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The activity of a large number of human erythrocyte antigens was monitored through a procedure which fractionates the erythrocyte membrane proteins. A significant amount of the erythrocyte "ghost" protein could be removed by aqueous extractants without affecting the activity of any antigen. Subsequent extraction of the residual "ghosts" with pyridine solution resulted in a separation of blood group M, N and Pr activities from blood group A, P1, P and I activities. A number of antigens including c, C, D, k, Kpb, Jka and Fya were labile to this treatment. Gel filtration in the presence of detergent showed that the MN and Pr activity was associated with the erythrocyte sialoglycoprotein. In agreement with Whittemore et al. (1969) two types of blood-group-A antigen could be distinguished on the basis of their partition properties on butanol extraction. Type OS-A antigen can be extracted into butanol, while type WS-A antigen is retained in the aqueous phase. The characteristics of blood-group-P antigen were similar to that of type OS-A antigen, while blood-group-I antigen and type WS-A antigen showed similar behaviour.

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH-value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6-phosphate or fructose 1,6-bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration. [ABSTRACT FROM AUTHOR]

The cross-reactivity of heterologous erythrocytes was measured at the level of the B and the T cell. B cells produced antibody with restricted cross-reactivity as compared with the cross-reactivity of T cells, assayed by their ability to help B cells in a primary anti-sheep erythrocyte immune response. Helper function was maximally elicited with low antigen doses. The data suggest that the T-cell antigen receptors for erythrocyte antigens differ in specificity from those of B cells and that the sensitivity of T cells to antigen may be very great. [ABSTRACT FROM AUTHOR]

Serial sera from nine volunteers, injected 10 years ago with sheep erythrocytes and showing heterophile antibody responses comparable to those seen in infectious mononucleosis, were retrieved from storage and examined for antibodies to Epstein-Barr virus (EBV) related antigens. Eight of the nine volunteers had preexisting antibodies to EBV, as determined by neutralization tests and by indirect immunofluorescence reactions with fluorescein-conjugated antibodies to human IgG, but not with anti-human IgM conjugates. The EBV-specific reactivities remained unchanged after immunization. The results indicate that: (a) EBV- or EBV antigen-containing lymphoblasts from carrier or freshly infected cultures contain no detectable heterophile antigen; (b) immunization with sheep erythrocytes does not evoke antibodies which interact with EBV or EBV-infected cells. These points are supported especially by the results obtained with the heterophile antibody-positive, anti-EBV-negative serum of one of the volunteers who subsequently sustained a primary EBV infection. It is unlikely, therefore, that immunization with heterophile antigen will induce immunity to infectious mononucleosis, apart from the fact that heterophile antibody responses are largely restricted to the IgM class. [ABSTRACT FROM AUTHOR]

Cells binding the antigen Keyhole limpet haemocyanin (KLH) were detected among human peripheral blood leucocytes and antigen-stimulated cultured lymphocytes. They were detected by their formation of rosettes with antigen-coated human O red blood cells. In the peripheral blood 0.13% of the lymphocytes of non-immune and 0.80% of the lymphocytes of immune subjects were antigen binding. In KLH-stimulated cultures of the lymphocytes of non-immune subjects there were 0.1% antigen-binding cells and in those of immune subjects there were 8.5% antigen-binding cells. Binding was specific. Thus, lymphocytes from phytohaemagglutinin- and streptolysin O-stimulated cultures did not bind KLH-coated red blood cells and did not form rosettes with KLH-stimulated lymphocytes. Incubation of KLH-stimulated lymphocytes with heterologous anti-immunoglobulin serum revealed that the KLH receptor was related to IgM. Addition of metabolic blocking agents (puromycin, cycloheximide, actinomycin-D and arabinosyl cytosine) to KLH-stimulated lymphocyte cultures revealed that the generation of antigen-binding cells required both DNA and protein synthesis. After primary immunization of normal human subjects, antigen-binding cells were detected in their appropriate lymphocyte cultures at 7 days and reached their maximum level at 21 days, in five of the six kinetically followed individuals and at 14 days in the other. This data represents the first report of the generation of antigen-binding cells in lymphocyte cultures demonstrated by the rosette method. These methods should be useful in the study of a variety of immunological problems in man. [ABSTRACT FROM AUTHOR]

The results suggest that the anaemia in mice associated with the injection of C. parvum is mediated by an immune mechanism. Survival studies of 51Cr-labelled red blood cells (RBC) in splenectomized and unsplenectomized mice show that mice immunized with C. parvum three times, clear RBC faster than unimmunized mice. However, hyperimmunized mice stabilized their haemoglobin concentrations above their minimum level and did not clear 51Cr-labelled RBC faster than unimmunized mice. All mice produced antibody against C. parvum but remained direct Coombs'-negative. Serum from immunized animals did not contain opsonins for -51Cr-labelled RBC. The reticulocytosis, normal osmotic fragility tests and normal survival times of 51Cr-labelled RBC from C. parvum-immunized mice all suggest that the main direct influence of the micro-organism is extrinsic of the RBC. Antigens from the micro-organism were adsorbed by erythrocytes in vitro. It is hypothesized that the anaemia is caused by destruction of RBC brought about by antibody reacting with bacteria that have been adsorbed by RBC. [ABSTRACT FROM AUTHOR]

A red cell autoantibody in low titer was demonstrated in one-third of Ugandan sera tested by performing the Combs test. on enzyme (bromelain) treated erythrocytes, The authoantibody was found Keith increased frequency in subjects over 40 years of age and in those with the haemocyte anaemia in pregnancy syndrome. But was not detected in medical students. Two subjects with autoantibody\ie's and normal haemoglobin values has na normal re dell survival The authoantibody was not associated with acute attacks of malaria. The possible relationships of the autoantibody to the he\haemolytic anaemia in pregnancy and the tropical spend meagerly syndromes are discussed. [ABSTRACT FROM AUTHOR]

