR8-treated cells (Tables XXXVI and XXXVII), but since such cells did not react with the Rh positive, NDV cell agglutinin, it was not removed. Because of its inhibitor effect on the Rh0-FR8 and, Rh0-mumps cell agglutinins, which appear in

Table XXXVIII after absorption with untreated Rh negative cells, the various treated cell3 did not react even though antibodies for them are probably still present. In fact, as indicated later, such antibodies are probably even taken up when virus- treated cells are added, since these cells seem to absorb them.

In contrast, trypsinized Rh positive cells removed all the 1UU

Rho-NDV cell type agglutinin (Table XXXIX), as did untreated Rh

negative cells, and the subsequent failure of Rho-FR8 and Rho~

muraps cells to agglutinate in the absorbed serum suggest that

trypsinized cells removed virus-treated cell antibodies as well

as Rh antibodies. This would mean that these cells are probably

preferentially agglutinated by Rh antibody, and at the same time,

trypsinization has enabled them also to take up the inhibiting

antibody and the virus-cell antibodies as well. They resemble

Rh0-NDV cells by absorption data, but are apparently not agglu­

tinated by these other antibodies, and also pick up trypsin cell

antibody better than Rho-NDV cells (Table XLIV).

Absorption with Rh negative, trypsinized cells (Table XL)

emphasize the foregoing statements with regard to Rho-NDV cell

agglutinins and antibodies to other virus-modified cells, both

as to the capacity of trypsinized cells to remove the former, so

that in addition, antibodies to PR8 and mumps cells can also be

removed. Thus, all these antibodies appear to be common to both

Rh positive and Rh negative cells. As noted with absorption by

untreated Rh negative cells (Table XXXVIII) this seems also to

be true of the saline agglutinin for normal cells by absorption

with Rh negative trypsinized cells (Table XL). At the same time,

although agglutination of Rho-trypsinized cells was not as strong

after this absorption, the titer was relatively unaffected (one

tube) indicating these to be mostly Rh0(D) antibody. 0U5

Absorption with Rho-mumps cells and Rho-PR.8 cells (Table

XLII) behaved about as Rho-trypsin cells, probably removing all

antibody. Similarly, Rh negative cells treated with mumps virus, while they removed most of the Rh0-NDV cell agglutinin and Rh positive saline agglutinin, weakened the trypsin cell agglutinin

(Table XLI) as' did Rh negative, trypsinized cells, were not quite

as effective in removing antibody to Rh positive cells treated

with mumps or FR8 virus. A comparison of absorptions with Rh

negative cells treated with either trypsin, mumps or PR8, with

the untreated negative cell absorption (Table XXXVIII) emphasize that treated negative cells as well as positive cells all reacted with these antibodies as well as to NDV-cell antibody, while untreated negative cells did not remove them. Thus, untreated

Rh positive and Rh negative cells differ only in the ability of the latter to remove the NDV-cell antibody and exposing the anti­ bodies to the other virus-treated cells. Thus antibodies for these cells are directed toward antigen (s) common to both types of cells.

In an attempt to determine whether the removal of an aggluti­ nin from this anti-Rh0 serum left the serum completely devoid of antibody capable of adsorbing to such a cell (the absorbing cell), the experimental date recorded in Table XLVT were obtained.

Trypsinized Rho(D) red cells were exposed to a non-agglutinating serum (anti-D, absorbed with normal Rho cells) as well as to an agglutinating serum (anti-D, absorbed with normal Rh negative cells) and such cells after sensitization were washed several times in saline to remove excess, non-specific protein and were made up in the usual 2 per cent test cell suspension and titrated in the non­ sensitizing serum. As indicated in Table XLVI, trypsin cells sensitized with the non-agglutinating serum, agglutinated to a titer of ls5>12 in the agglutinating serum in which trypsin-cells had formerly agglutinated at a titer of Is32. The sensitized- cell agglutination differed from the unsensitized cell agglu­ tination not only in this respect but also in the strength of agglutination. The sensitized cell aggregates were partial agglu­ tinations as opposed to those observed in Table XXXVIII. Thus, it is apparent that trypsinized cells are capable of adsorbing from an absorbed anti-Rh serum, a factor which diminished the strength of their agglutination, yet increased their reactivity in an Rh antiserum in which they had formerly agglutinated com­ pletely but less extensively. In the contrasting situation

(Table XLVI) trypsin cells sensitized with this agglutinating serum, exhibited a progressively stronger agglutination in the non-agglutinating serum. The non-agglutinating serum for tryp­ sinized cells, previously observed to exhibit agglutinability for both types of NDV-treated cells (Table XXXVII), when first adsorbed to the trypsin cell, interfered with the agglutination of this cell in saline dilutions of a serum which contained no TABLE XLVI

Effect of Virus-Treated Cell Antibody on the Agglutination of Rh Positive Cells by Rh Antibody

HTryp Test Cells Test Dilutions of Serum in Saline Sensitized with: Serum 2 U 8 16 32 61* 128 256 512 102U

Knickerbocker Saline Knickerbocker Saline Anti-D Absorbed 1 x Anti-D Absorbed 1 x 3 2 2 2 2 2 1 1 1 + with NH with N-H

Knickerbocker Saline Knickerbocker Saline Anti-D Absorbed 1 x Anti-D Absorbed 1 x 2 2 2 2 2 3 3 3 3 k with N-H with NH

* 1 part serum UBed as sensitizer to 2 parts packed HTryp cells incubated for 30 minutes at 37°C and cells washed three times in saline. ll*8

detectable NDV-cell agglutinin but did possess & strong agglutinin for the trypsinized Rho cell* Thus, again it appears that when the antibody most readily detectable with Rho NDV-treated cells was in the same serologic system as the classical anti-Rho anti- ? body, interference in reactivity was noted. However, in the absorptions previously studied, the preference for Rho antibody predominated in the reactivity of the Rho cells, with the excep­

tion of NDV-treated Rh0 cells, while in this system described in

Table XLVI when the NDV-trype agglutinin was allowed first pre­ ference in reacting with a strongly agglutinating Rho cell (HTryp)

subsequent reactivity in an Rh agglutinating serum was altered as has been described. Further evidence to support this observation also is contained in Table XLVI. When the Rho-trypsin cells were

first exposed to the agglutinating anti-Rho and subsequently ti­

trated in the non-agglutinating NDV-cell type serum inhibition

of the expected four plus type agglutination was evident up to

a dilution of 1:1021; in the non-agglutinating serum. Thus, the

interference between these two types of agglutinins for normal

red cell antigens indicated in the absorption studies (Tables

XXXVI through XLV) does exist though it is difficult to demon­

strate possibly because of the similarity of red cell antigens

present on both types of normal and treated erythrocytes and

also because of the peculiar nature of the antibody designated

as NDV-cell agglutinin. TABLE XLVTI

Titrations of Anti-Rh0(D) Albumin Agglutinating Antibody with Normal and Virus-Treated Red Cells

Test Dilutions of Serum Cells C 2 h 8 16 32 6k 128 256 512 102k

NH m m mm NH* - 1 + - — - N-H - - ~ - - - N-H* ------HTryp - S s h 3 3 3 1 MB -- HTryp* - s h h 3 3 3 3 1 + mm H-Tryp ------H-Tryp* - — ------mm m m XlilTIM HM* - 3 2 -- - mm D wPi H-M* m m mm m HPR8 mm - — •- mm - HPR8* _ 1 + mm — m m — H-PR8 mm — — - — - - H-PR8* - - mm - mm - -- HNEV - 1 + - mm - - -- - _ HNBV* - h I 3 2 + «• - - mm H-ND7 ------m m

H-NDV* a * mm • —

* 8 percent albumin (human) diluent. Ortho. 1$Q

These reuslts were obtained in an antiserum which contained both saline agglutinin and the incomplete form of anti-Rh^D).

An albumin agglutinating anti-Rh0(D) serum (Ortho) was similarly

examined. This serum was heated at 36°C for 30 minutes prior to

use to eliminate any possible interference from any unknown heat-

labile serum factors not previously removed in the commercial

preparation of the serum. As indicated in Table XLVII, this

albumin agglutinating anti-Rh serum was originally less complex

than the other serum characterized (Table XXXVI). The only

saline agglutinin present in the unabsorbed serum was that for

RIIq-trypsinized cells and was present in a titer of 1:128. NDV-

treated Rh positive cells agglutinated feebly (one plus) in a

1:2 saline dilution of serum. The albumin diluent test revealed

the following order of cell reactivity: 1:25>6 titer for HTryp,

1:16, for HNDV, 1:U> for HM, and a weak 1:2 for both HPR8 and NH.

Rh negative cells both normal and treated did not react in either

saline or albumin dilutions of this serum.

One absorption of this serum with normal Rh0 cells left only

a 1:8 albumin agglutinin titer for the Rho-trypsin cell (Table

XLVIII). After one absorption with N-H cells, the trypsin-Rho

cell titer in saline was diminished to a 1 :32, while the albumin

agglutinin for these same cells increased to as high as a 1:312

dilution. The saline and albumin agglutinins for the Rh0-NDV

cell appeared to be the same while the PR8-Rho cell reactivity 151

in saline was then evidenced at a serum dilution of XsU and in albumin was strongly agglutinated to a titer of 1:32, similar to the Rh0-trypsin cell reactivity* Thus, both FR8 and NDV, Rh positive cell saline agglutinins apparently increased after absorption with Hr cells. The Rh-mumps cell reactivity in saline exhibited a strong four plus agglutination in a 1:2 serum dilu­ tion, a non-agglutinating zone up to a 1:6U dilution, and partial agglutination to a 1:312 dilution (similar to the HTryp albumin titer). However, in the albumin diluent test, reactivity of these cells was evident in only the 1:2 serum dilution. This apparent decrease of titer in viscous diluents has been discussed pre­ viously.

Continued absorption of the serum with normal Rh negative

(Hr) red cells revealed quite different results. The Rho- trypsin cell saline reactivity was not greatly affected by one further absorption with N-H cells, the cells agglutinating in a 1:16 serum dilution. But the albumin reactivity for Rh0-tryp­ sin, cells was greatly reduced (to a titer of 1:32) and much the sane as the saline agglutinability. The FR8-Rh0 cell agglutinability in both saline and albumin remained unchanged, while the Rh0-NDV cell titer in saline was reduced to 1:U and the albumin reactivity one tube (1:8). There were no demonstrable saline agglutinins for either normal or mumps-treated Rh0 cells but both agglutinated in TABLE XLVIII

Titrations of Anti-Rh0 (iD) Albumin Agglutinating Antibody after Absorptions with Normal Red Cells

Absorb­ ing Test Dilutions of Serum Cells Cells C 2 k 8 16 32 6U 128 2^6 $12 10 2h

1 x with HTryp . __. NR HTryp# - 3 2 1 ------KNOT mm - - mm ------HNW* mm ------— - - H-NDV - — —- -- -- m - _ H-NW* ------*- - 1 x with Mryp - S h u 3 2 - mm -- - N-H HTryp* - h h h h h 3 3 2 2 - HM - k - - -- 1 2 2 2 + HM* k — ------HPR8 - 2 1 mm -- mm - mm HPR8* - h h 3 2 1 mm - - - - H-FR8 — mm — mm -— — -- — mm H-FR8* •- m. - — - - - — HNOT - k 2 2 2 ------tm HNBV* •" k 2 2 2 — — ■ » •

* 8 percent albumin (human) diluent TABLE XL7III (Continued)

Titrations of Anti-RhQ(D) Albumin Agglutinating Antibody after Absorptions with Normal Red Cells

Absorb­ ing Testq KLlutions of Serum Cells Cells C 2 h 8 16 32 6k 128 256 512 102i*

_. 2 x with NH mm _ N-H NH* - 3 2 1 ------N-H _ _ N-K* _ _ mm HTryp - k k k 2 - - - - - mm HTryp* - h k 3 2 1 - ---- H-Tryp ------H-Tryp* ------TJMXlrl a* HM* - U h 3 ------n—ri H-I4* mm HPR8 _ 3 1 «■ - - — _ — _ mm HPR8* - k 3 2 2 1 - - -- H-PR8 — -— - -_-_- _ - H-PR8* _ - --- - mm - -- mm HNW m m 3 1 ------HNW* - h 3 2 ------H-HOT ------H-NW* *• * * *• *

* 8 percent albumin (human) diluent. 15U

albumin diluent. The albumin reactivity of the NH cells was then evident in a titer of 1:8 as opposed to a 1:2 serum dilution reactivity in the unabsorbed serum. The mumps-Rh0 cell reactivity

(1:8) in albumin was increased over that observed when the serum was absorbed only once with normal Rh negative red cells. The Rh negative cells exhibited no agglutinability either in saline or albumin after normal Hr cell absorption. Thus, it appeared that there was present in this serum a small amount of antibody similar to that which has been referred to as HNDV agglutinin, only in this serum (Ortho), it is of such configuration that it is detected by the trypsinized Rh0 cell in albumin serum dilutions.

It is distinct from the Rho antibody as noted by the differences in Rho-trypsin cell reactivity in saline and albumin and by the fact that when the albumin reactivity was decreased upon con­ tinued absorption with normal Rh negative cells, the saline trypsin-RhQ cell titer was not greatly affected, nor was that of

Rh0-PR8 cells, while the albumin agglutinin for normal Rho cells was increased over that present in unabsorbed serum.

Absorption of the albumin agglutinating anti-D with RIIq -NDV- treated cells removed saline and albumin agglutinins for the virus- treated cells, diminished the trypsin-RhQ saline reactivity to a

1:U serum dilution and practically abolished the reactivity of albumin dilutions of this serum for these cells. However, normal

RhQ red cells then agglutinated weakly in saline at a titer of 1:8 TABLE XLLX

Titrations of Anti-Rh0(B) Albumin Agglutinating Antibody after Absorptions with NOT-Treated Erythrocytes

Absorb­ ing Test Dilutions of Serum Cells Cells C 2 h 8 16 32 6k 128 256 512 1021*

1 x with NH 1 1 1 _ HMOT NH* - + + ------mm HTryp - 2 l - - - - mm mm - HTryp* - 2 ------H-Tryp mm + mm ------mm H-Tryp* mm - mm ------HM ------mm HM* - -- mm —— -__ _ mm HFR8 .. mm _ _ _ - mm _ mm HPR8* ------mm 1 x with 'M --- - 3 2 2 2 1 1 - - -- H-NOT NH* - 3 3 3 2 1 1 - m m — • + + + + N-H - 1 1 - - - - N-H* —— « ■ «• mm •• — ■ ■ HTryp - U it it it 3 2 2 -— — HTryp* - it h it it 3 3 1 - - - H-Tryp - + + l ------mm H-Tryp* mm ----- m t - - - - HM mm l 2 1 1 1 1 •• mm - mm HM* - it it it 3 2 1 - mm - - H-M - - + + + 1 1 + + + + H-M* mm • mm •-— mm —— — «• HPR8 - - m m —— - mm -——

HPR8* - h 2 2 2

* 8 Percent Albumin (Human) Diluent. TABLE YT.TY (Continued)

Titrations of Anti-Rh0(B) Albumin Agglutinating Antibody after Absorptions with NW-Treated Erythrocytes

Absorb­ ing Test Dilutions of Serum Cells Cells C 2 h 8 16 32 6h 128 2^6 512 102U

1 x with H-PR8 mm H-NIflT H-PR8* ------_ — mm m m - 2 mm -- - « w -- - mm HMW* - 2 + ------mm - 7 "x with M. - 2 T 1 + ------H-ND7 NH* - k 3 3 1 1 - - - - - N-H ------N-H* __ ------mm - HTryp - k h 3 3 - - -- - HTryp* - h 1; h 3 3 1 - - - - H-Tryp ------H-Tryp* ------* - HM. - + -- - -— — - HM* - n 3 2 2 2 - - - mm - H-M - + + + + + 1 1 + + + H-M* —_ — — - — -- — — — HFR8 - _ — -—- — —- HPR8* — h h 3 2 2 1 —-- - H-PR8 _ -— — - -- H-PR8* -- «. mm - — - _* — _ m m - u 2 1 + - m -- - - HNDV* - 3 2 1 - --- - mm H-NEV mm — ------—- - H-NBV* ------* 8 percent Altumin (Human) t&luent 157

but reacted questionably in albumin diluent. The only negative type cell tested against this absorbed serum was the H-Tryp cell which did not show any definite reactivity whether the serum was diluted in saline or in albumin.

