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Grass pollen allergens are shown to remain associated with protein material and a yellow pigment during paper chromatography and during dialyses and ultrafiltrations of various types. Dialysable* allergens comprise only a fraction of I per cent of the total activity and the amount of activity extractable by diethylene glycol (DEG) and similar solvents is of the same order. Besides the allergens, the DEG and aqueous extracts contain large amounts of inositol, glucose and fructose, also some yellow pigments and phosphates. Larger amounts of free and combined amino acids are found in the aqueous than in the DEG extracts, but the reverse is true for sucrose. In addition the DEG extracts contain a yellow glucoside different from the dactylen of the aqueous extracts, a glucosan and an arabinose-galactose-pigment complex, only the latter being associated with any activity. The spontaneous release of the crystalline dactylen from originally clear aqueous pollen extracts is found not to be caused by enzymes. The washed crystals are found to be chromatographically and electrophoretically homogeneous and devoid of allergenic activity. [ABSTRACT FROM AUTHOR]

In the preceding paper a population of lymphoid cells was identified which (1) were derived from germinal centres in the appendix, (2) were localized in follicular structures elsewhere, and (3) could perform as antibody-forming cell precursors. The present paper presents evidence (1) that germinal centres in the spleen and lymph nodes perform the same function as germinal centres in the appendix, and (2) that germinal centres are dependent upon a stream of cells derived from the bone marrow. A new hypothesis is put forward regarding the origin and cellular kinetics of the B-cell system in mammals. It is proposed that germinal centres throughout the body function as an essentially antigen-dependent amplification system for the B-cell population of lymphocytes. Implications of this hypothesis are discussed. [ABSTRACT FROM AUTHOR]

Heat and pH stability studies and experiments with organic solvents show that the A-antigens discussed in the preceding paper (Augustin, 1959c) are much more labile than the I-(‘inner ring’) antigens. Breakdown products and/or aggregates are produced which no longer precipitate with antisera to the original extracts, but act as inhibitors. Solutions of pollen allergens, on the other hand, are found to withstand even autoclaving for 15 min. at 20 atm. and vigorous boiling over the naked flame of a bunsen burner. None of the carbohydrates tested has a demonstrable effect on skin reactivity which is, however, destroyed by crystalline pepsin, crystalline trypsin, a crystalline mould protease and a tissue protease (a partially purified extract from rabbit spleen). It follows that the bulk of the allergens—if not all—are proteins. The relation of skin reactivity, immuno-electrophoretic patterns, carbohydrate and protein reactions to the selective destruction of the pollen antigens is investigated. Pollen components prove to have a somewhat wider range of electrophoretic mobilities than serum proteins and are probably as complicated a mixture. The most and least highly negatively charged components are without skin reactivity in allergic subjects. The skin reactive allergens appear to have the mobilities of α- and β-globulins. Not all the hay fever subjects react equally to all the components, and Cocksfoot and Timothy activity patterns vary in different subjects. [ABSTRACT FROM AUTHOR]

A mean of 6.9 per cent of small lymphocytes in peripheral blood preparations and between 1.8 and 2.4 per cent of small lymphocytes in lymph node, spleen, bone marrow and thymus preparations from unimmunized guinea-pigs bound 125I-labelled purified protein derivative of mammalian tuberculin (mammalian PPD). The percentage of these cells fluctuated but did not alter substantially after immunization with BCG or with BCG emulsified with human thyroglobulin (HTg) in Freund's incomplete adjuvant (FIA). Blocking experiments indicated that the binding of 125I-labelled mammalian PPD was specific and there was tentative evidence that the lymphocyte receptors may be IgG. A comparison is drawn between the observed time course of 125I-labelled mammalian PPD- binding small lymphocytes and the response of lymphocytes sensitive to strong histocompatibility antigens, and it is proposed that the propensity of certain anti- gens to induce a delayed hypersensitivity-type response is related to the presence of substantial numbers of antigen-binding cells in unimmunized animals. A noteworthy incidental finding was an unexplained depression in the cellular and humoral responses to mammalian PPD in guinea-pigs that had been immunized with HTg-BCG-FIA emulsion. [ABSTRACT FROM AUTHOR]

The experiments described in this paper extend the observations of previous work in vitro demonstrating a decrease in the frequency of antigenreactive units specific for horse RBC in spleen cells from mice primed with KLH. It was observed that the addition of lymphoid cells having T-cell function reconstituted the anti-HRBC response to normal values. Significant enhancement of the response beyond the normal values could be evoked using two dissimilar methods, (i) addition of allogeneic thymus cells, and (ii) restimulation in vitro with the priming antigen. The latter type enhancement was elicited by the addition of small doses of KLH to cultures of spleen cells from mice recently primed with KLH, including cultures otherwise demonstrating antigen-induced suppression. The stimulus required to enhance the response to HRBC in these cultures was specific for the priming antigen, KLH. These results are discussed in the light of current theories of 'antigenic competition' and specific heterologous enhancement. [ABSTRACT FROM AUTHOR]