Plaque assays for derepressed autoimmunocompetent B lymphoid cells with specificities for two murine erythrocyte surface autoantigens have been developed. Using purified anti-HB and anti-X anti-erytlirocyte autoantibodies, fluid-phase complement fixation reactions were performed to determine the influence of pH and ionic strength on the efficiency of autoantibody-mediated cytolysis of murine erythrocytes. Anti-HB autoantibody exhibited optimal cytolysis of bromelin-treated erythrocytes at pH 7.0, ionic strength 0.15; haemolysis of intact erythrocytes by anti-X autoantibody was observed over a broad range with maximum sensitivity and specificity at pH 6.6, ionic strength 0.14. NZB spleen ceils secreting anti-HB autoantibody were detected in modified direct haemolysis-in-gel assays, were neutralized by soluble HB autoantigen, and appeared to represent derepressed B lymphoid cells of anti-HB type. Anti-X plaque-forming cells, not demonstrable using conventional assays, were detected under conditions corresponding to the pH and ionic strength optima determined for complement-dependent cytolysis using purified autoantibody. NZB spleen cells secreting anti-X autoantibody were detected in indirect assays employing monolayers of erythrocytes, were neutralized by soluble X autoantigen, and appeared to represent derepressed B lymphoid cells of anti-X type. The distribution of observed plaque diameters in both HB and X assays suggested that more than 95% of all cells secreting autoantibody were detected. [ABSTRACT FROM AUTHOR]

The effect of procarbazine and cyclophosphamide on the formation of antibodies to sheep red blood cells in mice is investigated. Cyclophosphamide was found to have the best immunosuppressive effect when given after antigen; recovery from drug treatment was relatively fast; under experimental conditions that favour cyclophosphamide action (antigen prior to drug) IgM formation was found to be depressed and prolonged; IgG formation was markedly depressed or altogether missing. Procarbazine was found to act best when given prior to antigen; recovery was slow; under experimental conditions that favour procarbazine action (drug prior to antigen) the induction period was prolonged. IgM titres were slightly and IgG titres markedly depressed. Under all conditions tested procarbazine had little effect on the onset of IgG production. [ABSTRACT FROM AUTHOR]

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr1 groups of cold agglutinins. The findings with serum Sher indicated that the Pr1 antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen. [ABSTRACT FROM AUTHOR]

Antigenic determinants of Australia antigen (AuAg) may be determined by haemagglutination inhibition with ease and high sensitivity. The reaction depends on the inhibitory effect of the test antigen-positive sample on the agglutination by specific antibody of glutaraldehyde-fixed sheep erythrocytes which have been tanned and coated with AuAg of different specificities. The standard monospecific antibody reactants are directed against one or other of the common antigens, a and Re, or against the subtypic determinants, d,y,w and r. Because it is more sensitive than conventional immunodiffusion or electrosyneresis, and more convenient than radioimmunoassay, haemagglutination inhibition is most suited to large-scale determinations of AuAg subtypes in samples of relatively low antigenic activity. [ABSTRACT FROM AUTHOR]

Guinea-pig erythrocytes are cleared very rapidly from the circulating blood of 10-day-old rabbits; in contrast, sheep and mouse erythrocytes are cleared twelve to thirteen times more slowly, while rat erythrocytes are cleared six times more slowly, The differential rates of blood clearance of these xenogeneic erythrocytes largely substantiate differences between low titres of haemagglutinins or haemolysins, either naturally acquired or of maternal origin. Interference by such antibody in induction of the plaque-forming cell response and the minimum time antigen must persist in the circulation for successful induction of a haemolytic antibody-forming cell response are discussed. [ABSTRACT FROM AUTHOR]

Thirty-five weak A samples including fourteen A3, eight Ax, seven Aend, three Am and three Am were studied in order to determine their A antigen site density, using an IgG anti-A labelled with 125I. The values obtained ranged between 30,000 A antigen sites for A3 individuals, and 700 sites for the Ae1 red cells. The hierarchy of values observed made it possible to establish a quantitative relationship between the red cell agglutinability of these phenotypes measured under standard conditions, and their antigen site density. [ABSTRACT FROM AUTHOR]

A saline solution of bovine serum albumin (40 mg/kg) readily stimulated the production of precipitins and passive haemagglutinating (HA) antibodies in adult chickens and pheasants, but not in quail, following a primary (day 0) and secondary (day 38) intravenous injection. In quail a primary injection of BSA in Freund's complete adjuvant produced low passive HA titres but not precipitins; a further injection of soluble antigen intravenously on day 25 resulted in greatly enhanced passive HA titres and the appearance of precipitins. A single intravenous injection of sheep erythrocytes (109/kg) promptly evoked an agglutinin response in all three species, with the chicken yielding the highest and the quail the lowest titres. A second injection, 21 days later, stimulated titres higher than those achieved during the initial response only in the quail. [ABSTRACT FROM AUTHOR]

Sheep erythrocyte (SRBC) antigen was delivered directly into the lymphoid component of the appendix or via the appendicular artery, followed by ligation or clamping of appendicular vessels. Antigen was also injected intravenously, into the spleen, or into the thymus. Counts of anti-SRBC plaque-forming cells (PFC) in the appendix were compared with counts in the spleen and thymus. None of the injection routes employed resulted in an elevation of PFC activity above background in the appendix. However, delivery of antigen via the appendicular artery caused a marked increase of PFC activity in the spleen. These results and other available evidence point towards a helper-cell function of lymphocytes in the appendix, and therefore suggest a central role of this organ in humoral immunity. [ABSTRACT FROM AUTHOR]