One absorption of this serum with Rh negative NDV-treated red cells revealed an amazingly high saline agglutination of normal Rh0 cells (l:6U). These same cells agglutinated more strongly but to the same titer in the albumin diluent test.

Even normal Rh negative red cells agglutinated feebly (one plus) at saline dilutions of 1:8 and 1:16. The Rho-trypsin cells agglutinated in dilutions of either saline or albumin as a classical agglutinin at a titer of 1:128, and oddly, the H-tryp cell, like the normal negative cell, agglutinated weakly only in saline and only in one dilution (1:8). Mumps-treated Rh negative cells exhibited this same peculiar spotty agglutination in saline dilutions of serum (1:32 and 1:6U) and likewise was not agglutinated in the plasma diluent test. The Rho-mumps cells agglutinated weakly in saline to a titer of 1:32 but were aggregated in the manner of a classical agglutinin to the same titer in serum diluted in albumin. Rho-PR8-treated cells agglu­ tinated only in albumin (1:16) but not in saline. The reactivity for the Rho-NDV cell was practically nil (1:2 titer in saline and albumin). Rh negative mumps and PR8-treated erythrocytes showed no reactivity in this serum. On continued absorption with NDV«- 158

treated Rh negative red oells, the spotty saline agglutination of the N-H and H-Tryp cells ■was removed but that for H-M cells remained essentially the same merely shifting one dilution higher

(1:61; and 1:128). The Rh0 normal cell agglutinated to a lesser degree in saline (1:8) idlereas the albumin agglutinin titer for these same cells was 1:32. The Rh0-trypsin cell reactivity was slightly diminished but both diluents and serum yielded similar reactivities (1:32 in saline and 1:61; in albumin). The Rh0-mumps cell no longer agglutinated in saline dilutions of this serum after the second absorption with these same cells (Table XLIX).

However, these mumps cells reacted similarly in the albumin test,

1:32 titer after two absorptions of the serum with H-NDV and a

1:61; titer after one such absorption. The PR8-Rho cell reac­ tivity was increased two dilutions in albumin but the cells still showed no agglutinability in saline serum dilutions. The Rh0-NDV cell reactivity was increased to a titer of 1:8 in both diluents.

Rh negative red cells treated with PR8 virus remained serologically inert in this serum.

Although this incomplete anti-D serum contained little if any agglutinating capacity for the NDV-cell treatments, absorp­ tions of the serum with both Rh positive and Rh negative red cells which had been treated with NDV rendered the serum capable of agglutinating normal Rho erythrocytes in saline which was not possible in the unabsorbed serum as well as increasing the agglu- l$9

tinability in albumin sarum dilutions of the same Rho cells.

This latter effect was evident upon absorption of the serum with

Rh positive and Rh negative red cells (Table XLVIII). Absorption of this serum with H-NDV cells left in the serum a slight but detectable reactivity for Rh negative normal, trypsinized, and mumps-treated cells similar to, but not nearly as marked as, that observable in the Knickerbocker anti-D serum when comparably absorbed (Table XLV). From the data obtained in these absorptions of the albumin anti-D, it seemed apparent that the incomplete anti-Rh antibody supposedly inagglutinable in saline dilutions of serum and normal Rho cells may be restrained from agglutinating in saline due to the presence of antibody to other cell structures and which is absorbable by NDV-treated red cells (either Rh positive or Rh negative).

Table L represents a further attempt to characterize anti­ body to modified red cells as opposed to that with Rho specificity.

An incomplete normal cell antibody was selected which agglutinated mumps-treated Rh0 cells to a higher titer than normal Rho cells

(Table III) and was used to sensitize normal Rh0 cells, cells sensitized in this manner were designated as I. The ill-defined mumps-cell agglutinin found in both anti-HPE8 and anti-HNDV

(Table II) and which was evident after these sera were absorbed five times with NH cells and subsequently twice with HNDV cells was used to sensitize red cells. Normal Rh0 red cells sensitized TABLE L

Inhibition and Enhancement of Agglutination in Anti-Globulin Serum of Red Cells Sensitized by Anti-Rh^D) Serum and Serum to Virus-l-Jbdified Cells

Developing Dilutions of Serum Antiserum Agent Test Cells C 2 U 8 16 32 6b 128 256 512 102it 201+8 1+096

Anti-HFR8 I _ _ _ Abs. 5 x CARG I 2 b h b it b b it 3 3 3 3 3 with NH - III - -- mm -- - -O* - _- - and 2 x CARG III 2 2 2 2 2 2 2 2 + - 1 2 3 with HNDV - HM-Rhsera ------_-_—_ Plasma HM-Rhsera _ — HFR8-Rhsera Plasma HPR8-Rhsera - HNDV-Rhsera - 2 1 + - mm - mm - mm •• • -

Plasma HNDV-Rhsera - 2 2 1 2 2 2 2 2 1 + --

Anti-ktffcB - III ------~ - - • Abs. 5 x CARG III 2 + 1 2 2 3 2 1 + + + _ with NH, - HM-Rhsera ------mm - - - 2 x with Plasma HM-Rhsera ------— * _ — HNDV, and - HPR8-Rhsera - 2 2 1 + mm 2 3 1 - - . _ 1 x with Plasma HPR8-Rhsera - 2 2 1 - + + it 1 - - - mm NH - HNDV-Rhsera - 3 2 1 mm - - •m - -- -- Plasma HNDV-Rhsera - It 3 2 2 2 1 Anti-KM - It ------Abs. 2 x CARG II 1 2 2 2 2 3 3 it h 1+ 3 2 1 with HPR8 - HM-Rhsera - + + --- -- Plasma HM-Rhsera - E 2 + — HPR8-Rhsera -- Plasma HPR8-Rhsera - - - HNDV-Rhsera -- Plasma HNDV-Rhsera - - TABLE L (-Continued)

Inhibition and Enhancement of Agglutination in Anti-Globulin Serum of Red Cells Sensitized by Anti-Rh (JD) Serum and Serum to Virus-Modified Cells o'

Developing Dilutions of Serum Antiserum Agent Test Cells C 2 h 8 16 32 6U 128 256 512 1021* 20l*8 1*096

Anti-NH HM-Rhsera Abs. 2 x Plasma HM-Rhsera - 2 1 + with HPR8 HPR8-Rhsera and 1 x Plasma HPR8-Rhsera with NH «r HNDV-Rhsera - 3 1 Plasma HNDV-Rhsera - 3 3 Incomplete - " T " ‘ ------mm Anti-Rho(D) CARG I 2 1* h 1* 3 3 3 3 3 3 3 3 1 Lederle - II m m mm ------mm — mm CARG II 1 - + + + + + + + + 1 1 1

Saline - I - Anti-Rho(D) CARG I 2 3 Ortho - II —- CARG II 1 - CAkG Nb + I - 2 1 II - 1 h i - 3 2 2 1 I NH 0 Rho Red Cells Sensitized with 2 parts 1:2 Anti-NH Absorbed 2x with HPR8. II NH 0 Rho Red Cells Sensitized with 2 parts 1:2 Anti-HPR8 Abs. 5x with NH and 2 x with HNDV. Ill NH 0 Rhc Red Cells Sensitized with J part Knickerbocker Saline Anti-Rho(D) CARG 1:2 Chicken Anti-Rabbit Globulin (0,1 cc/tube). Plasma Pooled human 0 Rho (0.1 cc/tube). Sensitized Virus-Treated Cells 1 part serum: 2 parts cells. Cells sensitized ^ hour and washed. 162

■with this agglutinin from anti-HFR8 serum were designated II. Rho positive red cells sensitized with anti-Rho were designated as III.

When the normal cells sensitized with the incomplete normal cell antibody, which was an agglutinin for mumps-treated cells, were subsequently exposed to the serum used to sensitize type II cells, there was an enhancement of agglutination in the antiglubulin test system (Table L). However, if the normal cells were first sensitized with the II sensitizer and subsequently with I, inhibi­ tion of agglutination in the antiglobulin titration was observed.

Likewise, it is obvious from the table that type I cells when added to an antiglobulin system in the presence of both saline agglutinating and incomplete types anti-Rh^D) demonstrated an enhanced agglutination over the control system, whereas type II cells, comparably titrated in the several anti-Rho sera, exhibited a decreased agglutination. Similarly, when anti-Rho was first added to normal Rh0 cells and then tested in the presence of type

II sensitizer, inhibition of agglutination again occurred. Thus, the incomplete normal cell agglutinins present in antiserum to both normal and mumps-treated red cells reacted with anti-Rh sera to yield an enhanced antiglobulin agglutination, even though no reactions were demonstrable in saline. The antibody, described above as type II sensitizer, in the presence of anti-Rh sera demonstrated inhibition of agglutinability of normal Rho cells 163

and since this inhibition occurred when either anti-Rho or type

II sensitizer was first exposed to the normal cell, this inhibi­ tion was considered competitive. This inhibition of anti-Rh agglutinability found in antiserum to virus-treated red cells

(PR8-treated) may be the same as, or similar to,the serum factor described in the Knickerbocker anti-fiho(D) serum. It differs from the NDV-cell type agglutinin in anti-Rh serum by not being absorbable with NDV-treated red cells though it is produced by these cells. However, such an antibody may be directed against subsurface antigens which lie quite deep in the stromata and thereby cannot be absorbed by whole cells whether treated or not.

It is obvious from the data that such an antibody was bound to normal cells though it did not agglutinate them, and hence it may be inferred that such a binding occurred only through one determinant grouping and the resultant inhibition of the Rh^D) antibody was merely a matter of steric hindrance. The fact that

HNDV cells when first sensitized with Rho antibody containing

the previously described NDV-cell type agglutinin, washed in

saline and then exposed to the type II sensitizer displayed an

enhanced agglutination over the control system demonstrated that

these two types of Rho serum inhibitors did not inhibit each

other, but exerted an additive effect when on the same cell.

This did not occur when mumps-or PR8-treated cells were sensitized

with anti-Rho and than added to the type II inhibitor. One 161*

absorption of the II inhibitor with normal Rho erythrocytes in­ creased the agglutinability of the NDV-cells sensitized with anti-

Rho and allowed the comparably sensitized PR8-treated cells then to agglutinate in the type II inhibitor. (PR8-treated cells also produce this inhibitor as evidenced in specific modified cell sera indicated in Table II). It is also evident that mumps- treated cells sensitized first with anti-Rh sera and then added to the type I sensitizer, acted as normal Rho red cells to yield an enhanced agglutination. PR8-and NDV-treated red cells simi­ larly sensitized and titrated did not do this.

To explain further this behavior of NDV- and PR8-treated red cells sensitized with anti-Rh sera and titrated in type II sensitizer, the HNDV cell may be considered to be altered to such an extent that it is able to bind both Rhc antibody as well as inhibitor antibody (either contained in the Rh antiserum or that designated as type II). This claim may be substantiated by the increased agglutinability of NDV cells so treated, and by the fact that in absorptions of the Knickerbocker serum it became obvious that Rho-NDV cells preferentially bound the modified-cell type of antibody but could also bind anti-Rho. Furthermore, in the absorption of the albumin agglutinating anti-Rho(D) with

HNDV cells almost all of the Rho reactivity was removed.

Thus, it would seem that the primary specificity in any

Rh antiserum is directed toward the Rho antigen, but that in the 16£

t process of immunization to the D antigen other cellular configura­ tions, among -which areaa variety of the antigens bared, upon viral treatment of similar cells, also serve as immunizing materials.

Since such antibodies are directed toward altered red cells or perhaps, more accurately, degenerative red cells, such antibody as can be detected by virus-modified cells is innocuous in the normal individual or, on the other hand, functions in the normal process of the removal of the older, degenerating cells.

Dodd, Wright, et al. (1953) showed that cells of many hemo­ lytic anemia patients were agglutinated by specific trypsinized red cell antibody. More recently, Crowley and Bouroncle (1956), corroborating the findings of others, have demonstrated that some patients with autoimmune hemolytic anemia may form antibodies against specific blood group antigens present in their own erythrocytes. In order to study further the presence of virus- modified red cell agglutinins in the sera of hemolytic anemia patients, the cells and sera of four such individuals were ob­ tained from Dr. C. A. Doan. All individuals were Rho positive.

These cells and sera were compared with Rh0 normal and treated cells as well as with Rh negative normal and treated cells and with the serum from a normal Rho individual.

In the erythrophagocytic test described by Bass (1953)» normal Rho red cells had a phagocytic index (P.I.) of 0. The

P.I. values for cells from the four hemolytic anemia patients TABLE LI

Modified-Cell Agglutination in Normal and Hemolytic Anemia Sera

Saline Diluent Titers Anti-globulin Developed Titers Test Cells DKSHNS DKSH NS

NH t m • » 1:32* 1:32* 1:256* 1:32* HTryp - 1:2 -- 1:32* - 1:32* - HPR8 - — - - 1:128* 1:256* 1:102k* 1:32* 1:6k* HM I:20k8* 1:20^8* - - - 1:512* 1:102k* 1:256* 1:128* 1 :32* HNDV 1:2 1:2 1:16 ith 1:102k* l:k096* 1:102k* 1:512* 1:128*

N-H m m 1 :32* 1:128* 1:6k* H-PR8 - - -- - —• — m m H-M 1:102k* 1:102k* - 1:102k* 1:102k* 1:128* 1:32* 1:6k* 1:32* - H-NDV 1:U 1:8 1:512* 1:2 1:102k* 1:32 1:8

* Pro-zones of non-agglutinability. NS Serum from a normal individual. D, K, S, and H Sera from hemolytic anemia patients.

H* ON O n 167

were elevated in this test to 27, 3>k, 1*2, and $6. Wright, Dodd,

at al. (1953) reported similar findings. Further evidence of red

cell alterations were indicated in titrations with virus-modified-

cell antisera. From these titrations and absorptions, it was

evident that these cells did not behave as normal Rho erythrocytes

but reacted in these sera more similarly as mumps-treated red

cells but not as PR8- or NDV-treated erythrocytes. Because these

data are too unwieldy tc present in complete form here, the most

characteristic reactivity will be described.

After normal Hho cell absorptions of anti-HFR8 (Table II)

in which normal cells were no longer agglutinable, the red cells

of two of the four hemolytic anemia patients were agglutinated.