Whilst the ability of isoantibodies to agglutinate mouse red cells in saline medium does depend in part upon the properties of the antibody molecules, the concentration of antigen upon the red cell and certain genetically determined properties of the cell surface are of even greater importance. The red cells of mice of any strain will give positive results in the human serum:dextran system whilst reactions in a saline medium are unusual. In this paper the term ‘incomplete antibody’ is used to denote antibodies that can only be detected in conjunction with other antibodies in the human serum:dextran system. The time of appearance of antibodies to homografts of an ascites sarcoma, an ascites leukosis and a solid mammary carcinoma has been studied. Incomplete antibody as defined above has been detected by two methods. In the blocking test incomplete antibody is allowed to react with red cells before contact with complete antibody, a positive result being observed as a fall in titre when the treated red cells are subsequently exposed to complete antibody. In the second type of test, the synergic test, incomplete antibody is mixed with suitably diluted complete antibody and fitrated against suitable red cells, a positive result being a significant rise in titre as compared with complete antibody mixed with normal serum. Antibodies produced by the antigen donors did not give significant blocking or synergic effects. Incomplete antibodies are sometimes detectible on the third day and with regularity on the fourth day. Antibodies ‘complete’ for A strain red cells may appear at any time from the fifth day onwards. There is sometimes a drop in fitre of varying duration on the sixth day. This corresponds with the commencement of marked inflammatory changes on the graft bed. The maximum titre is attained at about two weeks. Antibodies may disappear in under three months or persist as long as a year. The function of antibodies in homograft reactions varies with the target tissue. However, they appear before the onset of anatomical signs of homograff response regardless of the type of target cell. [ABSTRACT FROM AUTHOR]

The purpose of this study was to demonstrate the presence of exoantigens in the intestinal washes of mice infected with H. nana, to partially purify the antigen, and to show identity with heterologous antisera to H. nana soluble proteins. This paper describes the increase in specific activity to tapeworm exoantigens with increased purification both with serum obtained from infected mice and heterologous rabbit serum. [ABSTRACT FROM AUTHOR]

This paper describes the ultrastructural location of labelled antigens and carbon in the spleens of rats from 4 minutes to 5 days after injection. Particular attention was focused on the sites of deposition 4 minutes after intra-arterial injection of microgram quantities of 125I-labelled Salmonella flagellar antigens, crayfish haemocyanin and BSA, using colloidal carbon for comparison. The combination of radioautography with both light and electron microscopy showed the importance of antigen binding by lymphocytes in the marginal zone of the spleen. Macrophage sequestration of antigens was not prominent in the spleen, although it occurred in the liver with the flagellar antigens and haemocyanin. In the spleen marginal zone, avid antigen-binding cells were found in situ 4 minutes after the injection of labelled haemocyanin. These appear to be the counterpart in vivo of antigen-binding lymphocytes prepared in vitro. Such cells also occurred infrequently after the injection of labelled polymerized flagellin, but were not found with either BSA or carbon. The apparent movement of flagellar antigen from the marginal zone to the white pulp between 1 and 2 hours after injection was seen to involve lymphocyte-associated antigen. The follicular antigen localization occurring from 1 day onwards after injection was on the dendritic reticular cells of germinal centres, as has been described in lymph nodes after subcutaneous injection. Carbon particles were rapidly sequestered in macrophages of the spleen and liver, although some particles were found between cells in the marginal zone for as long as 2 hours after injection. By 2 and 5 days, however, all the carbon was in phagocytes, even in the white pulp. Differences between the localization of antigens and carbon were clear, even in the ultrastructural sites of their location in tingible body macrophages of germinal centres. The unexpected emphasis of lymphocyte association with labelled antigens in the spleen marginal zone has allowed a revision of the mechanism previously proposed for the movement of antigens within the microenvironments of the spleen. [ABSTRACT FROM AUTHOR]

This paper describes a new method for the morphological study of individual antibody-forming cells (AFC) on cell smears of the quality of normal haematological preparations. The early AFC response to polymerized flagellin of S. adelaide was studied in vivo using C57BL mice, which have very low background levels of AFC and in vitro using dispersed spleen cell cultures from CBA mice. AFC, arising as a result of in vivo or in vitro stimulation were found to comprise a heterogeneous population, including basophilic mononuclear cells, lymphocytes of most sizes, immature blast cells and occasional plasma cells. The earliest AFC detected comprised a high percentage (28 per cent in vivo, 31 per cent in vitro) of small lymphocyte-like cells. Studies of the incorporation of [³H]thymidine showed that most AFC arose by proliferation but that a proportion of AFC, the small lymphocyte-like cells, arose by differentiation of precursor cells not involving cell division. The effects of antigen concentration on the kinetics of AFC were investigated in vitro. Subtolerogenic antigen doses caused a delayed and decreased AFC response. [ABSTRACT FROM AUTHOR]