The ability of rabbits to respond to relatively high doses of sheep erythrocytes appears within 5 days after birth Young rabbits exhibited an apparent sequential maturation of responsiveness to sheep, mouse, rat and guinea-pig erythrocyte antigens. The delay in response to these antigens can be correlated with different levels of specific haemagglutinin, presumably of maternal origin, in the circulation of young rabbits. [ABSTRACT FROM AUTHOR]

HgA-positive red cells were treated in vitro with approximately 10 μmoles N-ethylmaleimide (NEM) per ml cells for 1 hour at 37° and then injected into the circulation of HgA-negative rabbits; all the cells were cleared within 24 hours After 1-3 months the animals were given a series of injections of untreated HgA-positive red cells at 3-monthly intervals. Five out of seventeen rabbits showed progressively diminishing survival of HgA-positive red cells with the development of anti-HgA. On the other hand, in eight rabbits red cell survival following three consecutive injections of untreated red cells was normal or almost normal and in four further rabbits survival was initially subnormal but became progressively longer and was almost normal following a third injection of cells. Thus, in as many as twelve out of seventeen animals (70 per cent) the survival of HgA-positive red cells following a third injection of untreated cells was normal or almost normal whereas this pattern was previously observed in only seven out of fifty-one animals (14 per cent) not given an initial injection of NEM-treated cells. It is concluded that the injection of NEM-treated HgA-positive red cells to HgA-negative rabbits induces a degree of immunological tolerance to the HgA antigen. [ABSTRACT FROM AUTHOR]

Successive injections of HgA-positive red cells were given at intervals of 2-3 months to HgA-negative rabbits. In forty-four out of fifty-one animals, red cell survival became progressively shorter and, in the majority of instances, anti- HgA was found in the serum. Characteristically, the first survival curve was initially normal but showed 'collapse' after a mean interval of 17 days; following subsequent injections, cells were cleared at an accelerated rate. In the remaining seven HgA-negative rabbits the survival of HgA-positive red cells was either repeatedly normal or was subnormal after the first one or two injections and became almost normal following further injections. In four out of six of these animals, following additional injections of HgA-positive cells given in with FCA, anti-HgA became detectable in the serum, Thus, some animals which appeared to he non-responders to HgA as judged by normal survival of HgA-positive red cells after i. v. injection alone showed some response when antigen was also given in, with adjuvant. [ABSTRACT FROM AUTHOR]

Surface immunoglobulin can be demonstrated on almost all sheep erythrocyte rosette-forming cells in CBA and AKR mice, either by inhibition of antigen binding by anti-immunoglobulin or by immunofluorescence. When surface immunoglobulin on a rosette-forming cell is `capped' by anti-immunoglobulin, the bound red cells migrate with the immunoglobulin, demonstrating that the receptor-combining site and immunoglobulin antigenic determinants share the same mobile unit in the membrane. These observations strongly support the concept that the antigen receptor is surface immunoglobulin (at least for B cells), and illustrate the usefulness of the `capping' technique for studying the associations between membrane elements. [ABSTRACT FROM AUTHOR]

The progeny of mothers subjected to marginal lipotrope or moderately low protein diets exhibit deficient humoral immune responses. Serum antibody titres and numbers of antibody-forming cells to sheep erythrocytes (SRBC) are lower in the offspring of mothers whose diets were limited in either percentage protein or percentage lipotropes than in offspring whose mothers were fed a complete diet. Immune lymphocyte stimulation by SRBC antigens was marginal and not measurably different between the progeny of malnourished mothers and controls. The in vitro response of' spleen cells from offspring of nutritionally deprived mothers to the T-cell mitogen phytohaemagglutinin (PHA) is considerably less than that of controls. Thymus cells from marginal lipotrope offspring respond well to PHA, suggesting that PHA-reactive cell migration may be inhibited in these animals. These results, obtained using a T-dependent antigen (SRBC) and a T-dependent mitogen (PHA), support the conclusion that maternal dietary deficiency can adversely affect T-cell function in rat offspring. [ABSTRACT FROM AUTHOR]

The regulation of the immune responses by antibody administered passively on peritoneal exudate cells (PEG) was studied in guinea-pigs. The immunogenicity of sheep red blood cells (SRBC) associated with PEC for the induction of delayed hypersensitivity (DH) to soluble erythrocyte antigen was enhanced when PEG were incubated with anti-SRBG antibody. In contrast, the antibody response to SRBC was depressed. This phenomenon was only observed with specific antibody and was partially blocked when PEG covered with anti- SRBC antibody were incubated with rabbit anti-guinea-pig globulin serum. Comparable amounts of anti-SRBC antibody injected separately had no enhancing effect. Anti-SRBC sera from which the cytophilic antibody activity had been removed were inactive. PEG-associated SRBC were usually more immunogenic than the same amount of `free' SRBC for the induction of delayed hypersensitivity and always more immunogenic for antibody production. These observations suggested that macrophage cytophilic antibody might be involved in the regulation of the immune response and play a role in the preferential induction of delayed hypersensitivity in the studied system. [ABSTRACT FROM AUTHOR]

Antibody reacting against syngeneic erythrocytes (RBC) was elicited in several strains of mice by immunizing the mice with rat RBC. In nearly all mice sustained production of antibody reacting against syngeneic RBC required that the mice be continually stimulated with rat RBC. We suggest that this is further evidence that naturally-occurring immunological tolerance can be terminated temporarily by immunization with antigens cross-reacting with self. The results support the concept that clones of antibody-producing cells potentially reactive against self, exist in the normal mouse. [ABSTRACT FROM AUTHOR]