In the comparable absorption sequence of anti-HNDV, only one of

these patient’s cells were reactive. After four absorptions of

this same anti-HNDV with trypsinized Rh0 red cells, the following

titers were obtained; NH, 1:32} HTryp, 1:U; HNDV, 1:102U} and

the erythrocytes of three of the four hemolytic anemia patients

gave titers of 1:128, 1:128, and 1:61*. Thus, these patients'

cells exhibited agglutinability in these sera over that observ­

able for normal Rh^ red cells and similar to that of virus-

modified erythrocytes.

With the incomplete normal Rh0 cell antibody present in

anti-HM serum, (anti-HM absorbed twice with HFR8 cells), in

which normal Rho cells were not agglutinable in saline, and 168

mumps-treated erythrocytes agglutinated to a titer of 1:1:096, FR8- and NDV-treated cell agglutination was negligible, while trypsin- treated Rho cells agglutinated to a titer of 1:16. However, in this serum the red cells of all four patients agglutinated strongly, as characterized by reactivity in saline-serum dilutions ranging from 1:61). to 1:256. It thus seemed that red cells from hemolytic anemia patients were antigenically similar to mumps-treated erythrocytes.

Further differences in the antigenic alterations of the red cells from these patients were observed when these cells were used as absorbing cells for anti-HM. They were unable to remove in two absorptions, the normal Rho cell agglutinability although they did greatly reduce it. This observation is in contrast to that observed after two absorptions with normal Rho red cells in which normal cells were able to remove virtually all homologous agglutinins. The erythrocytes from one of these hemolytic anemia patients removed from anti-HM, in two absorptions, all homologous agglutinins while normal and modified-cell agglutinins remained but were reduced as compared with the unabsorbed serum. However, the cells of this patient when trypsinized did agglutinate in

this homologously absorbed serum.

Ho antibody was detectable on the hemolytic anemia cells as negative agglutinations were observed when these cells were

incubated in the presence of a properly standardized antiglobulin 16?

serum.

Normal and modified-eell reactivity in the sera of these patients was studied and compared with that of the serum from a normal individual. These results are summarized in Table LI. The erythrocytes were first titrated in saline dilutions of the various sera, read for agglutination, and then washed three times in saline. To these washed red cells was added 0.1 cc. of a standardized and appropriately absorbed anti-human globulin rabbit serum. The cells were re-incubated for 30 minutes at 37°C and read for agglutination in this antiglobulin developed reaction.

The data in Table LI, in general, show that modified-cell agglutinability was more marked in hemolytic anemia sera than in normal serum. The titers obtained in the antiglobulin test, for the most part, were proceeded by zones of complete inagglutin- ability. The 1:32 anti-globulin titers recorded for normal RIiq and trypsinized RIIq red cells were mainly only one or two tube agglutinationsj namely, at 1:16 and 1:32 serum dilutions. All agglutinins were removed from these sera with normal Rho red cell absorptions.

Though inconclusive, these results with cells and sera of hemolytic anemia patients as well as those observable in normal serum are not incompatible with the basic premise of this investigation. The cells of hemolytic anemia patients showed altered antigenicity most distinctly with the incomplete normal TABLE H I

Titrations of Saline Anti-RhQ(l>)# after Absorptions with Pneumococcal Polysaccharides Types I and III and Their Respective Uronic Acids

Absorbing Test Dilutions of Serum Material Cells c 2 U 8 16 32 6U 128 256 £12 102U

0 WH 3 3 1 ___ HNDV - U 3 3 3 3 2 2 2 1 1

1 x with Pn. Polysac. NH m m 3 2 + m m _ m m m m «. Type I HNDV - 2 3 1 2 - - -

1 x with Pn. Polysac. NH 3 3 1 mm MB «M MB •> Type III HNBV - h 2 1 - - MB --

1 x with saturated NH h 3 2 *• MB mm tm BM solution of glucurone HNW *• it 3 2 MB * ** in saline

1 x with saturated NH MB solution of alpha-D- m m 2 1 galacturonic acid in saline

* Knickerbocker 171

Rho cell antibody of antiserum to mumps-treated Rho erythrocytes.

As formulated previously, this form of antibody is that which is to be expected in the serum of hemolytic anemia patients, i.e., reacting as a complete agglutinin for the antigenically altered cells but as an incomplete agglutinin for normal cells. As previously noted, though distinct, the serologic specificity for mumps-modified cells is more closely related to normal cell anti­ genicity than is that of the PR8-NDV type antigenic red cell alteration. Since red cells so altered and consequently bound

■with antibody are removed from the circulation at an increased rate it seems unlikely that much modified-cell antibody would ever be present in the serum in easily detectable amounts. For this reason, the results presented in Table LI are not unexpected.

Also, as mentioned before, a similar red cell removal process would function in normal individuals only at a much slower rate.

This is substantiated by the presence of principally incomplete normal and modified-cell agglutinins in generally lower titers in the normal serum. Though it is questionable and seriously doubted that viral and bacterial infections are the direct cause of autoimmune processes, it is not unlikely that the results of these pathogenic events, though ill-defined, may contribute to the establishment of an autoimmune state. If for no other reason than economy on the part of the body to remove the debris left in the wake of pathogenizing agents, it is feasible that antibody 172

formation directed against injured or degenerating tissues would greatly facillitate such a process. Thus. , all individuals should possess antibody for degenerative tissue structures which would be also incomplete for normal tissue, and which might be stimulated periodically by infections. Since the primary specificity of this

antibody would be directed against grossly altered tissue antigens

it would be relatively innocuous in the normal individual and most difficult to demonstrate serologically. Furthermore, since

this postulated antibody is incomplete for normal cells and

absorbable by them, it would be removed from the circulation also

by normal cells and return to the circulation only upon sub­

sequent tissue degeneration as an anmenestic antibody response.

Hexose structures are abundant not only as structural

components of blood group receptors of red cells but also in the

material removed from the erythrocyte surface during the process

of viral eluation (Gottschalk, 1953). Since the nature of the

eluates obtained, from treatments of red cells or their stromata

with mumps virus or periodate (Tables XIII through XXXIII)

indicated a possible carbohydrate specificity, as well as the

fact that virus-modified cell agglutinins were present in anti-

Rho(D) sera (Tables XXXVI through XLVIII), known carbohydrate

materials were used to absorb an Rh-antiserum (Knickerbocker

saline anti-Rho(D) ). These materials as shown in Table H I

were pneumococcus polysaccharides Types I and III and their 173

respective uronic acids. These uronic acids were selected not only for their hexose structure but also for their two constituent reactive groupingsj namely, the carboxyl group on the sixth carbon of the molecule and the aldehyde configuration on the first carbon atom. Both types of substances removed the agglutinin for NDV- treated erythrocytes. Pneumococcus polysaccharide Type I, did not remove the normal Rho cell agglutinin as did its homologous uronic acid (galacturonic acid). This galacturonic acid absorption re­ moved even more agglutinins for the HNDV cell than did its re­ spective pneumococcal polysaccharide. Both of these pneumococcal polysaccharides had previously been observed to precipitate with this particular anti-Rho serum as well as with several other Rh antisera. At the time, the possibility that such precipitation was the result of specific anti-pneumococcal antibody antigen interaction could not be excluded. However, these results recorded in Table LII indicated that such precipitates could also have been due to the reactivity of the pneumococcal polysaccharide and NDV-modified cell antibody.

It, therefore, became even more apparent that the possibility existed that simple tissue constituents once released in the processes of tissue degeneration, produced by viral and bacterial agents, may not be innocuous, but in conjugations with protein materials serve as antigenic stimuli for the production of anti­ body directed not only against the immunogenic conjugate, but also 17U

against the tissues containing the haptenic portion of such pos­ tulated complexes. In conjunction with this concept, it has been observed that a preponderance of mucopolysaccharide depolymers are liberated in the disintegration of tissue matrix in advanced eachexic diseases, neoplasms, hematologic dyscrasias, protracted shock, and collagen diseases* Hexuronic acids, especially glu­ curonic acid, are contained in such mucopolysaccharides and are widely distributed in the animal body as in the chondroitin of cartilage, mucin, heparin, hyaluronic acid, and platelets.

Glucosamine and galactosamine structures are found as integral parts of red blood cell receptors, brain and other tissues. The fact that antibody can be formed to such simple hexose configura­ tions is further substantiated by the recent evidence of Kabat

(1956) in which oligosaccharides were shown to inhibit a dextran- human 1* • • *6 antidextran system and that the dimensions of the complementary areas to some of the antidextran combining sites included areas complementary to tri-, tetra-, and hexa-, sac­ charides. Thus, antibody of carbohydrate specificity may act­ ually be directed against molecular units which arise in vivo from the liberation of degenerative tissue components. Further-

j more, the serologic specificity of molecular units such as the hexuronic acids would reside in the linkages of these compound either in the simple conjugated state or in biological materials of which they are structural components. As observed in the data 175

presented in Table LI pneumococcal polysaccharide types I and III, as well as their respective uronic acids, inhibited the agglutina­ tion of virus-treated erythrocytes in a non-Rho antibody fraction of an anti-fthoCD) serum.

For these reasons, this investigation contains preliminary observations of immunologic and histologic examinations of the response of rabbits injected with low molecular weight carbo­ hydrates, so as to create, if possible, an excess of these sub­ stances in areas of injured or degenerating tissue. For this purpose, doses of 0.25 to 5*0 g. of alpha-D-galacturonic acid or 1.0 to 3*0 g. of glucurone incorporated in as much as 10-20 cc. of adjuvants of the water-in-oil type, were injected intra­ muscularly in the thighs.

Twelve to twenty-eight days later all rabbits developed reactions ranging from small nodules to massive swelling of the area. Skin tests at this time with 0.1 ml. of a 0.5 per cent saline solution of alpha-D-galacturonic acid were typified 2h hours later by areas of erythema \ inch to 2 inches in diameter, the central portions of which were pale and hard and persisted in some instances for 96 hours. The reactions to glucurone were also maximal in 21* hours and characterized merely by erythema and edema. All rabbits became skin test positive in approxi­ mately three weeks. Rabbits previously injected with galact- uronic acid reacted only to the homologous uronic acid, while 176

glucurone-injected animals reacted to both substances. The skin-

sensitivity was passively transferred to normal rabbits -with serum in spite of the delayed nature of the response. Control, normal animals occasionally reacted to this concentration of galacturonic

acid but these differed in being only erythematous reactions, never more than 1cm. in diameter, with a flat, pale center, and which disappeared after 20 hours.

It must be admitted that the serologic examinations of_the

sera of these animals lack conviction since the antigens involved

are speculative. If specific reactions to the uronic acids

occurred, they would undoubtedly be directed toward conjugates,

and not unlikely to more than one such conjugate, possibly in­

cluding normal or abnormal tissue components. Thus, preliminary

serology was performed with the simple uronic acids or with red

cells treated with them. No reactions were observed in agglu­

tination titrations with these so-called "immune” sera and normal

or uronic acid-treated rabbit erythrocytes. Virus and trypsin-

treated human erythrocytes agglutinated in the sera of these

animals prior to and after injection of the carbohydrate materials.

The agglutination of these treated erythrocytes in the sera of

these animals were variable. Precipitation tests involving these

sera and the uronic acids, pneumococcal polysaccharide types I

and II and blood group antigens A and B were inconclusive. Thus,

no direct in vitro antibody demonstrations were obtained with sera 177

from these animals.

In complement-fixation reactions the sera of these animals exhibited an anti-complementary effect prior to and after swelling in the muscles was observed. The base-line and early sera of these animals did not show this anti-complementary activity. Such anti-complementary action of the sera may have been due to the presence of complexes of the uronic acid-protein and specific

antibody. Further substantiation of the existence of such com­ plexes were the observations of positive latex fixation in eluates prepared from the red cells of animals after symptoms had deve­

loped and not in the red cell eluates of unimmunized animals

(Table LIII), These eluates were prepared by incubating heavy

suspensions of washed rabbit red cells from both normal and in­

jected animals at 56°C for 30-60 minutes and removing the super­ natant after centrifugation. Table LIII indicates the results

for sera and eluates with latex ♦ glucurone (G), latex * galact­

uronic acid (ADG), and latex alone. The latter was used to detect

the presence of possible complexes. The sera of the pre-injection bleeding show some reactivity while the eluates of normal ery­

throcytes were negative. Sera from animals 39 days after injec­

tion of each uronic acid were negative in all three tests, but

samples obtained U6 and 57 days after injection of glucurone

were strongly positive in the presence of latex and either uronic

acid. Eluates, however, from red cells obtained 39 days after TABLE LIII

Latex Fixation Reactions with Serum and Eluates from Red Cells of Rabbits Injected with Uronic Acids

Time Uronic after Material Tested Acid Injection Red Cell Latex Latex Injected (days) Serum Eluate l£ G 1% ADG Latex

m m 1:20 * ♦ -— 1:20 ♦ * — - - 1:20 - —- -- 1:20 - - —

ADG 1:20 39 1:20 ♦ ♦ j 39 1:1:0 h* h+ u+ 39 1:1:0* — 39 1:1:0#* — - —

G • m 1:20 + 2+ 39 1:20 + * — 39 1:1:0 k* k * h* 39 1:1:0# - - 2+ 39 1:1:0#* -- _ U6 1:20 1:+ u* 5? 1:20 U+- u+ -

* Absorbed with 1# Glucurone ** Absorbed with 1% 2-D-Galacturonic Acid 179

the ill j action of either agent were positive in all three tests and this reactivity was removed by absorption with either com­ pound. Thus, it would seem that this latex-fixing antibody was present earlier and in greater amounts on cells than in sera. A further indication of the presence of complexes in the red cell eluates and sera of uronic acid-injected animals was the ob­ servation that red cells on which the surface charge had been altered by treatment with chromium chloride agglutinated strongly in red cell eluates while reacting in a lesser degree in the sera of these animals. This chromium-cell agglutination was entirely absent in red cell eluates and sera of normal enimals.

Evidence for antibody reactive with both normal and uronic acid-treated rabbit erythrocytes was demonstrated indirectly as indicated in Table LIV. The glucurone-treated cells, GR, and normal rabbit red cells, NR, were used in 2 per cent suspensions.

After incubation at 37°C with the globulins precipitated from the

serum of a rabbit injected with glucurone, the cells were washed, and incubated with a non-agglutinating dilution (l:102ii) of anti­ rabbit globulin chicken serum (GARG). At the intervals shown after injection, the serum samples increased the agglutinability

of both normal and treated cells in anti-rabbit globulin over

the low initial values of the pre-injection bleeding. At 18

days after injection, agglutination was stronger for treated

cells, while after 33 days, the reverse occurred. Some evidence TABLE LIV

Attempts to Demonstrate Agglutinins for Normal and Glucurone-treated Rabbit Red Cells in Serum Globulin of Glucurone-injected Rabbits

Globulin - ' ' (ORff------— CXHS (1:2) Cells Saline 1:102U Cells Saline 1j102U

Preliminary GR ♦ NR ♦ 12 days GR - - NR - ♦

18 days GR U+ NR 2+ Abs. GR GR - 2+ NR - -

+ 28 days GR NR 2+

33 days GR ♦ NR h * Abs. NR GR - 3+ NR - 2+

39 days GR h + NR 2+ U6 days • GR - 3+ NR - 3+ f>6 days (21 - h * NR - 3+

mm GR mm NR H § 181

that these are different reactions and may interfere with one another was obtained by absorption of the serum globulins. Absorp­ tion of the 18 day sample with treated cells removed the homo­ logous cell activity and to a lesser degree that for normal cells.