Data presented in this paper indicate that by column chromatography using an O acetylated galactan, a considerable degree of purification of specific antibody to O antigen 5 may be achieved. [ABSTRACT FROM AUTHOR]

When lymphoid cells from hypersensitive rabbits are incubated with antigen, biologically active substances are formed and released which are capable of inhibiting the migration of normal non-sensitized mesenchymal cells. In the present paper some basic parameters of their production were determined. These substances were regularly obtained after 6 and 18 hours incubation, but not after 2 hours. Under more favourable cultivation conditions (lower density of lymphocyte suspension) an increased activity in the cell extracts as compared with the supernatants was observed. Another critical factor in the production of these substances is the quantity of antigen added. Ten micrograms of PPD leads to the production and liberation of a highly effective substance. A lower dose of antigen results in the liberation of a substance into the supernatant which by itself is almost inactive, but becomes more active when more antigen is added. The efficiency of the released substances was determined by serial dilution. The inhibiting activity was maintained at 1:5 and 1:20 and sometimes at 1:100 dilutions. [ABSTRACT FROM AUTHOR]

The studies reported here indicate that tanned red cells coated with a mixture of chromatographically purified ovalbumin and conalbumin give agglutination titres which are directly related to the amount of antibody (as meas- ured by quantitative precipitation) present against either of these antigens in mixed sera. With whole (unabsorbed) mixed sera, these coated cells record the titre of the major antibody present, whether this is anti-ovalbumin or anti-conalbumin. There was no evidence of greater sensitivity to anti-conalbumin. A trace of ovalbumin in a preparation of conalbumin used to coat cells is fully sufficient to enable them to react with any anti-ovalbumin in a serum. Twice recrystallized ovalbumin still contains some conalbumin. Sera raised against it may contain unexpectedly high levels of anti-conalbumin and this could cause confusion in tanned red cell tests. [ABSTRACT FROM AUTHOR]

Although induced immunological tolerance of histocompatibility antigens is generally considered to be specific, cases of concomitant third-party unresponsiveness may occur. The present paper describes one case in point. Antigenic overlap between original and third-party donor, as well as runt disease have been excluded as possible explanations for the observed lack of specificity. It is hypothesized that an innate inability, of the host to respond to the relevant third-party antigen(s), or the ability of certain antigens to induce tolerance of themselves as well as of distinct but related antigens, may be responsible for the phenomenon. [ABSTRACT FROM AUTHOR]

Lymph node cells from immunized rabbits were cultured with varying concentrations of antigen in preheated (56°, 30 minutes) autologous serum which had been collected before immunization. [3H]Thymidine was present for the last 6 hours of the 24-hour culture period and the radioactive labelling of acid-precipitable material was then determined. Changes in labelling due to variations of culture conditions were interpreted according to whether they were specific for control or antigen-treated cultures or non-specific. Cell concentration and serum concentration were predominantly non-specific variables influencing the labelling in control and antigen-treated cultures to a proportionate extent. However, at serum concentrations below 5 per cent labelling was disproportionately inhibited in antigen-treated cultures; there were further minor disproportionate inhibitions at higher serum concentrations. Labelling was inhibited by increasing the concentration of serum from 25 to 50 per cent, mainly due to a non-diffusible competitive inhibitory activity. Isotope-dilution analysis of the effects of serum on labelling over a wide range of serum concentrations indicated that the relationship was a complex one with at least three step-wise stimulations of the maximum labelling rate being produced by increasing the serum concentrations from 0 to 25 per cent. Labelling in antigentreated cultures containing post-immunization serum was less than labelling in cultures containing an equal volume of preimmunization serum, but labelling in control cultures was enhanced by post-immunization serum. These results are shown to be compatible with the proposals (i) that labelling in control cultures reflects the response of cells to low concentrations of endogenous antigens, and (ii) that preimmunization serum and post-immunization serum contain ‘natural’ and ‘acquired’ antibodies respectively, which normally buffer cell-borne receptor sites against reaction with endogenous and exogenous antigens. [ABSTRACT FROM AUTHOR]

Histamine release by antigen from rat mast cells was strongly inhibited by prolonged anoxia. The inhibition was reversed by oxygen and by glucose. Thioglycollate in low concentration potentiated histamine release and in high concentration inhibited it. The effects of anoxia and thioglycollate are additive: inhibition was produced by a low concentration of thioglycollate combined with a short incubation in nitrogen. This inhibition was reversed by glucose and became a potentiation. The effects of anoxia, glucose and thioglycollate on histamine release by compound 48/80 were quantitatively similar to those on histamine release by antigen. These results are consistent with the view that two opposing mechanisms are at work: a freeing of tissue SH groups which potentiates the anaphylactic mechanism and a disruption of tissue S-S bonds which inhibits it. Oxygen lack may inhibit by a reduction of S-S bonds as well as by an exhaustion of metabolic stores. [ABSTRACT FROM AUTHOR]