An acidic polysaccharide which consists of D­glucuronic acid. D­glucose, D­galactose and L­fucose in a molar ratio of 1:1:1:1 and which contains 5.2% of O-acetyl groups was isolated from Escherichia coli 08:K 27 (A):H- cells. About 80% of the total polysaccharide present on the bacterial cell could not be extracted with saline at 60°. This polysaccharide fraction was solubilized with 45% aqueous phenol at 65°. Purification of the acidic polysaccharide was achieved with cetavlon precipitation. It was shown to be homogeneous by ultracentrifugation analysis. The acidic polysaccharide has a high molecular weight (mean value 3 × 106), gives an aqueous solution with high viscosity ([η] = 5.0 dl/g) and possesses a very large form factor (ƒ/ƒo = 6.5). Treatment of the acidic polysaccharide with dilute alkali produced smaller molecules of a molecular weight of about 100,000. Alkali treatment did not alter the sugar composition of the acidic polysaccharide nor did it impair its serological properties. The acidic polysaccharide was oxidized by sodium periodate with concomitant destruction of the galactose moiety. Glucuronic acid, glucose and fucose in the polysaccharide resisted periodate oxidation. When the periodate oxidized and sodium borohydride reduced polysaccharide was hydrolyzed, 0.8 moles of glycerol per mole of the oxidized galact, ose was detected in the hydrolyzate. The methylated acidic polysaccharide was subjected to methanolysis and the methyl glycosides of the methylated sugar constituents were investigated with the aid of gas liquid chromatography. Methyl-tetra-O-methyl-D-galactoside was found to be present in the methanolyzate. Acid hydrolysis of the acidic polysaccharide gave rise to the following charged oligosaccharides: (i) glucuronosyl-(l:3)-fucose, (ii) glucosyl-(l:3)-glucuronosyl-(l:3)-fucose and (iii) galactosyl-(1:3)-glucosyl-(1:3)-glucuronosyl-(l:3)-fucose. No neutral oligosaccharides were detected in hydrolyzates of the acidic polysaccharide. It is concluded that the acidic polysaccharide of E. coli 08:K27 (A):H- consists of subunits of a molecular weight of about 100,000. These subunits are probably linked through ester bonds between carboxyl groups of glucuronic acid and hydroxyl groups of sugar constituents. It is suggested that the polysaccharide chains of the subunits consist, of glucose, glucuronic acid and fucose in repeating order to which galactose is bound in 1:3-linkage, with glucose as the branch point. The acidic polysaccharide absorbes directly onto erythrocytes. In passive haemagglutination tests the polysaccharide-sensitized red cells reacted strongly with E. coli 08:K27 anti OK and anti K sera but very weakly with anti O serum. After absorption of anti OK serum with the purified acidic polysaccharide the antiserum no longer agglutinated the encapsulated strain. The acidic polysaccharide was precipitated by 08:K27 antiserum and by K27 antiserum. This precipitation was inhibited by the tetrasaccharide (iii) and to a lesser extent by the trisaccharide (ii), but not by disaccharide (i) and glucuronic acid. After periodate oxidation—which destroyed the galactose moiety—the acidic polysaccharide was no longer reactive in serological tests. From these results it is concluded that the acidic polysaccharide isolated from E. coli 08:K27 (A):H- is the K antigen of this strain. Galactose appears to be the immunodominant sugar constituent of the K27 antigen, but the whole determinant group consists (at least) of the galactose-1:3-glucose disaccharide. [ABSTRACT FROM AUTHOR]

In contrast to MN receptors, Pr1 and Pr2 antigens of isolated glycoprotein of erythrocytes are not inactivated by acylation of free amino groups. By introduction of easily leaving groups, e.g. trifluoroacetic residues, inactivation of the MN activity can be made reversible. No differences between Pr1 and Pr2 antigens could be found with the reactions described in this paper. Acylation m aqueous phase (buffer/anhydrous acetic acid) and m organic phase (pyridine/ anhydrous acetic acid) gave identical results qualitatively and quantitatively. It was not possible to split the N-acetic binding neither under mild alkaline conditions nor with aminoacylase. 4-5 amino groups out of 11 could be acylated, whereas succinylation in aquous phase (buffer/ anhydrous succinic acid) resulted in modification of only one free amino group, the calculations being based on a molecular weight of 30 000. Following succinylation MN activity was also lost, whereas the antigenic activity of Pr1 and Pr2 remained unchanged. The appearance of a negative charge instead of the positive charge of the free amino group on the glycoprotein did not affect Pr1 or Pr2 activity m any way. If glycoprotein was esterified under conditions which cause esterification of free sialic acid to a wide extent neither MN activity nor Pr1/Pr2 activity was altered. [ABSTRACT FROM AUTHOR]

Anti-erythrocyte autoantibodies of two distinct antigenic specificities can be recovered from immunoglobulin-coated NZB erythrocytes. These autoantibodies react with either exposed (X) or hidden (HB) antigenic determinants on murine red cells and both types can be neutralized by mouse plasma. Antibody neutralization by plasma is dependent on two distinct plasma activities which appear to be soluble analogues of the erythrocyte surface autoantigens X and HB. The soluble erythrocyte antigens SEA-X and SEA-HB can be separated by molecular exclusion chromatography, with approximate sizes of 2 × 105 for SEA-X and ⩾ 5 × 106 for SEA-HB. The presence of these soluble erythrocyte autoantigens in the plasmas of 'autoimmune' NZB mice has to be considered in relation to the immunologic homeostasis of these autoantibody responses and pathogenesis of both the autoimmune haemolytic anaemia and the autologous immune complex disease of these mice. Individual anti-erythrocyte autoantibodies of both anti-X and anti-HB types exhibit discrete and individually distinctive differences in affinity for autoantigen and also in respect to antigenic determinant specificity. These results suggest that factors which control the genetic selection of these responses in an inbred strain do not prescribe a single specific B lymphoid cell clone; but rather it appears that a number of different autoimmunocompetent B lymphocyte clones with differing affinity and determinant specificity for given autoantigen molecules may be derepressed by other events in the recruitment of the autoantibody responses. [ABSTRACT FROM AUTHOR]