"When the 33 day sample was absorbed with normal cells, the anti­ globulin test for normal cells was diminished but the reaction with treated cells was stronger.

Red cells from animals at various intervals after injec­ tion were also agglutinated directly by antiglobulin. Another altered aspect of untreated red cells from these animals was the abnormal sedimentation of dilute suspensions (less than 0.25 per cent) in saline into patterns suggestive of viral hemagglutination.

The abnormal sedimentation pattern also occurred occasionally with cells of control rabbits given Freund adjuvant alone, but such cells were never agglutinated by the same antiglobulin test.

Table LV demonstrates that these two phenomena were constant findings in the animals, but were subjected to considerable variation in individual animals at the various intervals tested.

Electrophoretic patterns of the sera of injected animals were abnormal and varied during the post-injection observations.

The electrophoretic patterns of the sera of an animal that had received 2.0 g. of galacturonic acid in Freund adjuvant were determined for the pre-injection bleeding and those 12 and 18 days after injection. The electrophoretic patterns of the 12 and 18 182

TABLE LV

Altered Reactivity of Erythrocytes from Rabbits Injected with Response of Galacturonic Acid

Time After Injection Anti-Globulin (days) Cells Pattern Test

Normal m m

iu 88U * 885 2* - 889 ♦ _ 891 * ♦ 893 k* — 896 k* -

28 88U * 2+ . 885 — 2+ 889 _ 3* 891 — 896 - -

35 881; 2+ 889 - - 891 u* +

Ul 88U 3* 889 - m m 891 k* -

U8 889 2+ 891 k* -

70 889 k+ 2* 891 2+ *

77 889 k * 2* 891 2+ 2+ 183

day 3arum samples showed an increase in globulin components with a concomitant decrease in the albumin content over that of the base-line serum. When either of these sera were absorbed with saline solutions of the uronic acids, the resulting electro­ phoretic serum patterns were altered. All serum components were reduced but the most marked reduction occurred in the globulin components (Figure 1). This did not occur when the pre-injec­ tion serum sample was comparably absorbed either with glucurone or galacturonic acid (Figure 2). It was also observed that the pattern of a serum obtained several days before death, from an animal injected f> months previously with $.0 g. galacturonic acid in Freund adjuvant demonstrated a marked depression of all com­ ponents, very similar to those patterns just described after addition of the uronic acids to the 12 and 18 day samples.

The changes occurring in the electrophoretic pattern of the serum proteins over a period of six weeks following the injection of a rabbit with 2.0 g. of galacturonic acid were studied (Figure

3). By one week after injection, the globulins were noted to increase while the albumin was slightly depressed. By k weeks,

the albumin fraction was markedly low, representing merely 23 per cent of the proteins present in contrast to 50 per cent

concentration seen in the base-line serum sample. However, in the sixth week increases in all serum components seemed apparent, although the albumin fraction was but 20 per cent of the total. /84

UNABSORBED ABSORBED WITH GLUCURONE ABSORBED WITH cx-D-GALACTURONIC ACID

/i «

/

r \ f

/ \ / U v ' y — v

FIGURE I. ELECTROPHORETIC PATTERNS OF SERUM -18 DAYS AFTER INJECTION -AD 185

UNABSORBED ABSORBED WITH GLUCURONE ABSORBED WITH OC-D-GALACTURONIC ACID

FIGURE 2. electrophoretic patterns of PRELIMINARY SERUM *-AD ELECTROPHORETIC PATTERNS OF SERA FROM A RABBIT INJECTED WITH (X-D-GALACTURONIC ACID INCORPORATED IN FREUND'S ADJUVANT

J

Preliminary 1 Week after injection p/sj

4 Weeks after injection 6 Weeks after injectionI

Fioune 3, 187

The patterns for rabbits receiving glucurone were even more striking, principally because of the fact that the globulin com­ ponents markedly exceeded the albumin. Sera from an animal receiving an equal amount of Freund adjuvant did not vary in the

same fashion and electrophoretic pattern of serum obtained 23

days after injection was essentially normal.

In these animals injected with either uronic acid, the

globulin increase was more marked in the beta and alpha globulin

components. In certain of these animals the increase in alpha-2

globulin was the most marked increase. According to Seibert,

Pfaff and Seibert (19U8), alpha-globulin is elevated in patho­

logical processes involving tissue destruction, while Banerjee

and Chatterjee (1957) attributed beta-globulin increase to the

stability of protein complexes present in the serum. These latter

authors also suggest that albumin depression may be the result of

liver dysfunction.

Coburn and Moore (19U2) studied the plasma proteins in 1$

patients with normal proteinemia and inversion of the albumin to

globulin ration (A/G) in most cases of lupus erythematosus. All

showed a fall of albumin and hyperglobinemia. In two cases which were studied electrophoretically, the hyperglobinemia could be

traced to the gamma globulin. They also observed that the gamma

globulin from cases of lupus erythematosus reacted in vitro with

the phospholipids of Wasserman and Kline antigens, which could 188

explain the oscillations of serologic tests in certain cases of the disease. Hasserick and Borst described an increased alpha-

2 globulin fraction in addition to the hypergammaglobinemia (1956).

According to the literature, all cases of lupus erythematosus are accompanied by hypergammaglobinemia. Baptista, Hoxter, Vellini, and Mungioli (1956) studied the electrophoretic patterns of the sera from 18 individuals with lupus erythematosus, 6 of which belonged to the disseminated and 12 to the fixed type. In all

cases, they observed a decline of albumin and of beta globulin with increases in alpha-2 globulin. The decrease of albumin and of beta globulin was more pronounced in the disseminated cases, whereas the increase of alpha-2 globulin and of fibrinogen was

equivalent in both forms. The gamma globulin increase noted by others wa3 observed only discreetly by these authors in 5 of the

6 disseminated cases and in 1 of the 12 fixed cases. Hyper- proteinamia was observed in one of the disseminated and in four

of the fixed cases. No correlation could be established between

the severity of the disease and the gamma globulin levels. Alpha-

2 globulin was increased in U of the disseminated and in 6 of the fixed cases. Their findings were in agreement with those cited

of Hasserick and Borst. Baptista, Hoxter, Vellini, and Mungioli reported that special attention should be given to the fact that

an increase of alpha-2 globulin is far more frequent than a rise

in gamma globulin. Fibrinogen increase was noted in all the 189

disseminated and in 11 of the 12 fixed cases. Thus, they felt that the simultaneous increase of alpha-2 globulin and fibrinogen is usually found in mesenchymopsbhies, and constitutes a point

■which ought to be considered of greater significance than hypergammaglobinemia. They also considered the constant decrease of beta globulin evident in all the disseminated and the greater part of the fixed cases to be significant and noted that this phenomenon occurred in the Nicolas-Favre disease and in leprosy.

Thus, the sera of these animals injected with uronic acids re­ sembled sera of humans with lupus erythematosus, in that alpha-2 globulin increase occurred but not in regard to beta globulin decrease.

Korngold and Lipari (1956) presented evidence that multiple myeloma proteins are antigenically deficient gamma globulins.

They showed that antisera against multiple myeloma proteins con­ tained antibodies specific for the homologous multiple myeloma globulin* antibodies specific for multiple myeloma globulins of the same group as the immunizing multiple myeloma globulins, and antibodies that cross-reacted with normal gamma-globulins as well as with all other multiple myeloma globulins regardless of their antigenic grouping. Furthermore, since Bence-Jones protein con­ tains determinants present in the multiple myeloma globulin but not in normal globulin, they assumed that it was more closely related to the former. Isotope studies indicated that the theory 190

that Bence-Jones protein is either a breakdown product or pre­

cursor of the multiple myeloma globulin as suggested by immuno­ logical data was improbable. Thus, they suggested that Bence-

Jones proteins are produced by cells that are no longer capable

of synthesizing complete multiple myeloma globulin. The incom­ pletely synthesized proteins, which are smaller and more deficient

antigenically than the serum proteins, are excreted in the urine

as Bence-Jones proteins. Hileller-Eberhard and Kunkel (1956) have

shown that multiple myeloma proteins represent abnormal glyco­

proteins as they were found to exhibit an intensive PAS (per-

iodic-acid-Schiff) reaction after paper electrophoresis and

staining. Pathological sera with marked elevation in gamma­

globulin showed a carbohydrate-protein ratio for the gamma­

globulin similar to that found in the corresponding fraction (7S)

in normal serum. This was only partially true of the myeloma

proteins with a mobility in the gamma-globulin region. There

were differences exhibited among these proteins. The beta-

myeloma proteins, those of faster mobilities, contained con­

siderably more carbohydrate. These two authors discussed the

possibility that these elevated carbohydrate-containing globulins

may contain sialic acid or even neuraminic acid. These sub­

stances have recently been crystallized from serum proteins. And

thus, the increase in gamma-globulin and the greater beta-globulin

increase exhibited in the sera of these animals, injected with 191

uronic acids, could be due to the presence of glycoproteins similar to or the same as these described by Mneller-Eberhard and Kunkel.

It is not unlikely that in these animals basic cellular processes particularly of mesenchymal tissues, as will be de­ scribed later, are disturbed. It is also probable that in addition to tissue degeneration as indicated by increased alpha-2 globulin levels, there also occurred a concomitant immunologic process involving the linkages of the uronic acids and similar carbohydrate materials liberated in the tissue degeneration as shown by the increased beta and gamma globulin levels. To substantiate further such a concept is the recent report of

Deutsch and Morton (1957) in which they discuss the recent evidence that macroglobulins have their antigenic counterparts in normal serum and suggest that these molecules might represent discrete aggregations of serum proteins of low molecular weight. They also present data which promotes their earlier hypothesis that macroglobulins may be aggregates of normal serum globulins of molecular weight near 160,000.

In conjunction with this concept and the skin test reactivity, previously described, are the observations of Aladjem, MacLaren, and Campbell (1957) that skin-sensitizing antibody is not demon­ strable in sera containing large amounts of precipitating anti­ body, while sera containing no precipitating antibody were found to be the most strongly skin-sensitizing. This finding is com­ 192

patible with the results reported here, for even though these animals elicited positive skin tests -with the uronic acids, pre­ cipitation tests with sera of these same animals were negative as formerly described. They also observed that the most frequent distribution of skin-sensitizing activity was that in which maxi­ mum activity was associated with alpha-2 globulins, although some activity was found in other components. Precipitating antibody against the various antigens used in their work was found to be associated only with the gamma globulins. They presented the con­ cept that there may be a circulating antigen-antibody complex in extreme antibody excess that is present in the serum of immunized animals which is responsible for skin-test reactivity with only the serum from such an animal when passively transferred.

Further indications that the injection of rabbits with either uronic acid produced marked disturbance of basic cell processes were the occasional observations of prolonged blood clotting, ab­ normally hemolytic serum, increased red cell fragility, and an increase in reticulocytes. At autopsy, the injection sites varied from mesenchymal-like tissue with either clear or bloody exudates in animals sacrificed U to 11 days after injection to discreetly walled-off, fibrous tissue observable in animals sacrificed later after injection. On gross examination the lungs and kidneys most frequently observed to be hemorrhagic. The other vital organs often appeared pale. 193

Histologic examinations of tissues removed from these animals indicated characteristic changes nhich varied chiefly in degree of reaction depending on time and dosage. The first histologic findings presented •will be those from a series of animals, each injected with 2.0 g. galacturonic acid in a total I.M. inoculum of 10.5 cc. and containing Freund adjuvant. In animals sacrificed il days after injection, there was marked calcification of the skeletal muscle at the injection sites, vascular changes in the hearts, glomerulitis of the kidneys, and patchy foci of pneumonia­ like areas with a minimum of interstitial involvement in the lungs.

The bronchii demonstrated a minimal amount of cellular reaction.

The early changes were most pronounced in the skeletal muscle and heart, as typified by the rheumatic nodule and histiocytic, round cell infiltration of the skeletal muscle, and Aschoff-like changes and myocarditis of the cardiac muscle. Eleven days after injection, the following cellular changes were observable: The skeletal musculature at the injection site exhibited areas of degeneration, as indicated by calcification, next to areas of repair? giant multinucleated cells were present, as was a thick, viscous material composed of histiocytes which was surrounded by mononuclear cells similar to a granuloma. Calcification was evident in the cardiac muscle, as was myocarditis, and early lesions in the blood vessels. Swollen medial cells were evident and the cytoplasm of the cardiac muscle fibers stained eosin­ 19U

ophilic, indicative of the earliest observable changes of endo­ carditis .

Twenty-one days after injection, the sites of injection in skeletal muscle showed improvement, calcification being absent and no acute inflammatory response was evident. A disseminated, homogeneous cell mass was present which looked much like scar

tissue. However, in areas farther away from the injection site in skeletal muscle multinucleated, syncytial masses were observed.

The cardiac muscles were noted to have undergone further degener­

ative changes as the capillary walls were fox the most part dis­

integrated. The myocarditis was no longer present in discrete

foci but was widespread. A multiplicity of nuclei were present

in the cardiac muscle cells which is not usual. Scattered foci were present in the lungs, consisting of cell members which were

larger than those noted earlier. Other areas of the lungs were

completely congested and the vessels showed fibrotic alterations.

The kidney changes were not much more extensive than those noted

after 11 days, although the glomeruli were slightly increased in

size and the cellular reaction was more marked. In all animals

there was a general tubular degeneration in the kidneys and there­

fore is not specifically mentioned*

Thirty days after the injection, the skeletal muscle again

appeared much worse histologically, similar to those changes de­

scribed at U days. Regenerative areas as well as multinucleated 19£

cell masses associated with degenerative changes were present.

Large granulomas composed of histiocytes were demonstrable. A myositis was present in which there was an infiltration of cells into vessels. This reaction differed from that described after

I4. days in that the character of cell filtrate, as well as that of the muscle, was different in that there was an inflammatory reac­ tion without calcification. The alterations previously noted in the heart were progressively worse. More perifocal collections of cells were evident around vessels and areas of necrosis were prominent. An intense cellular reaction occluded blood vessels were observed in the lungs. The glomeruli as well as the tubules were extensively altered, a solid proliferation of tubular cells suggestive of an adenoma being observed. Necrosis and infarcted areas of the kidneys were evident at hemorrhagic sites. In one animal sacrificed at this time (30 days after injection) early fibrinoid degeneration similar to that of scleroderma was seen in the intestine. Muscle fibers were pulled away and around the nucleus.

Forty-three days after injection, the skeletal muscle alter­ ations were much the same as those described at 30 days. There were present disseminated areas of inflammation in the normal muscle structure. An excised skin test site removed from one of these animals showed fibrinoid degeneration and large cell infil­ tration. The hearts showed severe pericarditis in which even the 196

pericardium was raised and appeared unattached in some places.

The heart seemed almost as though lupus erythematosus was present.

Kidney alterations continued and no glomeruli at all were evident.

The lungs still exhibited diffuse foci of histiocytic cells.

Animals receiving small doses of galacturonic acid were sacrificed 66 days after injection and showed similar alterations.