The oxygen consumption of isolated rat mast cells has been determined using a micro capillary, respirometer designed by Cunningham and Kirk and modified to permit stirring and addition of reactants. Uptake ranged from 0·5 pl. per cell per minute in 100 per cent O2 to 0·1 pl. per cell per minute in 1 per cent O2. It remained steady for at least 1 hour and was not affected by the presence of glucose. Under a variety of conditions neither histamine release by antigen added to sensitized cells nor by compound 48/80 added to normal cells resulted in any increase in the rate of oxygen uptake. Oxygen does not appear to be a rate limiting factor. [ABSTRACT FROM AUTHOR]

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13-27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen. The sedimentation coefficients of 131I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen. Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA. These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed. [ABSTRACT FROM AUTHOR]

The immunological responses of rabbits to HSA, HGG or BSA were tested at various times later in animals which had received the corresponding antigens before or shortly after birth. As judged by the criterion of failure to show immune elimination of antigen, a high proportion of the rabbits remained unresponsive at times when it was calculated that all the originally administered antigen would have been eliminated from the circulation. Furthermore, removal of antigen by passively administered antibody failed to restore the capacity to respond. It is concluded that, in respect of the antigens used, their persistence in the extracellular body fluids is not a prerequisite for maintenance of immunological unresponsiveness.
Further administration of the same antigen to rabbits which had escaped from a state of specific immunological unresponsiveness generally produced a very weak response, and in a few instances resulted in a return to the unresponsive state.
When the cross-reacting antigens HSA and BSA were administered adsorbed on alum to rabbits made unresponsive by neonatal contact with BSA and HSA respectively, and at the same time a further dose of the original antigen was given, antibodies were formed which were specific for the second antigen and did not cross-react with the first. In only 1/9 animals was responsiveness to the first antigen restored. The significance of these results is discussed. [ABSTRACT FROM AUTHOR]

An adaptation of the technique of disk electrophoresis is described. It allows combination of antigen and antibody, as well as the separation of the free antigen from the complex, in a single electrophoretic step. An Iodine-131 labelled human growth hormone : antihuman growth hormone system was used to demonstrate this combination which was expressed as per cent radioactivity bound to immune γ-globulin. Dilution of antiserum gave reproducible titration curves when run at three different temperatures: 10°, 20°, 35°. The curves obtained at 35° showed increased binding. The reduction of bound radioactivity on addition of unlabelled growth hormone to the sample was a linear function of the logarithm of unlabelled growth hormone. The standard curves obtained are suitable for a highly sensitive assay of human growth hormone. [ABSTRACT FROM AUTHOR]

Rabbits were immunized with 2,4-dinitrophenyl (DNP)-protein conjugates. Spleen cell suspensions were prepared and incubated in the presence of various DNP protein conjugates, the proteins alone, and DNP-lysine. The antigen dependent stimulation of DNA synthesis was used as a measure of the antigenic ‘activity’ of the DNP preparations. It was found that the cells were strongly stimulated by the DNP-protein conjugates used for immunization, and weakly stimulated by the protein alone. Highly substituted DNP protein conjugates were markedly more effective than lightly substituted conjugates. DNP-conjugates with proteins other than the one used during immunization were inactive. DNP-lysine alone was inactive but inhibited stimulation by the DNP protein conjugate used for immunization. The significance of these findings is discussed. [ABSTRACT FROM AUTHOR]

In continuation of the previous work on organ specificity of bovine adrenal, rabbit antisera against human, porcine and equine adrenal were obtained. It was demonstrated that, in addition to ox, man, pig and horse contain adrenal specific antigens that can be associated with organ preparation obtained by extraction at 100° followed by precipitation at a 72 per cent ethanol concentration. The present study allowed identification of two thermostable adrenal-specific antigens, one of them being species-restricted and the other crossing the species line. The former antigen was serologically dissimilar in man, ox, pig and horse. It could be detected only by homologous anti-adrenal sera and was represented by a thicker precipitation line. The latter antigen could be detected by both homologous and heterologous anti-adrenal sera and it was represented by a thinner precipitation line. This antigen was closely related, thought possibly not identical, in man, ox, pig and horse. [ABSTRACT FROM AUTHOR]