Naturally occurring titres of heterophil haemagglutinins against rat erythrocytes are related to ABO blood groups, being higher in people of blood groups O and A and lower in people of blood groups B and AB. Comparable titres of haemagglutinin activity can be demonstrated against human AB erythrocytes as well as rat erythrocytes. Activity against rat erythrocytes can be absorbed with human AB erythrocytes and vice versa. Furthermore, after absorption of heterophil antibody with human A or B erythrocytes, heat elution allows partial recovery of the antibody which reacts with rat erythrocytes. A rise in titre of heterophil antibody comparable to that occurring after renal transplantation was demonstrated after surgical operations but only where blood transfusions were given. It is postulated that these rises in antibody titre result from exposure to non-HL-A isoantigens which crossreact with human A and B substance and rat erythrocyte antigens. These antibodies can be distinguished from Forsmann and infectious mononucleosis antibodies, are absent in placental cord blood, occur naturally as IgM and respond to a secondary challenge as IgG, except in immunosuppressed renal transplant patients, where both IgG and IgM responses occur. [ABSTRACT FROM AUTHOR]

A significant increase in titre of human heterophil antibodies against rat erythrocytes was demonstrated in eighteen of twenty-eight patients after renal transplantation. Although this rise in titre preceded an acute rejection episode in the renal graft in some instances, it is considered that this relationship was coincidental. There is no relationship between the numbers of such rejection episodes or the degree of incompatibility for HL-A antigens between donor and recipient and the titres of heterophil antibodies after transplantation. [ABSTRACT FROM AUTHOR]

Homocytotropic antibody induced with ragweed and timothy pollen extract has been produced in non-atopic dogs. Characterization of the antibody was carried out by direct skin tests, passive transfer, tanned red cell haemagglutination, Sephadex filtration and immunoelectrophoresis. Two types of antibody were induced. A 7S antibody which had skin sensitizing characteristics and a 19S antibody which was the haemagglutinating antibody and 2ME sensitive. The findings are similar to that seen in spontaneous canine atopy except that the dogs did not respond with respiratory or systemic symptoms after antigenic challenge. [ABSTRACT FROM AUTHOR]

Blood group HgA negative rabbits were injected with 2 x 109 HgA positive erythrocytes from a donor rabbit. Assortative matings were set up between those rabbits which produced detectable anti-HgA (responders) and those which did not (non-responders). The progeny were injected with 2 x 109 HgA positive erythrocytes 3 months after birth and their anti-HgA response measured. It was found that 20/24 (83 per cent) of the offspring of responder parents, but only 4/26 (15 per cent) of the offspring of non-responder parents, produced detectable anti-HgA. No difference was found between the ability of responders and non- responders to produce haemagglutinins to various doses of sheep erythrocytes. In contrast, non-responder rabbits rarely responded to non-HgA blood group isoantigens on the donor erythrocytes whereas rabbits which responded to HgA after injection of the donor erythrocytes often responded to non-HgA isoantigens. Typing the cells of non-responder rabbits with the sera from responder rabbits containing non-HgA antibodies showed that the non-responder cells were antigenically more like the donor cells than were the cells from responder rabbits. The ability to respond to HgA was not associated with any particular heavy or light chain immunoglobulin allotype specificities. It is considered that HgA has an important helper function in regard to the antibody response to non-HgA antigens but the latter probably also have a similar but less marked effect in relation to the HgA antibody response. The genetic control observed is thought to operate through T cell recognition and reaction with HgA and other blood group determinants. [ABSTRACT FROM AUTHOR]

The injection of sheep red cells and capsular antigen of Pasteurella pestis leads to increase in the absolute and relative number of plasma cells in the draining lymph node and spleen. An equation is proposed which is compatible with the linear relationship which is observed between the logarithm of the antigen dose and the square root of the number of plasma cells. An index of homogeneity is derived from this equation which is related to the number of different antigenic determinants and different antigenic molecules in the immunogen. This index varied in the predicted way when mice were immunized with relatively pure and crude preparations of capsular antigen. [ABSTRACT FROM AUTHOR]

The quokka (Setonix brachyurus) a macropod marsupial endemic to Western Australia forms circulating antibodies to bacteriophage ΦX174, Salmonella adelaide flagella, sheep erythrocytes and bovine serum albumin. The kinetics of antibody production in the primary and secondary responses suggest that the immunological capacity of the quokka is between that of eutherians and the lower vertebrates. Three classes of antibody, macroglobulin, IgG and a low molecular weight antibody (80,000 daltons) were produced. However, the production of low molecular weight antibody was dependent upon the nature of the antigen. Evidence is presented to suggest that while the IgG and low molecular weight classes of antibody share common antigenic determinants on the heavy chains, they are independent entities. A low molecular weight degradation product of IgG accumulated in immune sera but the low molecular weight antibody is neither a degradation nor an anabolic byproduct of IgG immunoglobulin. [ABSTRACT FROM AUTHOR]

Peritoneal exudate lymphocytes (PEL) of guinea-pigs injected with Freund's complete adjuvant (FCA) were capable of liberating skin reactive factor (SRF) and macrophage migration-inhibitory factor (MIF) when cultured for 24 hours in the presence of sheep red cells (SRC) preincubated with lymph node cells (LNC) of guinea-pigs immunized with SRC in FCA. Similar release of soluble mediators was found in the following situations: (a) incubation of non-immune PEL with SRC sensitized with the exclusion peak of Sephadex G-200 fractionated immune LNC supernatant; (b) incubation of non-immune LNC with complexes of SRC and anti-SRC IgG, prepared from serum of animals immunized with SRC in CFA, and (c) incubation of non-immune LNC with a mixture of SRc urea extract and anti-SRC IgG. SRC incubated for 24 hours with immune LNC and freed of lymphocytes by selective filtration through a Millipore filter, were able to provoke an inflammatory reaction in the skin of normal guinea-pigs, which was delayed in character. These findings are interpreted as suggestive evidence for the non-identity of the mediator-producing and antigen-reactive cells and as support for the hypothesis that a complex composed of antigen and an antibody-like material, secreted by stimulated antigen-reactive lymphocutes, is responsible for the release of soluble mediators in cell-mediated immune reactions. [ABSTRACT FROM AUTHOR]