An animal that had received 0.25 g. of this hexose in a total of

6 cc. of inoculum containing Freund adjuvant was injected in one

thigh. At this injection site, no calcification reaction was evident. Evidence of auto-destructive processes of the blood vessels in the skeletal muscle was present. Superior myocarditis was present in the heart, as was a bleeding lesion, and no attempt had been made to wall of the injured area. Walls of the various blood vessels could not be seen. The kidney demonstrated only first degree alterations and marked tubular proliferation. The blood vessels of the lungs were occluded and dense focal masses of cells were seen. In an animal receiving 0.5 g* of galacturonic acid in a total of 12 cc. of inoculum containing Freund adjuvant, marked vascular changes were evident in the skeletal muscle of both injection sites as indicated by the occlusion of blood vessels and on the in growth of connective tissue. Large histio­

cytic cells were present. The changes occurring in the arterioles of the skeletal muscle are best described as being compatible with

those of malignant nephrosclerosis. Degenerative changes were 197

paramount in the heart. Actual holes, or clear areas, were evi­ dent, giving the tissue a "moth-eaten" appearance. The heart alterations were compatible with those of widespread myocardial disease and the pulmonary artery was well occluded. Vascular occlusions were also evident in the kidneys, and the glomeruli were affected in the same manner as previously described. A tendency for focal cell collections wa3 seen in the lungs.

Animals injected with glucurone usually showed more marked kidney alterations than described previously for galacturonic acid-injected animals. One animal, injected with 1.0 g. glucurone incorporated in Freund adjuvant was sacrificed over seven months after the initial injection.NoEndomycium was observed on the heart and the same "moth-eaten" appearance as just described was evident in the cardiac musculature. Also, an ellipsoidal,

Aschoff nodule typical of rheumatic fever was present in the heart.

The skeletal muscle alterations were typified by a histiocytic response with round cells (multiple syncytial cells). Calci­ fication was also evident at the injection sites in skeletal muscle.

Eight of twelve animals injected with 2.0 g. galacturonic acid in Freund adjuvant and one which received U.O g. in a saline- mineral oil emulsion showed definite symptoms of encephalomye­ litis characterized by weakness and paralysis of one or more limbs, muscle tremors, and irritability. The symptoms appeared as early as 6-8 days post-injection in some, and not for 9-10 198

weeks in others. Two animals showing symptoms in 6-8 days were skin tested and developed the characteristic response as described previously. Weekly serum samples of one of these two animals from the seventh day until it was sacrificed on the 26th day were positive in complement-fixation tests with saline-extracts of normal rabbit brains. Sections of areas of the brains of these animals showed diffuse disseminated perivascular round cell in­ filtration of the intercerebral vessels, and some round cell in­ filtration of the medium sized intercerebral vessels, as well as beginning dissolution of myelinated fibers in the areas of in­ flammation indicating allergic encephalomyelitis. Animals in­ jected with glucurone showed similar symptoms of central nervous system involvement although these examinations have not been, as yet, carried out as extensively as with animals injected with galacturonic acid.

The animal described above whose sera when mixed with saline extracted rabbit brain fixed complement and whose brain upon i neuropathological examination showed evidence of allergic en­ cephalomyelitis was sacrificed 26 days after injection with 2.0 g. of galacturonic acid in Freund adjuvant. Further histologic examination of the tissues of this animal showed that in the skeletal muscle the changes were essentially the same as those described for animals sacrificed at 30 days after injection with this compound. No calcification of skeletal muscle was seen. 199

Cellular foci composed of large histiocytic cells were plainly evident. The blood vessels of the cardiac muscle had been dis­ integrated. Multi-nucleated cells were next to what formerly had been blood vessels. Also present in the heart were connective tissue changes involving mucopolysaccharide or fibrinoid degener­ ation. An Aschoff process was present which followed the entire course of a blood vessel. A giant Aschoff nodule occupied the area where a blood vessel had formerly been situated. The reac­ tion in the kidneys of this animal was not as extensive as noted in animals 30-143 days after injection. However, the same process seemed in progress. The glomeruli were in the early processes of degeneration and the previously indicated tubular prolifer­ ation was present. A mixture of diffuse and focal cellular involvement was apparent in the lungs of this animal.

Animals were also injected with these compounds in saline solutions intraperitoneally with no apparent abnormalities. This would be expected since these uronic acids once adsorbed into the circulation are detoxified in the liver and eliminated through the kidney. This route of injection apparently did not afford the opportunity of either tissue injury or the resulting postulated conjugation of these substances with tissue components.

Furthermore, biological materials containing the glucuronic acid configuration (glucurone in solution is actually glucuronic acid) were injected with Freund adjuvant intramuscularly. Such 200

substances were heparin (Connaught Medical Research Laboratories,

Toronto) and hyaluronidae (Wydase, Wyeth... Laboratories, Inc.,

Philadelphia, Pa.). As yet, these studies have not been com­ pleted. However, animals injected with these substances were skin tested with both uronic acids 2-3 weeks after injection and gave the typical skin reactivity as formerly described. The animal injected with k50 USP flocculating units (based on turbo­ metric readings) of hyaluronidase in a total 6.0 cc. inoculum containing Freund adjuvant, was sacrificed 80 days after injec­ tion. At this time, skin tests in this animal were positive only for galacturonic acid, which was characteristic of the Arthus type reaction with a hemorrhagic, raised central portion sur­ rounding a blanched area. The animal had gradually grown list­ less and exhibited occasional tremors and disorientation. Seven to ten days prior to sacrifice noticeable hair loss over the entire body of this animal was apparent. By the time of sacrifice large, depilated areas predominated. Histologically the tissues of this animal were the most altered of any studied with glomer­ ular changes in the kidney extremely marked and greatly resembling those of lupus erythematosus. The electrophoretic serum patterns were similar to those described for glucurone and galacturonic acid-injected animals with the exception that a marked alpha-2 globulin increase was obvious. The brain of this animal also

showed changes typical of allergic encephalomyelitis. Red cells 201

from both the heparin and hyaluronidase-injectad animals fixed antiglobulin and sedimented in abnormal patterns as described with regard to Table LIII. Thus, hyaluronidase when injected

intramuscularly with adjuvant, and liberating both acetyl gluco­

samine and glucuronic acid from hyaluronic acid polymers in the

connective tissue of the skeletal muscle, apparently can bring

about the same changes described for the injections of rabbits with uronic acids. This fact further substantiates the claim

that uronic acids, per se, are capable of conjugating with tissue

protein to initiate essentially an autoimmune process.

Histologic examinations of animals injected with large

amounts of Freund adjuvant and saline (10-12 cc.) did not show

these histologic alterations as described, nor did they show any

evidence of encephalomyelitis either symptomatically or upon

histologic examinations of brain.

Animals were injected with sera from rabbits that had been

injected with these uronic acids in adjuvants as well as with

these sera mixed with the uronic acids. Since this phase of the

work is far from completed, only those observations that, at

this time, have some meaning will be reported here. An animal

was injected I.V. with 0.9 cc. serum obtained from an animal

injected two weeks previously with 2.0 g. galacturonic acid, and

mixed with 0.9 cc. of the homologous uronic acid. This animal

did not elicit skin test reactivity for galacturonic acid until 202

nine days after the intravenous injection of the serum-uronic acid mixture. Eighty-five days later this animal was injected intramuscularly with 2.0 g. galacturonic acid in Freund adjuvant.

Within 20 hours both thighs (injection sites) were swollen ex­ tensively and the animal showed some signs of shock, such as irritability and deep, abdominal breathing. This was not noted in an animal that had previously had been sensitized only with this serum and injected similarly with the uronic acid-adjuvant emulsion intramuscularly.

A female rabbit was injected with 0.1+ cc. serum obtained from an animal which had been injected twelve days previously with 2.0 g. galacturonic acid-Freund adjuvant intramuscularly.

One-hundred-fifty-seven days later this female animal gave birth to a litter of young. All died excepting one which by three weeks after birth was observed to be abnormal with noticeable tumor-like growths present in the spine, abdomen, and thighs.

The structure of the head of this young rabbit was grossly ab­ normal and enlarged. This abnormal infant was extremely weak and thus was sacrificed. Histologic examination indicated abnorma­ lities in the development of mesenchymal tissues. The female was sacrificed 178 days after the serum sensitizing injection and her brain was found to show pathology indicative of allergic en­ cephalomyelitis .

An animal was injected intrademally with the following sera: 203

0.1 cc. amounts from an animal injected 28 and 38 days previously

■with 2.0 g. glucurone in Freund adjuvantj 0.1 cc. from an animal injected l£ days previously with 2.0 g. galacturonic acid in

Freund adjuvantj and 0.1 cc. from this latter animal 27 days after injection. Nineteen hours later these skin injection sites were skin tested with the respective uronic acids. No skin test reac­ tivity was evidenced either immediately or in 2U to U8 hours.

However, if the sera contained complexes of uronic-acid-protein- antibody, no skin reactivity should be expected. Twenty-two days later this animal was observed to be emaciated and near death.

The animal was sacrificed and histologic tissue examinations per­ formed. It was difficult to find normal cardiac tissue in this heart. It appeared that the blood supply had been diverted from the heart. The kidneys did not show the usual tubular reaction but the glomeruli were swollen. The lungs of this animal were essentially normal.

According to the histologic data, marked tissue alterations were in progress as early as the fourth day after injection of the uronic acids. For this reason, animals were injected intraven­ ously with sera from these U-day animals. The sera of the serum- injected animals initially did not precipitate with the "antigen” sera. However, nine days after the injection, the serum of an animal injected with U-day serum precipitated with this "antigen” serum. Sera of the serum-injected animals taken at 16 and 31 20U

days after injection did not. Therefore,' it would seem that the

U-day sera, which showed greatly increased alpha and beta globulins over the base-line sera, contained carbohydrate-protein complexes that were immunogenic. As discussed previously, it is probable that antibody specific for the hapten portion of such complexes would bind with the carbohydrate wherever it was available either as tissue components or in the serum. For this reason, anti­ body of this specificity would not be expected to be present for any time in the circulation as it would be readily removed by tissues containing the hapten specificity. DISCUSSION

In riew of the various findings in acquired hemolytic anemia it is generally conceded that this condition is associated with autoimmunization. Stats and Wasserman (1952) pointed out that antibody which is formed in acquired hemolytic anemia

. . . could be looked upon as the abnormal product of antibody synthesis by a diseased or derranged reticuloendothelial (or lymphocyte or plasma cell) system. ...The other point of view about the origin of a hemolytic anemia placed the primary abnorma­ lity upon the red cells. It can be conceived that minor alterations in the constitution or structure of the red cell may impart new antigenic properties to it. Such a new antigen could then immunize the patient against the altered red cells. It seems entirely reasonable to expect that an antibody produced in response to altered or partially-damaged red cells might have marked effects on normal cells.

Evidence of virus modified cells as well as enzyme altered cells in hemolytic anemia can be included among the alterations postu­ lated for the initiation of this immunologic phenomenon (Wallace,

Dodd, and Wright, 1955)* Although no attempts to induce auto- immunization as such were undertaken in this investigation, evidence was presented in the preceding section which affords greater insight into the nature of erythrocyte alterations. In addition, the possible implications of both the modifying process as well as the fate of altered tissue cells, typified here by virus or enzyme treated red cells, will be discussed.

Red cell alterations produced by the mumps-Newcastle

2Q5 206

disease-influenza group are typified by alterations in electro­ phoretic mobility, receptor activity, antigenic specificity, and serologic reactivity, the latter two being most pertinent in this investigation. Furthermore, virus-treated Rho positive erythro­ cytes have been observed to agglutinate in saline dilutions of incomplete anti-Rh0 antibody. This fact coupled with the recent reports that in some patients with hemolytic anemia, autoanti­ body with Rh and Hr antigenic specificity is produced, when considered along with the observations reported here involving the interaction of the erythrocyte stromata and mumps virus, suggest a possible explanation for the formation of such antibody.

In antiserum to Rhg erythrocytes which have been treated with mumps virus, an incomplete antibody for normal Rh0 red cells was detected which agglutinated mumps-treated Rho or Hr erythrocytes, as well as normal Hr red cells and periodate-treated Rho cells.

An antibody such as this would be the type expected if modified red cells served as the incitant in the establishment of hemolytic anemia, i.e., an agglutinin for the antigenically altered red cell but an incomplete antibody for the normal, unaltered cell.

Both the normal and modified red cells should absorb an antibody of this type from the serum. This was actually the case with the incomplete normal cell antibody in antiserum to mumps- treated Rho red cells, for absorption either with mumps-treated or normal red cells removed all antibody. 207

The fact that mumps treatment involves subtle, yet defini­ tive, alterations of red cell antigens is evident when the fol­ lowing observations are considered* In serologic comparisons, the reactivity of antiserum to mumps-treated red cells with normal and treated cells more closely paralleled that of antiserum to normal, untreated red cells. Furthermore, this reactivity was different from that observed in antisera to red cells treated with either influenza (FR8) or Newcastle disease virus (NDV).

Likewise, mumps-treated Rho red cells were not as reactive in

Rho(D) antisera as were PR8- or NDV-treated erythrocytes, or as agglutinable as were normal Rho red cells in saline anti-

RhQ(D)* In addition, treatment of red cells with mumps virus did not alter the electrophoretic mobility of the red cells in comparison with that observed with the other viral treatments.

It is obvious that treatments of erythrocytes with either PR8 or NDV were distinctly severe enough to cause marked antigenic changes in the stromata antigens affected, and thereby normal cell antigens are not those which primarily register in the formation of antibody to these red cell treatments. For this reason, the appearance of the incomplete normal cell agglutinin in antiserum to mumps-treated red cells after two absorptions with PR8-treated red cells is more readily explained. Since the FR8-treatment of red cells would share in common with the mumps treatment alterations of the viral receptor material, 208

absorption with PR8-treated red cells should remove antibody common to the altered viral receptor. This is apparently the case for PR8-cell absorption removed essentially all homologous agglutinins and those for the NDV modification. As has been observed in the treatments of either human, chicken, or chick embryo erythrocytes (Wallace, 1953 and Bigley, 1955), the NDV- red cell modification is such that it encompasses those red cell alterations produced by PR8 virus. This greater antigen modi­ fying capacity of NDV has been illustrated further when NDV- treated red cells were used to absorb normal cell or mumps- treated cell antiserum. All modified-cell agglutinins were re­ moved for the homologous treated cells as well as for PR8- treated cells but agglutinins were left for mumps-treated cells and for normal red cells. If the antigenic mosaic of NDV- treated erythrocytes is pictured to resemble more closely that of a normal red cell after the initial in vivo degradation of the cell has occurred in the immunized animal then it (the NDV

cell) should not be expected to remove antibody to the prominent normal cell antigens for it is simply lacking them. This parti­

cular strain of NDV has been shown to alter red cell structure more than comparable treatment with the FE8 virus because of its marked hemolytic activity, its greater alteration of elec­

trophoretic mobility, and most importantly its more distinctive serologic alterations (Wallace, 1953). 209