A state of immunological paralysis has been induced in adult CBA mice by intrapertioneal injections of small quantities of bovine γ globulin (BGG). The minimum paralyzing dose of BGG has been found to be between 50 and 200 μg. A dose as small as 2 μg, has been found to have a slight paralyzing effect. The time necessary for the induction of paralysis by 50 μg to 2 mg of BGG in CBA mice is 3-4 days. Paralysis is induced by only one component of BGG; this component is incapable of inducing an antibody response unless an injection of adjuvant is made at the same time or slightly before the injection of the antigen. The BGG is centrifuged at an RCF of 20,00-30,000 g to remove particulate matter. Failure to remove the particulate matter leads to sporadic immune responses in groups of mice injected with the protein. Mice given a paralyzing injection of BGG were subsequently challenged by an injection of BGG in Freund's adjuvant. The result of this challenge was tested by an injection of radioactively-labelled antigen and the elimination of this antigen from the circulation of the challenged mice was followed for several days. 'Immune elimination' can easily be distinguished from 'non-immune elimination'. The presence of antibody to the non-paralysing components of BGG in sera from paralysed mice was confirmed using the Ouchterlony gel-diffusion technique. [ABSTRACT FROM AUTHOR]

A ring precipitation has been found when saline is layered on sera from certain patients with abnormal globulins. Positive reactions were found mainly, but not entirely, in the sera from patients with chronic liver disease. The precipitate formed appeared to be a gamma globulin. The phenomenon may be confused with a true precipitin test but does not give false positive results when studied by the agar diffusion or tanned cell techniques. [ABSTRACT FROM AUTHOR]

Dried ceils of Pasteurella multocida type I were extracted with 2.5 per cent aqueous solution of sodium chloride. Acidification of the extract with HCl to pH 3.8 yielded a fraction containing protein with some polysaccharide and lipopolysaccharide. This was removed by centrifugation. Addition of ethanol to the supernatant precipitated a polysaccharide containing fructose, mannose, glucose and glucosamine. This polysaccharide could be further fractionated into products containing varying proportions of glucosamine and fructose. It is produced by both ‘blue’ non-capsulated and ‘fluorescent&lrsquo; capsulated phases of the same strains. From the former it is almost entirely released into the surrounding medium instead of remaining bound to the surface layers of the bacteria. The purified (heterogeneous) polysaccharide fraction is precipitable and fixes complement with homologous sera. When added in repeated amounts to rabbit and cattle sera against whole bacteria until no further precipitate formed it reduced but did not abolish the mouse-protective power of the sera. It did not immunize mice to challenge when injected subcutaneously or intraperitoneally in doses ranging from 2 to 100 μg. [ABSTRACT FROM AUTHOR]

Rabbit spleen cells, which have been treated in vitro with certain rabbit or guinea-pig antisera (e.g. against human serum albumin) and washed, are capable of specifically adsorbing radio-isotope labelled antigen. The antibody responsible for this effect appears to be distinct from the main precipitating anti-body in the serum and the term ‘cytophilic antibody’ has been suggested. The activity of the cytophilic antibody is not destroyed by heating at 56° C. for half an hour. The cytophilic antibody is not adsorbed by red cells. Spleen cells which have been treated with methanol before treatment with antiserum take up less antigen than non-methanol-treated cells tested in the same way. However, the addition of flesh normal spleen cells to the methanol-antibody-treated cells before the addition of antigen increased the uptake of the latter. [ABSTRACT FROM AUTHOR]

The distribution of antigen and antibody in radial double diffusion systems was studied by means of materials labelled with 131I or 14C. These studies showed that, once a precipitate begins to form, the assumption that antigen and antibody obey the laws of free diffusion is invalid. They also showed that, in the systems used, no antigen and very little antibody diffused past the zone of visible precipitation. It was found that accurate estimates of the diffusion constants of antigens could be obtained by allowing antigen and antibody to diffuse from troughs set at right angles and by measuring the angle of the precipitin line. Examples of the use of this method, and a theoretical treatment are given. An alternative method for estimating the size of antigens is to use the ‘molecular sieve’ properties of gelatin gels, which are sharply graded with the concentration of the gel. [ABSTRACT FROM AUTHOR]

The value of sera containing immuno-conglutinin or conglutinin in demonstrating complement-binding by human blood group iso-antibodies was investigated. Provided that a suitable complement was used, positive results were obtained with all human sera which had been shown by other methods to contain complement-binding antibodies. When human red cells were sensitized with human complement-binding blood group antibody and then treated with horse serum as a source of complement, the strongest reactions were obtained with rabbit immuno-conglutinin; the reactions with bovine serum (containing conglutinin) were distinctly weaker, and the reactions with human sera containing immuno-conglutinin were weaker still. When rabbit complement was used instead of horse complement, stronger reactions were obtained, but it was difficult to avoid ‘false positive’ reactions. When human complement was used, good reactions were obtained with rabbit immuno-conglutinin, but the reactions with bovine conglutinin and human immuno-conglutinin were completely negative. [ABSTRACT FROM AUTHOR]