The onset of ability to produce haemolytic antibody to sheep erythrocytes does not appear in hamsters until 11 days after birth. However, when mouse erythrocytes were used as antigen an earlier onset of immunocompetence at 6 days after birth was detected. The hamster can make haemolytic antibody only 9.5 days after the appearance of lymphocytes in the thymus—this is sooner than any other species. This capacity is compared with other species on the basis of physiological development by age-equivalence. [ABSTRACT FROM AUTHOR]

Guinea-pigs immunized with sheep red cells (SRC) in Freund's complete adjuvant (FCA) developed delayed type hypesensitivity to a SRC urea extract 7 days after sensitization and showed combined Arthus and delayed reactivily from the 14th day on. Animals immunized by an intracardiac injection of SRC in saline and skin tested 4-5 days later with SRC urea extract, responded with an erythematous and indurated lesion reaching its peak 24 hr after testing and resembling a typical delayed hypersensitivity reaction. Lymphocytes from lymph nodes, peritoneal exudate and peripheral blood of guinea-pigs immunized with SRC in FCA released skin reactive factor (SRF) and macrophage immunized inhibitory factor (MIF) when cultured for 24 hr with SRC. No SRF was formed in the absence of divalent cations in the cultured medium. No SRF or MIF was produced by lymphocytes of guinea-pigs immunized by thc intracardiac route and cultured under identical conditions SRF obtained after in vitro exposure of sensitized lymphocytes to SRC was elutd from Sepbadex G-200 in the region of serum albumin. Lyric anti-SRC antibody of the IgM class was liberated into the supernatant of spleen cultures after intracardiac immunization and antibody of the IgG class was produced by lymph node cultures after immunization with SRC in FCA, in both cases in the absence of antigenic stimulation in vitro. It is considered improbable that the latter antibody is involved in the release of soluble mediators. [ABSTRACT FROM AUTHOR]

A graft-versus-host reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later cultures for primary immunization in vitro against sheep red cells were initiated with the spleen cells of the recipients. It was found that the antibody response against sheep red cells was considerably inhibited in spleen cells from mice subjected to a graft- versus-host reaction as compared to the response of spleen cells from untreated mice. Furthermore, when spleen cells from animals subjected to a graft-versus-host reaction were cultured together with spleen cells from normal animals or animals preimmunized to sheep red cells, the antibody responses of the latter two cell populations were inhibited. The relation between these findings and the antigenic competition phenomenon is discussed and it is suggested that antigenic competition is caused by an non- specific inhibition of the antibody response, caused by secretion of locally acting factors or by inhibition of normal cells by direct contact with ‘active’ cells, stimulated by antigen. [ABSTRACT FROM AUTHOR]

Sephadex will adsorb protein non-covalently and this can be detected by mixed agglutination reactions with antibody and antigen coated red cells. Rheumatoid factor will adsorb to Sephadex and can be demonstrated by immunoglobulin coated red cells. BioGel particles show considerably less nonspecific adsorption of protein and this makes them more suitable for the demonstration of antibody on antigen coated particles by means of antiglobulin reagents. Using a two step antiglobulin reaction it was possible to detect IgG, IgM and IgD anti-insulin antibody. [ABSTRACT FROM AUTHOR]

Tissue culture techniques were combined with cell separation procedures, antimacrophage serum and soluble and particulate forms of sheep red cell antigen to investigate the cellular requirements for a secondary antibody response. By using the highly efficient active adherence column separation method of Shortman which almost completely removes phagocytic cells, it was found that the secondary response to SRC was macrophage dependent. This conclusion was verified by the use of specific antimacrophage scream and by a combination of both methods. Critical tests were used to verify that these separation methods acted on phagocytic and not on other cells, for example, thymus or bone marrow derived lymphocytes. In contrast, the secondary immune response to POL and solubilized SRC antigen was not dependent on the presence of phagocytic cells, as highly purified lymphocytes responded normally to these antigens. Antimacrophage serum did not depress the immune response to these soluble antigens. These results indicate that the requirement for macrophage activity depends on the physical size of the antigen. These findings, obtained in the secondary response in vitro, were closely analogous to previous findings from this laboratory on the cellular basis of the primary response to SRC and POL. The reasons for the different cellular bases of the secondary immune response to various molecular forms of antigen are discussed. The similar cellular basis of the primary and secondary response in vitro suggests that the basic mechanisms of immunization in these responses are the same. [ABSTRACT FROM AUTHOR]

The method of preparation of partially purified [125I]IgM anti-D and of subunits of 180,000, 90,000 and 40,000 molecular weight with anti-D activity is described. It was found that approximately 11,500 whole IgM anti-D molecules would bind to each red cell of phenotype R1R2, On the other hand, approximately 31,000 molecules of both whole IgG anti-D and an IgM anti-D subunit of molecular weight 40,000 would bind to the same cell. The following possibilities are proposed to explain these findings: (1) IgM anti-D molecules bound to the red cell sterically hinder binding of other IgM antibody molecules to closely adjacent D-antigen sites and (2) some D-antigen sites are too far below the surface of the red cell to permit binding by the large IgM molecule. The average values of the equilibrium constants (K0) of two different examples of IgM anti-D were 1.7 × 109 and 2.5 × 109 litres/mole, and of the IgG antibodies obtained from the same plasmas were 9- and 32-fold lower respectively. The K0 of the IgM subunits was on average 23-fold lower than that of the whole IgM and it is concluded that IgM anti-D binds bivalently to each red cell but that the binding energy of one of the sites is relatively small. [ABSTRACT FROM AUTHOR]