The fact that mumps involvement of the viral receptor material was minimal in comparison with that of the other two viruses was

further substantiated by the observation that neither chicken or

chick embryo red cell receptor antigens were much affected by it

serologically (Bigley, 1955). It was then evident that mumps

virus affected most markedly those antigens peculiar to human

erythrocytes. Since periodate-treated red cells reacted to the

same extent with the incomplete Rho cell antibody contained in

antiserum to mumps-treated human erythrocytes as did normal Hr

red cells, the examination of the possible alterations of the

Rh0(D) configuration by mumps virus was indicated. Furthermore,

the similarity of the actions of the periodate ion and mumps

virus on viral receptor material of red cells was substantiated

by the observations that both involved oxidative processes. The

elution-hemolysis caused by mumps virus was observed by Gardner

and Morgan (1952) to be favored by oxidative conditions while

Fasekas de St. Groth and Graham (19U9) had observed that the

periodate ion, in low concentrations, altered oxidatively these

same viral receptors, both on red cells and on susceptible tissue

cells•

Morgan and Watkins (1951) observed that oxidation of red cells

destroyed, partially or completely, the Rhesus D (Rho) antigen

as evidenced by reduction or absence of agglutination in spe­

cific antiserum. Coffin and Pickles (1953) reported that speci­ 210

fic agglutination of periodate-treated cells may be restored if periodate treatment has not been too vigorous. Presumable D anti­ gen sites, previously unaltered by periodate, are made available by the action of trypsin. Mumps-treated red cells reacted mini­ mally or not at all in incomplete anti-Rho(D), in a manner similar to periodate-treated cells. Upon subsequent trypsinization, mumps- treated erythrocytes were observed to agglutinate in incomplete anti-RhQ just as did trypsinized red cells and periodate cells treated with trypsin. The reversal of this did not occur} namely, trypsin-treated red cells upon subsequent treatment with mumps virus did not lose their agglutinability in incomplete anti-Rh serum. These observations moreover fit rather well into the postulated action of mumps virus on red cell surface antigens, i.e., that a mumps-treated Rho cell is virtually, on the surface, a mumps-Rh negative cell. This is further substantiated by the fact that normal Rh0 negative (Hr) erythrocytes were agglutinated by apparently incomplete normal Rh0 cell antibody, described to be present in specific antiserum to mumps-treated Rho red cells, to the very same extent as were periodate-treated Rho cells, the surfaces of which were also devoid of reactive Rho antigen con­ figurations. Furthermore, mumps-treated Rh negative cells agglu­ tinated in this same incomplete normal RhQ cell agglutinating serum to the same titer, only stronger, than did the immunizing mumps-treated Rho cell, which indicates that the mumps-treated 211

Rh0 cell, upon primary degradation in the immunized animal, registers firstly as an Rh negative-virus-treated cell. Mumps virus and periodate treatments of erythrocytes are similar only with respect to their actions on the Rho(D) antigen, for it is evident in specific mumps-cell antiserum that periodate and mumps-

treated cells react differently with the ultimate serologic spe­

cificity residing in the immunizing, HM, cell.

The fact that Rh0(D) reactivity still resides in red cells

treated with either periodate or mumps virus is demonstrable by

their agglutinability in anti-Rh0(D) after trypsinization.

Trypsin treatment must merely remove other configurational units which block Rh0 antigen lying deeper in the stromata. The fact

that trypsin-treated cells subsequently treated with mumps virus did not lose their agglutinability in Rh antisera also is to be

expected, for the trypsin-treated cell is already an altered cell

and cannot possibly present the same configurational groupings

of a normal Rho cell to agents such as the periodate ion or mumps virus. Thus, tryptic action may expose too many Rho(D)

sites for complete inactivation or removal by these agents.

This explanation is doubtful since the reactivity of trypsin-

treated red cells treated with mumps virus for prolonged time

intervals' did not change in anti-Rho(D). However, the pos­

sibility exists that trypsinized cells are inert to the oxi­ dative actions of either mumps virus or periodate ions, possibly 212

because the combining sites of attachment of these agents are either blocked or are no longer complementary in configuration so that attachment is not possible. This latter possibility is negated by the fact that trypsinized red cells, either Rho or

Hr, agglutinated in viral hemagglutination titrations with mumps virus to the same titer as did normal Rh0 or Hr erythrocytes.

In investigations concerning the nature of the virus recep­ tor of susceptible red cells, Gottschalk (1953) demonstrated that as a result of the enzyme-like action of the viral particles during elution, certain chemical substances were released from the cell into the supernatant. Such materials were found to be various hexoses, hexosamines, and a compound identified as 2- carboxypyrrole. These materials were found after treatments of both viral hemagglutination mucoid inhibitors and red cells with influenza and Newcastle disease viruses. There was no reason to believe that mumps virus did not exhibit a similar action during the elution process. It, therefore became apparent that eluates from red cells or their stromata treated with mumps virus or periodate, may contain Rh0(D) antigen configurations and that these treatments of red cells did not involve destruc­ tion of the D antigen but literally removed these Rh0 hapten structures from the cell surface. This proved to be the case.

As shown in the preceding section, eluates from Rho cells or stromata treated with either periodate or mumps virus inhibited 213

Rh0 agglutinability in anti-Rho(D) sera. As predicated by the reactivity of trypsin-, RD3-, FR8-, or NDV-, treated red cells in anti-Rh0(D) sera, eluates from these treatment mixtures did not exhibit anti-Rh0 activity in any manner. Thus, it seems that either periodate or munrps virus treatment of Rho stromata involves the removal of the Rh0(D) hapten from the available surface structures.

Furthermore, these Rho inhibitory eluates were shown to inhibit specifically only Rho activity and not that of the other

Hr and Rh antigens. The specificity of this Rh0 inhibitor was further demonstrated by the formation of specific precipitates when anti-RhQ serum and inhibitory eluate were mixed together.

This Rho inhibition was not due to the virus, per se, as perio­ date eluates demonstrated an equally potent Rho inhibition.

Moreover, eluates in which the mumps virus was removed by absorp­ tion with Hr (cde/cde) red cells, or in which the viral activity was destroyed by heat, exhibited anti-Rh0 activity in undiminished potency. In addition, it was shown that if periodate oxidation wa3 prolonged, the Rh0 nature of the eluate was destroyed. Simi­ larly, when mixtures of Rho stromata and mumps virus were incu­ bated over extended periods with eluate samples removed period­ ically, the Rho inhibitor seemed to be present only at certain intervals as measured by inhibition of Rho agglutinability. As a consequence of these apparent bursts of inhibitory activity of these eluates, a secondary reaction was postulated to involve virus-RKq hapten-cell protein complexes. Once the virus had eluted from the cell surface with the consequent loss from the stromata of surface Rh0 configurations as well as other undefined substances, it is quite likely that the virus adsorbed to com­ plementary structures present in these eluates and, since such structures possess Rho antigen material, the Rh0 activity of the eluate was masked as was observed to be the case. To substan­ tiate further this secondary reaction was the observation that non-inhibitory eluates upon continued incubation could be shown to inhibit the same Rho agglutinating system that formerly had been inhibited weakly or not at all.

The reaction involving the viral enzyme-like activity was found to be essentially that of an enzyme in that it functioned optimally in a narrow pH range (pH 6.8 - 7»l) and that it reacted best at certain temperatures, namely, 37°C. Also, concentration of reactants were demonstrated to be of importance as measured by the extent of inhibition produced. (Chandler, 1936).

The Rh0-like material released from its stromal position only on treatment with either mumps virus or periodate further resembled Rho antigen in that once it was removed from the cell surface it was relatively unstable, withstanding storage at refrigerator temperature for only several days during which time its potency as an Rho inhibitor diminished. Storage at -$0°C was effective (Chandler, 195>6), further indicating a highly labile 215

material.

From these considerations it is possible to postulate the probable mechanisms of the release of Rho antigen by virus or periodate. As a result of the enxyme-like action of the viral particles, an amide bond of the receptor during the elution process, certain chemical entities are removed from the cell to the super­ natant. With the influenza viruses and NDV such substances were found to be complexes of various hexosamines and the 2-carboxy- pyrrole structure previously mentioned, (Gottschalk, 1953)•

Assuming that mumps virus possesses amidase activity as do the influenza viruses and NDV, the action of the viral particle may be thought of as breaking an amide bond, releasing an amino acid carbohydrate complex which acts serologically as Rh0(D). It may be postulated further that the periodate ion released the complex by breaking an alpha-glycol bond in the sugar between the amide bond and the protein, and, in contrast to the virus, would con­ tinue to act on the carbohydrate of the complex, destroying the specificity of the Rho(D) configuration.

During prolonged treatment of the Rh0(D) red cell stromata with mumps virus, maximum anti-Rh inhibition was detected in eluates only at certain intervals, which correlated with the appearance of titratable virus in the eluate. This observation suggests an association of the release of anti-Rh inhibitor with the elution of viral particles from the cell surface. This 216

does not rule out the possibility that the anti-Rh inhibitor may be released while the virus is still attached to the cell by the "browsing" phenomenon of Ada and Stone (1950) in which the virus may be thought of as continually eluting from some recep­ tors while still attached to and acting on other receptors. The periodic reduction of anti-Rh inhibitor occurring between peak titers during prolonged treatment of stromata with virus seemed to represent a "masking" of antigen probably from some secondary action of virus. When the virus was essentially eliminated as it was eluted from the stromata by including anti-mumps serum in the reaction mixture, or when the virus enzyme was inac­ tivated by heat when inhibitor was at peak titer, the charac­ teristic decreases in titer were absent, strongly suggesting that virus was directly involved in the postulated secondary reaction

(Chandler, 1956).

Two possibilities are evident as to the mechanism involved in this "masking" effect of virus on anti-Rh inhibitor. The

similarity of the action of periodate and of mumps virus on red

cells indicates that the virus as well as periodate may actually destroy the Rh antigen by a similar process. However, the sub­

sequent periodic reappearance of inhibitor in almost the exact

titer and at a time when red cells are known to be inagglutinable,

and as a consequence not available for further enzyme action,

strongly favors the suggestion that the interval peak titers are 217

due to the same substance which is masked at other intervals by a further readsorption of virus. In addition, these fluctu­ ations in titer continue to occur during virus treatment, where­ as the phenomenon occurs only early during periodate treatment resulting in the complete loss of inhibitor. The addition of fresh active virus to a heat-rinactivated eluate (Chandler, 1956) very quickly resulted in the characteristic decrease in anti-Rh inhibitor titer followed, in time, by a return to peak titer.

Further explanation of the 'basking" of inhibitor by virus may be postulated by reference again to the original assumption of an association between virus receptor and Rh antigen, such as the existence of the latter as some portion of the carbo­ hydrate configuration of the virus receptor. The loss of such a receptor-antigen complex from Rho(D) cells by oxidation of the carbohydrate portion with periodate, or the enzymatic action of virus during elution, is indicated by the loss of viral and anti-Rh agglutinability by treated cells. At the same time, anti-Rh inhibitor appeared in both types of eluates, accompanied by free virus in the mumps-cell eluate. However, it is quite likely that the anti-Rh inhibitor, or Rho antigen, exists in different forms in the two types of eluates, since, as mentioned previously, periodate oxidation and virus enzyme probably remove the amino acid-carbohydrate fraction by severance of different bonds at different sites in the receptor complex. Perhaps only 218

that portion of the receptor carbohydrate corresponding to the antigenic Rh configuration exists in the periodate eluates which in the absence of periodate inhibitor is subsequently'- destroyed by the continued oxidative action of periodate . In the mumps­ cell eluate, the antigen may exist as part of a larger complex, perhaps in that part of the receptor carbohydrate which also acts as the adsorbing site for virus by reason of complementary configuration. This form may be more stable, or may not be destroyed, in contrast to the previous situation, simply because the virus does not reporduce the oxidative action of periodate.

To substantiate this postulate further, the mumps-stromata type eluate formed stable precipitates in diluted anti-Rho(D) sera in contrast to periodate eluates.

It is only necessary then to assume that the virus comple­ mentary portion of the larger carbohydrate complex in the virus- treated cell eluates was still intact and contained at least part of the antigenic determinants, to provide a possible explanation of the periodic '•masking" of anti-Rh inhibitor. Thus elution of virus was accompanied by the release of a complex with a dual receptor-antigen function. With continued incubation at 37°C, virus combined again with this soluble receptor, at the same time

"masking" a portion of antigen determinants simply by making them unavailable serologically. Elution may occur again without destruction of antigen, either because of the lack of oxidative 219

action on the part of the virus, or because "masking” of virus

is incidental, perhaps steric hindrance. It may be assumed that

the receptor-antigen complex is present in excess, since rarely,

if ever, does the inhibitor disappear completely; or perhaps

only a portion of antigenic determinants is involved by virus

adsorption. Thus, when eluate was heated, inactivated virus which had lost the "browsing" capacity of enzymatically active

virus, was not capable of masking as much antigen. This excess,

in turn, provided the substrate for the "masking" action of

active virus added to heated eluate. Since trypsin exerted no

detectable effect upon heat-stabilized eluates (Chandler, 1956),

it was concluded that peptide linkages were absent from the

determinant portion of the inhibitor. Obviously, the enzymatic

action of the mumps virus must differ in some respects from the

amidase action of the influenza viruses and NDV. This difference

is indicated by the fact that mumps virus affects the Rh0(D)

antigen, while the other two do not.

The rapid disappearance of inhibitor in periodate-treated

cell eluates and the slower decline in the mumps-cell eluates

indicate that the substance is labile on release from cells.

Since Morgan and Watkins (1951) found that several concentrations

of periodate destroyed Rho(D) antigen over a relatively wide pH

range , it seems logical to conclude that the lability of the

eluate factor must be due to continued oxidative degeneration. 220

In either case, this may be 11 auto-oxidation”, or possibly in the mumps-cell eluates, other substances were present which reacted slowly with the antigen. It is also possible that the postulated complex in which the antigen exists in the latter eluate may influence its degradation. This lability is also compatible with the proposed identification of inhibitor as

Rh0(D) antigen in view of the belief that the failure to isolate this substance is related to some inherent instability when re­ leased from cells. (Bigley, Chandler, and Dodd, 1957)*

According to the findings of Makinodan and Maoris (1955) that antibodies to red cell antigens other than Rh0(D) were present in anti-Rh0 sera,was the observation that enzyme and virus-treated red cells were reactive with antibodies other than anti-Rho in several commercial Rh antisera. It is reason­ able to assume that in the immunization of an individual with

Rh0(D) antigen that other stromal structures present in the immunizing cell may register. The fact that normal and treated

Rh negative (Hr) erythrocytes reacted equally well with the non-Rh antibody present in Rh antisera further substantiates

this. This non-Rh antibody could well be directed against the protein carrier material of the Rh hapten and in that event both Rh0 and Hr cells should react with it as they did.

The fact that mumps-treated chicken erythrocytes reacted with one Rh0 antiserum in albumin dilutions of serum indicates 221

that the non-Rh antibody in Rh antiserum is probably directed against material in the altered viral receptor. This suggests a possible relationship between viral receptor material and

Rho antigen configurations as they are situated in the red cell stromata. Either the Rho antigen is part of this viral receptor area or is in close proximity to it, especially that portion to which mumps virus specifically adsorbs.