By immunizing rabbits with bacteria agglutinated by corresponding immune rabbit sera group iso-precipitins were obtained. Using the iso-precipitins thus obtained the rabbit sera could be divided into two serum groups: D (a+) and D (a−); for grouping the gel diffusion technique was applied. The serum groups are heritable; probably group D (a+) mothers can transfer Da protein non-genetically. The substance thus obtained is then gradually eliminated from the bloodstream of the young animal. Da antigen has γglobulin electrophoretic mobility as shown by means of immunoelectrophoresis. The incidence of group D(a+) in 92 unrelated rabbits was 27·2 per cent. The authors discuss the relationship between iso-precipitins and anti-bodies and the practical significance of rabbit serum groups analogous to human Gm serum groups. [ABSTRACT FROM AUTHOR]

EXPERIMENTS have been carried out to test the predictions of a new theoretical approach to the Oudin technique (Spiers and Augustin, 1957). The graphical method employed for this purpose is described in detail. The antigen (Ag)-antibody (Ab) systems used were crystalline Bovine serum albumin (BSa) and crystalline egg albumin (Ea), with the corresponding rabbit antiserum globulin concentrates. The experimental results fitted the theoretical curves best when both Ag and Ab were incorporated in gel. With Ag in solution the same theoretical curves fitted as for Ag in gel (except for the highest Ag/Ab ratios), but there was a slightly greater scatter of experimental points. The results supported throughout the assumption that the Ag diffused towards the interface and that the Ag concentration was not maintained homogeneous, as claimed by Becker et al. (1951). Conditions for the formation of a stationary line at the gel-gel or liquid-gel interface agreed with those predicted by the new theory rather than with those predicted by Becker et al. By means of the new method it is also possible to obtain estimates of Ag-Ab combining ratios (in the gel) together with a rough estimate of the ratios of the diffusion coefficients of Ag and Ab, neither of which had previously been possible. Diffusion coefficients determined by this new graphical method were about 15 per cent below the accepted values, and the combining ratios R had values in excess of equivalence ratios. Diffusion coefficients calculated from our experimental results according to the theory of Becker et al. (1951) were below those found by the new method. The reasons for this, and results obtained by previous workers, are critically discussed. The effect of variations of temperature and of absolute concentration of the reactants are also examined. [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 effect of a single batch of horse anti-mouse thymocyte globulin on the immune response to type III polysaccharide antigen bas been investigated in 2–3-month-old male A/HeJ, C57BI, BALB/c, DBA/I, CBA and C3H mice. In almost all cases the intraperitoneal administration of 5 mg of this material on days -4 and -2 significantly suppressed the immune response to 0·1, 1·0 and 5·0 μg of antigen injected i.v. on day 0. Further studies undertaken in BALB/c mice indicated that effective suppression of the immune response to type III polysaccharide antigen could also be achieved by injecting 5 mg of this product (i.p.) some 15–30 minutes prior to antigenic challenge. Preliminary cell re constitution studies in antilymphocytic antibody-treated CBA mice indicate that the ability to respond to type III polysaccharide can be partially restored by the injection of syngeneic thymocytes, bone marrow cells or spleen cells. [ABSTRACT FROM AUTHOR]

Bone marrow cells and thymus cells were observed in cell transfer expehments to collaborate in the production of anti-BSA antibodies. The target cells for tolerance induction either with a 'low dose' of antigen (100 µg of deaggregated BSA once a week × 5) or with a 'high dose' (a single injection of 5000 µg) were identified by the same technique. In 'low dose' tolerance, some indication was obtained that thymus-derived cells in peripheral lymphoid systems were the target cells; bone marrow-derived cells appeared not to be so susceptible, and the cells residing in thymus or bone marrow seemed to remain unimpaired, in contrast, the injection of a 'high dose' of tolerogen rendered both types of cells in spleen, thymus cells and bone marrow cells, unresponsive or hyporesponsive in parallel with one another. [ABSTRACT FROM AUTHOR]

Nephrotoxic antibody-inducing antigen was extracted in a water-soluble form with adequate ultrasonic treatment of rat glomeruli. Antiserum against this antigen induced severe proliferative glomerulonephritis when it was injected into rats. This mechanically extracted antigenic material had the character of glycoprotein. Moreover, the immunological and biochemical properties of this antigen were strikingly similar to those of the nephrotoxic antibody-inducing antigen (glycoprotein) which has been obtained with tryptic digestion of GBM. This glycoprotein seems to be an important membrane-unit of normal GBM. [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]