Immunosuppression was observed with dehydroheliotridine (DHH), a recently isolated and identified metabolite of certain pyrrolizidine alkaloids. The primary response to sheep red blood cells (SRBC) and to influenza virus vaccine (IVV) was significantly depressed. The secondary response to SRBC was suppressed only if the DHH was given at the time of the secondary stimulus. DHH is active when given before or with the antigen but not when given 6 or 24 hours after the antigen. There was some evidence that the withdrawal of blood for haernagglutination inhibition tests derepressed the immunological response of DHH treated mice. [ABSTRACT FROM AUTHOR]

The concept that antigen has a continuous role in the recruitment and differentiation of immune progenitor cells was tested with optimum and suboptimum doses of heterologous erythrocytes in mice. These studies further evaluated an immune cell maturation scheme in which continuous antigenic stimulation is required for both the recruitment of ‘antigen-sensitive units’ and the expansive proliferation of a distinct sensitized cell compartment, which undergoes irreversible differentiation to functional antibody-forming cells. Haemolytic plaque-forming cell capacity during both the primary and secondary immune reactions were studied, both in the intact animal and with the spleen cell transfer technique. This in vivo culture technique was used to measure the sensitized cell compartment in the absence of existing antibody regulatory mechanisms. The results clearly demonstrate a higher detectable secondary immune capacity in the suboptimum antigen dose group than in the optimum antigen dose group. This was demonstrated for both the 19S and 7S cellular responses, as well as with humoral antibody levels measured in the spleen cell recipient mice. It can be concluded that in the presence of a suboptimum dose of antigen, which rapidly diminishes during the early intervals of the primary response, there is adequate recruitment with subsequent preservation or rescue from antigen-mediated depletion of the sensitized cell compartment, at the expense of the detectable primary response. [ABSTRACT FROM AUTHOR]

The effect of a single subcutaneous dose of hydrocortisone (730 mg/kg ∼ 21-day LD50) on the haemolysin response of mice to sheep erythrocyte antigen was examined. Hydrocortisone was administered once at times varying from 5 days before immunization with sheep erythrocytes to 9 days after antigen. Total suppression of the 7S haemolysin titre was brought about by treatment with single dose of 750 mg/kg in the period 5 days prior to antigen until 2 days after antigen; at the same time, the titre of 19S haemolysin exceeded the control 19S titre. Microplaque assay on the 5th day failed to confirm total suppression of 7S antibody synthesis, as 4 per cent of the splenic plaque-forming cells produced 7S. The same assay failed to verify the augmentation of 19S production on a cellular level, as the number of 19S plaque-forming was significantly decreased. Hydrocortisone could be shown to influence the _morphology of the 19S antibody producing cells by increasing the percentage of mature cell types. A selective depressing activity by hydrocortisone on 7S memory was found. The theoretical implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Lepisosteus osseus possesses high titres of natural agglutinins to human group A (HuA) erythrocytes and to Salmonella typhosa 'O' (STO) antigens. Moderate natural haemagglutinin titres to sheep red blood cells (SRBC) were noted. Cold agglutinin (4°) titres exceeded agglutinin titres recorded at 25°. Immunization with STO antigen resulted in a marked increase in corresponding anti-STO agglutinins, as well as increases in anti-HuA and anti-SRBC agglutinins. Animals immunized with SRBC antigen produced elevated anti-SRBC agglutinin titres, while immunization with HuA erythrocytes produced no changes in agglutinin levels. Immunization with STO following previous injections with HuA resulted in increases of corresponding anti-STO agglutinins, as well as marked increases in anti-HuA agglutinins. Absorption studies indicated that anti-STO agglutinins were not cross reactive against HuA antigens. Natural and immune antibodies were all of the same macromolecular class. Rises in antibody titres were reflected in both cold and warm agglutinins. Animals maintained for long periods at low temperatures exhibited preferential ability for increased synthesis of cold agglutinins to HuA. Natural haemolytic titres, as measured by an SRBC indicator system, were also elevated as compared to mammalian systems. These titres increased with stimulation with SRBC. Results suggest that the high levels of natural haemolytic and agglutinin activity in this species are of immunogenic origin. [ABSTRACT FROM AUTHOR]

Strains of Escherichia coli with sufficient K antigen to resist killing by complement were poorly phagocytosed when injected intravenously into mice. Phagocytosis was markedly increased by anti-OK but not by anti-O sera. In contrast anti-K sera had little or no effect on the bactericidal reaction. This was not because K antigenic sites were scarce but may have been because their position was such that complement was activated at a distance from its substrate. Red cells coated with K antigen were poorly lysed by complement and anti-K serum, suggesting that the K antibody did not activate complement very effectively al- though again the sites may have been too superficial. The effect of K antigens on phagocytosis and complement killing or lysis could all be explained by their ability to impair protein binding. Strains of E. coli rich in K antigen were resistant to phagocytosis and complement killing and were virulent for mice on intracerebral injection. The significance of K antigens in animal and human infections is discussed. [ABSTRACT FROM AUTHOR]

Initiation of an immune response to sheep erythrocytes by the transplantation of spleen cells into irradiated recipients is thought to involve an interaction between two functionally different lymphoid cells. If such populations actually exist, it should be possible to separate them from each other and to study the properties of each type of cell. To this end, mouse spleen cells have been fractionated using equilibrium centrifugation in density gradients of Ficoll. Two functionally different populations of spleen cells were obtained. Neither population was active in initiating an immune response by itself on transplantation into irradiated mice, but mixtures of the two populations were. One population of spleen cells was also active when transplanted together with normal mouse bone marrow and the other population was active when mixed with thymus cells from normal mice. These results indicate that two different spleen cells must interact to initiate an immune response in vivo. The specificity of the cell that synergizes with thymus ceils was investigated further and shown to have antigen-specific receptors on its surface. These cells can be recognized by their ability to make rosettes with erythrocyte antigens. Thus, the ‘background’ rosette-forming cells found in unimmunized mice are actually progenitors of antibody-producing cells. [ABSTRACT FROM AUTHOR]