In the one anti-Rh0(D) serum (Knickerbocker) mumps-treated red cells removed all agglutinins, both the saline and plasma type antibodies. As previously discussed, the mumps treatment removed Rh0 activity from the surface of the erythrocyte, and that the mumps-treated Rh0 cell upon primary degradation re­ gistered as an Rh negative virus-treated cell, both of which indi­ cate the absorption behavior of the mumps-treated Rh0 cell in anti-Rh0 serum. The most logical explanation for the complete removal of the Rho antibody as well as modified-eell agglutinins in one such absorption of anti-Rho serum is dispecificity. It is assumed, as previously, that the mumps cell possesses a surface devoid, for the most part, of Rho configurations as shown by the inagglutinability of these cells in saline dilutions of Rho sera. That Rho activity is removed from the cell is substan­ tiated by the fact that eluates from such treated cells inhibit anti-D antibody. That more Rho groupings are left masked or deeper in the stromata framework of the cell is obvious upon the 222

restoration of Rh0 activity when such cells are subsequently trypsinized. Thus, the Rho mumps-treated cell when in the pre­ sence of anti-Rho serum has several reactive possibilities: firstly, the virus alteration may be such that the modified anti­ gens adsorb the virus-modified cell antibody and antibody to the protein carrier of the Rho(D) hapten simultaneously. However, if this were the case a preferential absorption of Rh antibody should be expected and virus-cell antibody left in low, but detectable amounts. Secondly, the possibility exists that Rh antiserum contains antibody mainly for the Rh0 hapten-protein carrier combination and that the Rh0 mumps cell surface bares a preponderance of the carrier antigen to this antibody and thereby removes all agglutinins from the serum. This concept demands that modified-cell antibody possesses a primary spe­ cificity for the Rh0 protein carrier and would be one example of a dispecific antibody.

And lastly, the antibody contained in Rh antiserum could be of the nature of a mixture containing antibodies for the Rh0 specificity, the protein carrier, for the cell structures bared only upon extensive red cell degradation, and combinations of these. The mumps-treated cell according to its postulated sur­ face could adsorb to it antibody specific for the Rho carrier and at the same time for antigens exposed during viral elution, or to the Rhc antigen, itself, (though masked in some manner) and 223

to either the masking groups or some viral-altered antigen. This, again predicates an antibody of dispecificity. To strengthen this argument are the experimental observations that in this un­ absorbed serum trypsin-treated Rh0 cells agglutinated directly while mumps-treated Rh0 cells did not. This difference between

the mumps- and trypsin-treated cells may be that incomplete anti­ body for normal negative cells (designated in the results as the

HNDV agglutinin) which, when present, blocked the Rho antibody from the mumps-treated Rho cells but not from the trypsinized

Rho cells. Since mumps-treated Rho cells removed all agglutinins

from the anti-Rho while trypsinized Rho cells removed all anti­ bodies excepting an incomplete antibody for both types of NDV

cell, it is obvious that the enzyme treatment does not involve

the same antigenic alterations of the red cell as do the viral

treatments. Wallace (l9!?3) formerly had demonstrated this in

antisera to specific virus and enzyme red cell treatment. The

significance of this observation in anti-Rh0 serum of the

similarities and differences between mumps virus and trypsin

treatments of Rho red cells further illustrates the ability of

mumps-treated cells to absorb incomplete normal cell agglutinins.

From the agglutination-absorption studies of an anti-Rho

albumin agglutinating system, it became more apparent that in-

agglutinability of normal Rho cells in the incomplete anti-Rho, may be due to the presence in the serum of antibody to other 22k

cell structures which is absorbable by either RhQ- or Hr-NDV treated red cells. This would easily explain the nature of the matured antibody response in the process of Rh immunization for the matured antibody is supposedly an incomplete one. Thus, the speculated monovalent antibody is not really monovalent but a divalent antibody, the direct demonstration of which is inter­ fered with because of the presence in the serum of antibody to other erythrocyte antigenic configurations. It is reasonable to assume that in the immunologic response to red cells, as to bacterial cells, that the continued antigenic stimulus and cell degradation promotes the formation of antibody to subsurface structures and consequently the specificity of the primary sti­ mulus appears lost in the myriad of antibody specificities pro­ duced. Thus, in the demonstration of antibody to Rho(D) antigen that has been submerged by numerous other cell antibodies, all with progressively dissimilar specificities, there are two alternativesj namely, 1.) enhancement of the antigen concen­ tration as is done in the trypsinization of cells. Trypsinization undoubtedly removes some of the antigens, antibody to which interferes with Rho antibody. And, 2.) by removal of the inter­ fering antibody with cells either devoid of Rh0 or cells the antigenic make-up of which has been so altered that deeper cell antigens are exposed. Thus, these newly bared antigens will remove first the antibody to themselves as, having been reg- 225

istered more recently than that of the Rho, they will be more specific. This second alternative is best typified by the ab­ sorptions of Rho antisera with both Rh0 and Hr NDV-treated red cells, in which normal Rho cells became agglutinable in saline dilutions of incomplete Rho(D) antibody.

Thus, it would seem that the primary specificity in any

Rh antiserum is directed against the Rho antigen, but that in the process of immunization to the D antigen other cellular configurations, among which are a variety of the antigens bared upon viral treatment of similar cells, also serve as immunizing materials. Since such antibodies are directed toward altered red cells or perhaps, more accurately, degenerative red cells, such antibody as can be detected by virus-modified cells is in­ nocuous in the normal individual or, on the other hand, functions in the normal process of the removal of the older, degenerating cells.

Dodd, Wright, et al. (1953) showed that cells of many hemo­ lytic anemia patients were agglutinated by 5) ecific antiserum to trypsin-treated red cells. More recently, Crowley and Bouroncle

(1956), corroborating the findings of others, have demonstrated that some patients with autoimmune hemolytic anemia may form antibodies against specific blood group antigens present in their own erythrocytes. Though inconclusive, the results ob­ tained with cells and sera of four hemolytic anemia patients as 226

wall as the observations noted in normal serum were not incom­ patible with the basic premise of this investigation. The cells of hemolytic anemia patients showed altered antigenicity most distinctly with the incomplete normal Rho cell antibody of anti­ serum to mumps-treated Rh0 erythrocytes. As formulated pre­ viously, this form of antibody is that which is to be expected in the serum of hemolytic anemia patients, i.e., reacting as a complete agglutinin for the antigenically altered cells but as an incomplete agglutinin for normal cells. As previously noted, the serologic specificity of mumps-modified cells, though dis­ tinct, is more closely related to that of normal cell antigen­ icity than is the alteration produced by PR8-NDV treatments.

Since red cells, so altered and consequently bound with antibody, are removed from the circulation at an increased rate, it seems unlikely that much modified-cell antibody would ever be present in the serum in readily detectable amounts. This was observed to be the case in the serologic examinations of the reactivity of the hemolytic anemia sera with normal as well as virus-mod­ ified red cells. A similar red cell removal process would function in normal individuals only at a much slower rate. This is substantiated by the presence of principally incomplete normal serum. Though it is questionable and seriously doubted that viral and bacterial infections are the direct cause of autoimmune processes, it is not unlikely that the results of these patho- 227

ganic events, though ill-defined, may contribute to the estab­ lishment of an autoimmune state. If for no other reason than economy on the part of the body to remove the debris left in the wake of pathogenizing agents, it is feasible that antibody for­ mation directed against injured or degenerating tissues would greatly facillitate such a process. Thus, all individuals should possess antibody for degenerative tissue structures which would also be incomplete for normal tissue, and which might be stimu­ lated periodically by infections. Since the primary specificity of this antibody would be directed against grossly altered tissue antigens it would be relatively innocuous in the normal individual and most difficult to demonstrate serologically.

Furthermore, since this postulated antibody is incomplete for normal cells and absorbable by them, it would be removed from the circulation also by normal cells and return only upon sub­ sequent tissue degeneration as an anmenestic antibody response.

Thus, the possibility presents itself that not only altered red cells but products removed from them in the modifying process may serve as autoantigens. Such products as Rh0 antigen removed from the erythrocytes surface could be easily bound to more stable factors either present in the serum or in relation to intact cells.

The fact that a substance is highly labile does not predicate its complete inactivation in vivo and thereby becoming entirely in­ nocuous. Because the term in vivo signifies the presence of a 228

myriad of unknown factors it also carries with it the implication of dynamic interactions and for this reason it would seem that products, even simple units containing relatively few carbon atoms, resulting from cellular degradations of the type in­ volving virus and tissue cell or bacteria and cell would be bound to any number of substances, particularly protein as it is so readily available. Usually, this type of mechanism would be considered a detoxification type process and likewise in the usual circumstances not much of such material would be present in the body. There is no reason why simple substances resulting from cell breakdown could not conjugate with larger proteins and thus immunize. Antibody directed against such determinants would not necessarily predicate pathologic events or injury from an autoimmune condition but on the contrary, might prevent auto­ immune destruction of the tissues containing the corresponding antigenic determinants. For antibody of this nature would facilitate the rapid removal of dead or degenerating tissues and would be most difficult, for this same reason, to detect serologically. However, the difficulty would arise when an abnormally abundant amount of tissue degenerative products was being liberated from a rather constant source, for then an un­ usual amount of antibody to autoantigens would be produced, and consequently combine with normal functioning units possessing the hapten material against which its primary specificity was 229

directed.

That such a process is credible is even more evident when toxicity resulting from burns is considered. Toxicity reactions are not considered immunologic in character nor is there time enough after the Hshocktt for the formation of antibody as class­ ically defined. But, if the antibody postulated to be present normally is capable of combination not only with the injured tissue components, but also with normal tissue antigens, then perhaps when the processes of elimination used ordinarily to remove tissue component bound to antibody are overloaded, elim­ ination is slowed or completely stopped thus affording an oppor­ tunity for the antibody-bound complex to bind either specifically to, or non-specifically adsorb to, normally functioning cells.

Likewise, the functions of these normal cells are then impaired.

This may be an exaggerated case of what actually happens during the rapidity of toxic reactions. On the other hand, when such

circumstances progress more slowly and are thus prolonged, as in protracted shock, large amounts of mucopolysaccharide depolymers have been observed to be liberated (Barnard, 195U). These

substances could quite conceivably conjugate to serum proteins and register as antigens.

The fact that antibody may be directed against small molecular units was recently demonstrated by Kabat (1956), who showed that oligosaccharides inhibited a dextran-human 1...... 6 230

antidextran system and that the dimensions of the complementary areas of some of the antidextran combining sites included areas complementary to tri-, tetra-, and hexa- saccharides. Similarly, in this work the inhibitor of periodate oxidation, a d-glucose- citrate mixture, was shown to inhibit slightly the agglutination of Rh0 cells in specific anti-Rh0 sera. Thus, it is feasible that antibody of carbohydrate specificity may actually be di­ rected against molecular units which arise in vivo from the liberation of degenerative tissue components.

The precipitations observed with mixtures of Rh antiserum and various carbohydrates such as pneumococcal polysaccharides may be due to antibodies to these substances acquired in the usual way. Reactions of low molecular weight carbohydrates such as alpha-D-galacturonic acid and glucurone, which are components of pneumococcal polysaccharides I and III respectively, have been observed with anti-Rh sera, and may also be due to cross-reactions of these substances with pneumococcal antibody. At the same time, consideration must be given to the possibility that antibodies to these two substances are blood group antibodies, even anti-

Rh, since sugars of these carbohydrates and their amines compose a portion of the structure of the major blood group antigens, and will react to some degree with their antibodies. These sub­ stances likewise may be related to the Rh antigens and to virus receptor. 231

Gottschalk (1957) found that the action of the various in­ fluenza viruses and NDV on a urine mucoprotein inhibitor resulted in an altered mucoprotein with liberated products composed of a carboxypyrrole structure and various hexose residues similar to the products liberated from a salivary viral inhibitor or from red cell-virus interaction. Glucose and galactose structures and their amines are consistently mentioned in the composition of various hemagglutinating virus inhibitors whether the inhibi­ tory substance be of animal or plant origin. A preponderance of mucopolysaccharide depolymers are liberated in the disintegration of ’’ground substance” or tissue matrix in advanced cachexic diseases, neoplasma, hematologic dyscrasias, protracted shock, and collagen diseases. Hexuronic acids, especially glucuronic acid, are contained in such mucopolysaccharides and are widely distributed in the animal body as in the chondroitin of cartilage, mucin, heparin, hyaluronic acid, and platelets. Glucuronic acid not only functions in certain detoxification processes but also is implicated in the inactivation-removal of insulin and steroid hormones. Glucosamine and galactosamine structures are found as integral parts of red blood cell receptors, brain and other tissues.

The recent observations of Kabat (1956) that the dimensions of complementary areas of some antibody combining sites include areas complementary to tri-, tetra-, and hexa-, saccharides further illustrates that carbohydrate specificity is finitely 232

registered in the immunizing process to even carbon chains of

three units. Thus, antibody of carbohydrate specificity may act­ ually be directed against molecular units which arise in vivo from the liberation of degenerative tissue components. Furthermore,

the serologic specificity of molecular units such as the hexuronic

acids would reside in the linkages of these compounds either in

the simple conjugated state or in biological materials of which

they are structural components. Such conjugates might possibly

include normal or abnormal tissue components.

Animals injected with glucuronic or galacturonic acid; with heparin which contains glucuronic acid structural units; and with hyaluronidase which releases glucuronic acid and acetylglucosamine from hyaluronic acid polymers - all gave evidence of altered

reactivity to the uronic acids, per se. This altered response was measurable by skin reactivity to the uronic acids; the presence of incomplete agglutinins for both normal and uronic

acid-treated rabbit erythrocytes in the sera of these animals; positive antiglobulin tests with erythrocytes from these same

animals; as well as abnormal sedimentation patterns of these

same cells indistinguishable from those of virus hemagglutinated red cells; and markedly altered electrophoretic patterns of the

serum proteins. Furthermore, the presence of complexes present

in the sera of these animals was exemplified by the anti-comple- mentary activity and complexes adsorbable from the sera to latex 233

particles. In addition, the latex detectable complexes were strongly evident in eluates prepared from erythrocytes of these animals prior to the time of their detection in the sera. The immunologic nature of the altered reactivity of these injected animals is upheld by the time interval required before modified reactivity could be detected. Also, the skin-sensitivity was observed to be transferrable with serum. Furthermore, the fact that symptoms of encephalomyelitis were manifested frequently in these animals as well as the findings of allergic type lesions in the brains of some of these uronic acid and hyaluronidase- injected animals indicates some form of auto-sensitization.

It is interesting to note that the histopathology of these animals was most marked in tissues or organs whose structural components were rich in hexose structures such as the red blood cell, brain, cardiac and skeletal muscle cells, lung, and kidney.

Also, worthy of mention is the fact that glucosamine and galacto- samine structures are not only primary units of erythrocyte antigens but are the very same materials which are components of the brain glycosides which are added to the brain even after birth. Thus, it appears that the manner of injection of these low molecular weight hexose units is the key to their irnnuno- genicity. Free in the circulation they are completely innoc­ uous j but when injected in an adjuvant into the muscle several events may be postulated. Firstly, the adjuvant base predicates 23U

a slower removal from the injection site, i.e. affords a time period for the uronic acid material to conjugate with body protein.

Secondly, the amount of material injected, as well as the injec­ tion site, insures a certain amount of tissue trauma so that these hexose units may also conjugate with degenerative tissue components. The hyaluronidase merely affords the opportunity for this type conjugation to occur with the body's own uronic acid, glucuronic acid, which is released upon hyaluronic acid depoly­ merization and hydrolysis by the enzyme. High concentrations of this enzyme were used to insure that an adequate quantity of glucuronic acid would be liberated within a short time interval.

Heparin, being biologically active and containing glucuronic acid, upon similar injections would be expected to conjugate in vivo as just described but the resulting antigen would differ in that it would be composed of not merely the glucuronate portion of heparin but, perhaps, the whole molecule in conjugation with degenerative tissue protein. Heparin possesses numerous sulfate groupings as it is a mixture of polysulfuric esters of mucoitin.