Use of an intra-articular route of injection (a single, hind limb knee joint) and a protein antigen (human serum albumin)in water-in-oil emulsion with added mycobacterial peptidoglycolipid (or Wax D) failed to induce an adjuvant effect upon the resulting immunological response observed three weeks later. Serum antibody levels as measured by the Farr Test (ABC30 μg/ml) and cell-mediated immunity as measured by a quantitative corneal test were consistently lower than those which followed use of a footpad route for the same injection mixture. [ABSTRACT FROM AUTHOR]

The enhancement effect of some antigens on in vitro colony formation by normal mouse bone marrow cells is mediated through a normal serum αmacroglobulin (αM) which is present in newborn animals and shows no immunological specificity. The proportion of antigen and αM affects colony enhancement, and it has also been shown that specific antibody successfully competes with αM for the antigen. It seems likely that antigen-αM complex acts directly on the colony forming cell, rather than indirectly through release of colony stimulating factor. The possible relevance of this phenomenon to immune induction and, to the effect of antigens in promoting irradiation survival is discussed. [ABSTRACT FROM AUTHOR]

Five tissue specific antigens in guinea-pig epidermis were characterized by heat treatment, enzyme digestion, DEAE-cellulose chromatography, (NH4)2SO4 precipitation, ethanol precipitation and gel filtration. These antigens appeared to be proteins although one was fairly resistant to proteinases. Two antigens (Sp2a and Sp2b) were heat-stable and precipitated at high (NH4)2SO4 concentration (3.0 M—saturation): Sp2b was also precipitated at very high ethanol concentration (50-90 per cent). On electrophoresis, Sp2a was shown to have three distinct molecules, while Sp2b showed two. The molecular weight of one component of Sp2a was 71,000 and that of one component of Sp2b was 13,500, as determined by gel filtration. A third antigen behaved electrophoretically and chromatographically like γ-globulin, but was separated from it by (NH4)2SO4 precipitation. The other two antigens did not show any characteristic features and behaved like many other non-specific tissue antigens. [ABSTRACT FROM AUTHOR]

Rabbit antisera to calf collagen were investigated using columns of denatured rabbit collagen insolubilized by coupling to diazotized p-aminobenzyl cellulose. Only antibodies with general collagen specificity were bound onto the immunoabsorbent. Species specific antibodies passed through the column and were isolated in the effluent. Column-bound antibodies could be desorbed by elution with collagen peptides. The separated antibody fractions exhibited in haemagglutination-inhibition experiments with calf and rabbit collagen and collagen peptides a characteristic behaviour allowing the differentiation between them. The results support the assumption drawn from serological experiments that most antisera contain mixtures of antibodies with different specificities. [ABSTRACT FROM AUTHOR]

Spent culture media of all the human cell lines tested have been found to contain the antigenic activity present on the ll,000-Dalton HL-A common portion fragment of the HL-A antigen molecule that appears to be a characteristic, invariant portion of HL-A antigen molecules. From the culture medium of one of these lines, RPMI 1788, a lymphoid cell line, the substance carrying HL-A common activity was isolated, which was shown to be identical to the HL-A common portion fragment with respect to molecular size, electrophoretic mobility, isoelectric focusing patterns, and certain antigenic characteristics. By an isolation procedure involving differential ultrafiltration, gel filtration, and column electrophoresis, 8 litres of the culture medium yielded 1.5-2.0 A280 units of the substance representing 15-20 per cent of the HL-A common antigenic activity originally present. A single protein band with a Rf of 0.47 was obtained by disc electrophoresis. The molecular size was shown to be about 11,000 Daltons by gel filtration and by sodium dodecyl sulphate acrylamide gel electrophoresis. Upon isoelectric focusing two bands were obtained which corresponded exactly to those obtained with HL-A common portion fragment prepared from papain-solubilized HL-A antigen preparations by acid dissociation. The isoelectric point of the major band was 5.0. The reactions of this substance with rabbit antisera against human lymphoid cell membrane and against the substance were essentially identical to the reactions of HL-A common portion fragment with these same antisera. [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]

Intestinal immunoglobulins and antibodies in the local immune response to E. coli O somatic antigens have been studied in young fistulated pigs. Antibody levels in intestinal secretion were raised for approximately 2-3 weeks following a single local antigenic challenge with a heat-killed aqueous suspension of E. coli. A second challenge provoked a similar response suggesting a lack of immunological memory. Antibody activity in the secretions was predominantly associated with IgA and immunofluorescent studies of biopsy specimens from young fistulated animals indicated that intestinal synthesis and secretion of IgA had commenced by the 10th day of life. Studies of piglets reared with the sow indicated that oral immunization with E. coli antigen after 10 days of age stimulated intestinal antibody secretion before weaning at 3 weeks. The response of gnotobiotic pigs to oral immunization and infection was evaluated by immunofluorescent histology of the intestinal mucosa. Repeated oral administration of heat-killed E. coli O8 resulted in an immunocyte response in the lamina propria numerically comparable with that produced by infection with the live organism. The early response was dominated by cells of the IgM class whereas after 3 weeks IgA cells predominated. In the germ-free animal very few immunoglobulin-containing cells were detected. In vitro studies of antibacterial activity indicated that the most probable mechanism of immunological control in the alimentary tract is bacteriostasis. [ABSTRACT FROM AUTHOR]