(1) Immunological reactivity to sheep red blood cells was tested in three congenic resistant strains of mice and was found to vary according to the H-2 locus and its alleles. The reactivity of the strains in decreasing order is: A. CA, A/Sn, A. BY. (2) Non-specific immunostimulation with Brucella increases the immune response to sheep red blood cells in the A. CA and A/Sn strains but not in strain A. BY. This immunostimulation with Brucella is H-2 dependent. (3) Methylcholanthrene has an immunodepressive effect on the immunological reactivity to sheep red blood cell antigens in the three strains of mice, each strain being equally affected. [ABSTRACT FROM AUTHOR]

The increase in splenic plaque-forming cell (PFC) responsiveness which occurs in mice following the injection of a primary dose of 108 sheep red blood cells was measured using the spleen cell transfer system. As for grafts of normal spleen cells, the peak direct (19S) and indirect (7S) responses obtained with grafts of primed spleen cells were found to be related to the number of cells transferred in a near-parabolic fashion. Splenic direct PFC responsiveness as measured by the regression defining the corresponding graft-response relationship was increased about five times 4 weeks after priming; focus-forming capacity was also increased by a similar factor. At the same time indirect PFC responsiveness was increased almost twenty times. These results are discussed in terms of antigen-induced changes in the populations of PFC precursors and antigen-reactive auxiliary cells in the spleen. [ABSTRACT FROM AUTHOR]

Normal serum samples from the Australian freshwater catfish, Tandanus tandanus (Mitchell), were shown to have haemolytic and haemagglutinating activity for a variety of animal erythrocytes. Specific agglutinins were present for the erythrocytes of most animals tested but some cross-reactivity of agglutinins was demonstrated. Evidence was obtained that the greater part of the human erythrocyte agglutinating activity was directed toward antigens of the ABH system. Whereas some sera clearly showed anti-H or anti-A and B agglutination patterns, others agglutinated equally erythrocytes of different groups. ABO erythrocytes were agglutinated to higher titres than were erythrocytes of the 'Bombay' phenotype, salivas from secretors inhibited the agglutination of erythrocytes carrying appropriate ABH antigens, whereas salivas from non-secretors did not and absorptions of sera often revealed ABH specific agglutinins. While some sera agglutinated equally ABO erythrocytes because they contained a mixture of agglutinins with different ABH specificities, other serum samples containing one predominating species of agglutinin caused equal agglutination of O, A, AB and B erythrocytes. The specificity of this latter type of agglutinin is discussed. [ABSTRACT FROM AUTHOR]

Solubilized sheep erythrocyte stroma was found to be antigenic in rats. Spleen and lymph nodes of rats injected with this antigen contained more 7S than 19S plaque-forming cells throughout the primary and secondary responses. When compared to the primary response, secondary immunization with this antigen elicited increased numbers of both 19S and 7S plaque-forming cells. Antibody synthesizing leucocytes in the blood during the primary and secondary responses were predominantly 7S producers during the first few days. Later 19S producers predominated. Intact sheep erythrocytes elicited the same pattern of 19S and 7S antibodyforming cell development in the lymph nodes and blood of intravenously injected rats, but during the early primary response in the spleen there was a predominance of 19S over 7S plaque-forming cells. The 7S cells were in a majority during the entire secondary response of the spleen to intact erythrocytes. The secondary response of spleen and lymph node to intact erythrocytes showed an elevated 7S plaque forming cell response but the number of 19S cells was similar to that detecrred after primary immunization. The appearance of hemolytic and haemagglutinating 19S and 7S antibody to sheep erythrocytes or solubilized stroma generally reflected the cellular picture of the spleen. By using an anti-7S globulin it was found that 19S and 7S antibody appeared simultaneously in the serum. After immunization of rats with intact erythrocytes or solubilized stroma the number of lymphoid cells that took up tritiated thymidine was about one hundred-fold greater than the number of antibody-forming cells as determined by localized haemolysis in gel. The number of lymphoid cells positive in an immunocyto-adherence assay was more closely related to the number of cells taking up tritiated thymidine. The passive transfer of spleen cells from rats immunized to sheep erythrocytes showed the number of circulating antibody-forming cells in the normal and irradiated recipients to be related to the concentration of antibody-forming cells localizing in the recipient spleen. The number of antibody-forming cells in the peripheral blood was greater in splenectomized recipients. Irradiation had no effect on the number of antibody-forming leucocytes in the circulation of the splenectomized recipients. [ABSTRACT FROM AUTHOR]

Adult mouse spleen cells were transferred into irradiated adult mice with xenogeneic erythrocytes. Antibody-producing cells were measured by the localized haemolysis in gel (LHG) assay. Anti-allotype serum directed against the γG2a allotype of the donor cells, given to the recipients at the same time as antigen and spleen cells, suppressed the plaque-forming cells (PFC) producing antibody of that allotype, and had little effect on the other classes investigated. [ABSTRACT FROM AUTHOR]

Quantification of the parameters in an antigen-antibody fluorescent reaction in which it was possible to vary the amount of the antigen, was carried out in a model system. Formalinized erythrocytes were coated with different amounts of 131I-labelled human IgM. The cells were then exposed to fluorescein-conjugated antiserum to human IgM. The fluorescence intensity of individual cells was measured with a microspectrofluorimeter. As the cells were coated with varying amounts of protein it was possible to investigate the relationship between the quantity of protein antigen and the intensity of fluorescence. [ABSTRACT FROM AUTHOR]