Mucoitin, in turn, is a complex polysaccharide composed of acetylated glucosamine and a hexuronic acid which formerly was thought to be galacturonic acid but is now tentatively identified as glucuronic acid. Thus, both the acetylated and sulfonated type linkages could readily be envisoned as those by which the heparin was bound to protein in the formation of postulated anti- 235

gen. It would then seem that antigens such as these should be properly designated as autoantigens.

With ,fautoantigensrt of this nature, all antibody produced should be bound to tissues with relatively little free in the circulation at any one time. That this may be the case is sub­ stantiated by the fact that direct serologic demonstrations, according to the classical techniques for antibody detection, were not obtained. Moreover, indirect serologic methods showed reactivity in the sera of these animals for not only altered red cells but for red cells from normal animals. Since this reac­ tivity was essentially absent from sera of the pre-injection bleedings, it is feasible to assume that autoimmunization had occurred. Furthermore, antigen-antibody eomplexes were removable from erythrocytes of animals injected with the uronic acids as shown both in adsorption to untreated latex particles and by the aggregation of normal rabbit red cells on which the charge had been altered by treatment with chromium chloride. In addition, the kidneys in all of these animals showed gross his­

tologic alterations. Glucuronates and glucuronides resulting from the normal processes of detoxification are eliminated through

the kidneys and comLd quite feasibly be held there by antibody directed against the hexose unit and thus elimination of toxic moieties would be impaired.

The fact that extremely basic cellular processes in these 236

animals, so injected, were deranged was evidenced by the rapid change in the electrophoretic patterns of the serum proteins which were different by one day after injection and markedly different by the fourth day. At this time (four days after injec­ tion) Aschoff-like lesions, typical of those classically described in the rheumatic processes, were found at the injection site in the skeletal muscle and in cardiac muscle. Furthermore, Aschoff- like processes were noted in the brain of an animal thirty days after injection with serum removed from an animal on the fourth day after injection with galacturonic acid.

Thus, it would seem that wherever these uronic-acid-protein complexes might lodge, after being removed from the injection site through the circulation, they effected a similar response in the tissue. The Aschoff-like lesion may be a particular tissue response to such complexes. Consequently, more host tissue is altered and the whole immunogenic picture is 'boosted11.

Thus, it is possible in some tissues for the release of antigen

(degenerated tissue protein linked with hexose structures from the cells or ground substance, or both) and the binding of anti­ body to intact cells further triggers degeneration is a moot point, but certainly the cell would not function as normally.

These preculations are substantiated by the histologic findings in which the heart was affected early in the "immunization1' and progressively degenerated into a "sieve-like structure" while the 237

muscle at the injection sites showed, later in the process, simul­ taneous repair and degeneration, and finally showed progressive degenerative alterations. The lungs and kidneys were affected more slowly but underwent a similar, progressive degeneration.

Just what event must occur for the breakdown of the brain is another nebulous and speculative point. But galactosamine func­ tions as a brain glycoside, there is no reason why the same pro­ cess could not function in the central nervous system.

Moreover, glucuronic acid has been observed to inhibit vac­ cinia virus, by combining with the viral particle. Since glu­ curonic acid is a constituent of hyaluronic acid and its amine is also found in the white matter of the central nervous system, a possible mechanism for the establishment of post-vaccinal en­ cephalomyelitis suggests itself. The time period between the vaccination experience and central nervous system difficulties corresponds generally with that between injection of uronic acids and central nervous system disturbances.

The immunologic ramifications of this theory are broad.

Furthermore, as observed in these uronic acid-injected animals a process similar to the rejection of tissue grafts occurs. At the injection sites in skeletal muscle, after primary degenerative changes have occurred, repair appears evident. Following this event, simultaneous degeneration and repair processes were evi­ dent. Finally, degeneration seemed to predominate accompanied 238

by occlusion and dissolution of the new blood vessels. This breakdown of blood vessels was observed also in the lungs and heart. The time interval between apparent repair in the skeletal muscle and degenerative dissolution of the blood vasculature corresponds to that time interval between the apparent take of a graft and its ultimate rejection. Furthermore, skin sensi­ tivity as well as marked histopathology were effectively trans­ ferred by serum injected intradermally.

Further indication of the presence of autoimmunogenic mat­ erial or complexes in the sera of these animals early in the pro­ cess of ,,autoimmunizationtt was borne out by the fact that pre­ cipitating antibody was demonstrable in the serum of rabbits nine days after injection of serum obtained from animals which, in turn, had received uronic acids four days previously. However, as previously postulated, as immunization progressed, no pre­

cipitates were detectable, for by then the antibody formed with

specificity for tissue structures (hexose, protein, etc.) would

have been absorbed by those tissues. If the time of these serum

immunizations had been prolonged, perhaps the postulated tissue

degeneration process would have been more marked. However, these

animals received but one milliliter of serum and the resulting

changes were adequate to uphold the theory.

In human3 in which rheumatic processes or collagen degrad­

ation is occurring, the process is essentially a chronic one. 239

It is seriously doubted whether many individuals would ever ex­ perience an infection or trauma of any sort which would release a proportionate amount of uronic acid-protein complexes as occurred in these rabbits. However, the parallels of the serum proteins of these animals with those of humans with lupus erythematosus and multiple myeloma are quite suggestive of a similar process occuring in humans. The only difference should be that in humans the diseased state is classified according to the organ or tissue affected and its ramifications are modified by the individual response. If time, it is appropriate to assume that these rabbits, injected as previously described with uronic acids, hyaluronidase, and heparin, did not have ua chance’* nor did the body as a whole, have much selectivity as to which system would be most extensively affected. The alpha-2 globulin increase noted by Baptista et al. (1956), the carbohydrate containing gamma and beta globulins of multiple myeloma (Mfteller-Bberhard and Kunkel, 1956), the skin sensitizing activity of non-pre­ cipitating antibody-antigen complexes present in alpha-2 globulins as recently described by Aladjem, MacLaren, and

Campbell (1957)* the increased alpha-globulin resulting from tissue destruction (Seibert, et al., 19U8), the depression of albumin due to liver dysfunction (Banerjee and Chatterjee, 1957) all are indicated here in the electrophoretic serum patterns of these uronic acid, hyaluronidase, and heparin-injected animals. 21*0

Robinson and Roseman (1957) in a recent report, in -which serum proteins, glycoproteins, and mucoproteins in rheumatic diseases were discussed observed

. . . The greatest degree of alteration in the elec­ trophoretic pattern is frequently seen in patients with the so-called collagen diseases, particularly in systemic lupus erythematosus where the alpha-globulin and gamma-globulin may comprise as much as 50 per cent of the total serum proteins. Changes to a lesser degree have been observed in periarteritis and in scleroderma. Although such changes are greater in degree than those observed in rheumatoid arthritis, there is no qualitative difference which serves to distinguish them from the changes seen in other forms of chronic inflammation.

Furthermore, these same authors noted that protein-bound hexose, hexosamine and sialic acid are consistently elevated in active rheumatoid arthritis while the protein-bound hexose and hexosamine were elevated in active rheumatic fever. In 17 of 19 cases of lupus erythematosus serum hexosamine levels were observed to be elevated. In addition, the increase in serum glycoproteins in rheumatoid arthritis was attributed to both increases in carbo­ hydrate-rich globulin fractions relative to the carbohydrate- poor albumin fraction and increases in the carbohydrate content of both alpha-1 and alpha-2 globulins.

Thus, it is interesting here to note that the same hexoses and sialic acid which Gottschalk (1957) recently described as part of the viral receptor material are found in the serum com­ plexes with the globulin components in collagen diseases and 2 k l

rheumatoid, processes. According to Gottachalk, the sialic acid or neuraminic acid of the viral receptor, both are acetylated, and readily degrade to 2-carboxypyrrole. The acetylation of these structures affords them a great facility with which to form complexes.

And thus, it seems that from two directions the same end is met; namely, that the eluates from virus-mofified cells and the uronic acids similar to hexose structures contained in such eluates both have the capacity to incite essentially an auto­ immune process. More specifically, the eluates from trypsin- treated rabbit red cells (Smith, 1953) and the in vivo modified cell which resulted from infections of chickens with NDV

(Gardner, Wallace, Dodd, and Wright, 195k) both induced auto- immunization as detectable by modified red cell antibody present in these animals. Similarly, the sera of these uronic acid-in­ jected animals showed altered reactivity with human red cells treated with either trypsin or NDV over the pre-injection sera.

However, no constant pattern could be established in these animals as the agglutinability of virus or enzyme treated human cells in theie sera varied considerably among the individuals tested.

Furthermore, the C-reactive protein, evident in chronic in­ flammatory states and in normal stresses such as extreme fatigue and pregnancy, and which reacts with antipneumococcal C substance, 21+2

could be but one expression of the formation of carbohydrate- containing serum factors. Apparently, C-reactive protein is the result, not the cause, of such processes, and would indicate by its very presence that the metabolism of some cells must have been altered for its production. Thus, the changes in the symptoms of a rheumatoid patient as a result of pregnancy may be further understood.

Also, in the establishment of rheumatic fever the time factor involved between the infectious process and the rheumatic symptoms is comparable to that in these uronic acid-injected animals be­

tween the first type of pathology described and that evident later by such fulminating degeneration. The group A, beta hemo­ lytic streptococci involved most frequently in the establishment

of rheumatic fever and glomerulonephritis, early in their de­ velopment possess a hyaluronic acid capsular material, which

later in growth stages is removed by hyaluronidase elaborated by the organisms. Thus there is no reason why in the infectious process, once established, that the hyaluronic acid capsules of

the young bacteria could not conjugate with body protein and

thus produce the antigenic material previously postulated.

Since a bacterial population would be far from static in such

a situation, the possibility also exists of the bacterial protein

conjugated with degraded hyaluronic acid due to streptococcal

hyaluronidase action. Thus, again could exist the protein-glu- 2U3

curonic acid conjugate. Both processes could be operative.

Bemfeld and Fishman (1953) showed that DNA possessed the ability to activate beta-glucuronidase and that the degree of activation of a given sample of the pujbified enzyme by DNA de­ pended on the enzyme concentration. Fishman had earlier noted that the relationship of beta-glucuronidase to neoplastic disease was indicated by the finding of markedly enhanced activity of this enzyme in the majority of human tumor tissues compared to normal tissues. Barakan, Barker, Gulland, and Parsons (19U8) reported that the two purine nucleotides, adenylic and guanylic acids, exerted an inhibitory effect in tumor growth in mice. Of the pyrimidine nucleotides, cytidylic acid exerted a negligible effect while the uridylic acid demonstrated a growth-promoting effect on sarcoma development. In considering these findings in view of these uronic acid-injected animals, it seems plausible that degenerative tissue components containing the uronic acid structure can in certain circumstances stimulate the production of antibody. Antibody of this hexose specificity could then compete with the beta-glucuronidase for the glucuronide structure which is abundant in tissues. This inhibition of the enzyme, beta-glucuronidase, would, in turn, affect the particular con­ centration of DNA whose function is to activate this enzyme. The excess DNA (perhaps in excess of the amount DNA-ase normally produced to degrade the DNA safely), could then function either as an intact molecule or through its degradation components, in an abnormal manner as evidenced in altered cell structure and function. This process, if true, might explain the mechanism whereby abnormalities in basic cellular processes such as altered globulin synthesis are initiated. SUMMARY

Antiserum to human Rho erythrocytes treated with mumps virus

contained an antibody which agglutinated the homologous red cells, mumps-treated Hr cells, periodate-treated Rh0 cells, and normal

Hr erythrocytes, but was an incomplete agglutinin for normal Rh0 red cells. Furthermore, the mumps alteration of normal Rh0 red c

cells was shown to be similar to, yet subtly distinct from, that

of the normal erythrocytes upon in vivo degradation of the cells

in immunized rabbits. These normal cell type agglutinins reacted most extensively with mumps-treated erythrocytes but were not

detectable in antisera to more pronounced red cell alterations, namely to influenza (FR8) virus or Newcastle disease virus (NDV)

treatments of red cells.

Mumps and periodate ion treatments of Rho(D) erythrocytes both removed anti-Rh0(D) inhibitory material from the cell surface

to the solution of the reacting mixture. The Rh0(D) specificity

of this inhibitory substance was indicated by inhibition of

agglutination of Rh0(D) cells in anti-Rho(D) sera and by the

formation of precipitates when mixed with the same Rh antisera.

The differences in the modes of liberation of the Rh0(D)-like

substance from erythrocytes by mumps virus and periodate ion are

discussed.

When the primary Rh0 specificity of anti-Rh0 serum was

2\6 removed by absorption with Rh0(D) cells, antibodies of other

specificities were demonstrable by agglutination of modified and normal cells.

Serologic examinations indicated that the red cells from

four hemolytic anemia patients were antigenically similar to mumps-

altered erythrocytes and that their sera possessed incomplete

agglutinins of greater reactivity for normal and treated Rh0 and

Hr red cells than did a serum from a normal individual.

Rabbits injected with uronic acids, hyaluronidase, and

heparin showed altered red cell reactivity, incomplete normal

and uronic acid-treated cell agglutinins, altered skin sensi­

tivity to the uronic acids, and markedly altered serum electro­

phoretic patterns. Symptoms of encephalomyelitis were fre­

quently evident in these animals and some of the brains showed

lesions characteristic of allergic encephalomyelitis. Histo­

logic examinations of the tissues of these animals indicate

hypersensitivity with fibrinoid changes, endothelial proli­

feration, and Aschoff-like nodules, some of which were similar

in certain respects to changes described as characteristic of

the collagen diseases. The immunologic response of the body

to immunization with simple hexose structures is further

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Weiner, W., Battey, D. A., Clgghorn, T. E., Mar son, F. G. W., and Meynell, M. J. 1953. Serologic Findings in a Case of Hemolytic Anemia. Brit. M. J. 2*125.

Wheeler, W. E., Duhby, Ai L., andScholl, M. L. 1950. The Action of Enzymes in Hemagglutinating Systems. II. Agglutinating Properties of Trypsin-Modified Red Cells with Anti-Rh Sera. J. Immunol. 30;291. 256

Wright, C-S, JDodd, M. C., Brandt, N. G., Elliot, S. M. and Bass, J. A. 1953. Erythrophagocytosis: Standardization of a Quantitative Tissue Culture Test and Its Application to Hemolytic, Malignant, and Infectious Idseases. J. Lab. and Clin. Med. Ul:l69.

Wright, C-S, Dodd, M. C., Bouroncle, B. A., Doah, C. A. and Zollinger, R. M. 1951. Studies in Hereditary Spherocytosis and in Acquired Hemolytic Anemia; Their Relationship to the Hypersplenic Mechan­ ism. J. Lab. and Clin. Med. 37:165. AUTOBIOGRAPHY

I, Nancy Jane Bigley, was born in Sewickley, Pennsylvania,

February 1, 1932. I received ray secondary school education in the public school of Tarentum, Pennsylvania. My undergraduate training was obtained at The Pennsylvania State College, from which I received the degree Bachelor of Science in 1953. I obtained the degree Master of Science in the winter of 1955 from The Ohio State University, during which time I served as a graduate assistant in the Department of Bacteriology.

Ullhile completing the requirements for the degree Doctor of

Philosophy I served successively as a graduate assistant and an assistant in the Department of Bacteriology and as a Research

Assistant in the Ohio State University Research Foundation.

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