The target cell of in vitro stimulation of primed spleen cells by hapten-carrier complexes was studied. The antigens DNP28-bovine serum albumin (DNP28-BSA), which gives only 2,4-dinitrophenyl (DNP) specific responses and DNP14-mouse immunoglobulin (DNP14-MIg), which probably reveals DNP as well as carrier specificity (new antigenic determinant) were used. Educated T cells could be stimulated with the antigens used for activation. A similar experiment with educated B cells, however, gave no indication that these B cells could be stimulated with antigen in the absence of T cells. Cortisone treatment of primed mice yielded spleen cells which had a higher activity than spleen cells from unprimed, cortisone-treated mice. This also points to stimulation oft cells by antigen. Treatment of primed spleen cells with anti-thymocyte serum (ATS) and complement (C) abolished stimulation by phytohaemagglutinin (PHA) completely, by lipopolysaccharide (LPS) slightly, while the antigenspecific activity was 90 per cent reduced. This indicates a mainly T cell-specific stimulation by the antigen. A corresponding experiment with anti-plasma cell serum (APCS) and C revealed a complete reduction of LPS activity and a small impairment of the PHA and conavalin A (Con A) activity. However, the antigenspecific activity was reduced by one-third to a half for the different antigens. This is an indication for a specific B-cell stimulation by the antigen, although it is on a lower level than the T-cell stimulation. The role of the hapten in the T-cell stimulation is discussed. [ABSTRACT FROM AUTHOR]

The repeated administration of bovine serum albumin by stomach tube to Charles River and Black Norwegian rats resulted in a state of partial tolerance to the antigen. A very small amount of antibody was detected in the serum at the end of the oral regime but anti-BSA-producing cells could not be demonstrated in the lamina propria, in the Peyer's patches of the small intestine, in the mesenteric nodes or in the spleen of these animals. Antibody was not demonstrated in the small intestinal contents or in the faeces of the same animals. The antigen was absorbed in the native form and not as constituent peptides bearing antigenic determinants in common with the native protein. Preliminary data, using a radioimmunoassay, indicate that the serum concentration of BSA after the administration of 25 mg of the protein by stomach tube, is in the range 1-10 ng/ml of serum. Preliminary experiments indicate that the state of partial tolerance could not be abrogated by syngeneic spleen cells or peritoneal exudate cells from normal rats. This form of tolerance therefore has some features in common with the state of tolerance induced by the parenteral administration of small amounts of bovine albumin (low zone tolerance). [ABSTRACT FROM AUTHOR]

The in vitro assay of inhibition of Ieucocyte migration was used in two forms for the study of the specificity of transplantation immunity in mice. In one system thymus cells (antigen) were mixed with peritoneal exudate cells (PEC) from mice immunized with skin grafts and inhibition of migration was detected only when the antigenic cells were of donor origin or were from a third-party strain which shared an H-2 region (K or D) with the graft donor. However, in another form of the assay, in which sensitized lymph node cells (LNC) were mixed with antigenic PEC, inhibition of migration was detected also using antigenic cells from third- party strains which did not share an H-2 region with the donor and possessed only few H-2 specificities of the donor, The inhibition of migration mediated by LNC was abrogated with the use of anti-θ sen. Transplantation immunity was studied in vivo with an assay of resistance to the growth of a third-party tumour (SaI) in mice pre-immunized with skin grafts from congenic strains having different H-2 antigens. Resistance was found in cases where the tumour cells did not share an H-2 region with the graft donor, but was weaker than the resistance obtained upon immunization with grafts from strains sharing an H-2 region with the tumour donor. [ABSTRACT FROM AUTHOR]

The experiments described here form part of a series carried out to determine the conditions of antigen presentation which dispose to the production of IgE antibody in the rat. We have found that Hooded Lister rats in comparison with rats of some other strains have an exceptional ability to produce reaginic antibodies responses can be consistently induced with very small doses of antigen and are boosted to high levels with a second dose of antigen. The effect of the dose of antigen on these responses is as follows. [ABSTRACT FROM AUTHOR]

Prednisolone causes a dose-related inhibition of antigen-evoked histamine release from IgE-sensitized human skin in vitro. The effective concentrations are of the same order as are achieved in plasma therapeutically. Analysis of prednisolone inhibition shows that it acts on the second histamine release stage, antigen-antibody combination being unaffected. In contrast with the traditional view, our results show that, at least in human skin, glucocorticoids can inhibit antigen-evoked histamine release [ABSTRACT FROM AUTHOR]