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The antigenic components of adrenal extract were fractionated by chromatography on DEAE-cellulose. Using a stepwise elution procedure, it was found that a component of major interest, termed Pk3b, was eluted by a buffer of pH 6.70 and 0.09 M in phosphate-chloride. This antigenic preparation reacted with several rabbit antisera that had been prepared by isoimmunization with rabbit adrenal homogenate, in contrast to several other fractions, which reacted with only one (or two) of the antisera. Pk3b also showed a high degree of homogeneity, as shown by immunodiffusion, paper electrophoresis, and analytical ultracentrifugation. By the latter technique, an extrapolated sedimentation coefficient of 4.45S was determined. The further comparison of this antigen in immunodiffusion, along with an antiserum and the adrenal extract from the antiserum-providing animal, confirmed again that this is an autoantigen, and that it is one of five autoantigens of the adrenal gland. [ABSTRACT FROM AUTHOR]

In the previous paper we showed that the immune response to sheep red cells (SRBC) was degenerate; serum antibodies showed increasing loss of specificity, signalled by cross-reactions with horse RBC (HRBC), with time after immunization and with hyperimmunization. In the present report we have analysed the role T cells may play in this process. To do this we studied the antibodies made by mice deprived of T cells by irradiation as well as those made by normal mice immunized with SRBC in distilled water, which was shown to depress the DNA synthetic response of T cells. In both instances the mice with fewer responding T cells made less antibody which could agglutinate HRBC than did the appropriate controls. This was true even when no difference in anti-SRBC titre was apparent. In addition we showed that the antibody made by mice with reduced numbers of T cells was less effective at passive suppression of the immune response to SRBC than was similarly titred (against SRBC) antibody made by normal mice. Thus, certain sub-populations of antibodies, normally made in response to SRBC, immunization, are particularly thymus dependent. We have discussed why we think they are those of particularly high affinity. [ABSTRACT FROM AUTHOR]

Lymph node and spleen cells from normal mice were cultured for 3 days with polyoma virus-induced tumour, Ehrlich's ascites turnout or leukaemia L 1210 cells. This resulted in in vitro immunization of the lymphocytes, which were then transferred to irradiated target cells labelled with 51Cr. Normal, i.e. non-immune thioglycollate-stimulated peritoneal macrophages were also added to some tubes. Non-immune macrophages mixed with immunized lymphocytes showed a significantly increased ability to destroy tumour ceils as compared with macrophages in the absence of immunized lymphocytes. The immunized lymphocytes were almost entirely inactive alone. When the number of macrophages was kept constant the cytotoxicity was dependent on the number of viable immunized lymphocytes placed on the target cells. Immunized lymphocytes, in the presence of macrophages, only exhibited strong killing of the target cells against which they had been immunized; some lysis of ‘bystander’ cells was, however, seen provided specific target cells were present. Macrophage monolayers exposed to immunized lymphocytes upon contact with specific antigen became ‘armed’ and showed a significant cytotoxicity for specific target cells. When immunized lymphocytes and normal macrophages were treated with actinomycin D and puromycin, cytotoxicity was inhibited in the immunized lymphocytes but not in the macrophages. The possible mechanism of normal macrophage cooperation with immunized lymphocytes in the cytotoxic killing reaction is discussed. Results presented in this paper favour the view that immunologically specific cytophilic factor (presumptive cytophilic antibody) is involved in the macrophage-mediated cytotoxicity in the system studied. [ABSTRACT FROM AUTHOR]

An earlier paper showed that 4 days after immunization with the contact sensitizing agent 'uxazolone' there is a peak in the percentage of cells in the draining lymph nodes that move to sites of inflammation. This was assessed by dissociating the lymph nodes, labelling them with 51Cr and injecting them into mice whose ears were painted within an hour with an unrelated contact sensitizing agent or with croton oil. The ears were then removed at about 18 hours and their radioactivity used as a measure of cell arrival. The cells that move to sites of inflammation are not macrophages as they are not removed by filtration through cotton wool. They are θ-positive and control studies show that the activity of the anti-B serum was indeed due to antibody to the θ-antigen. The cells are large and can be labelled both in vivo and in vitro with [125I]-iododeoxyuridine which is incorporated in the DNA of cells during the S phase of the cell mitotic cycle. It was concluded that the cells in immunized lymph nodes that move to sites of inflammation are T blasts. The unitary hypothesis is put forward that following immunization for delayed hypersensitivity a particular class of T cells proliferates and gives rise to large pyroninophilic blast cells; and that these cells or their immediate descendants possess the properties, either at the same time or at closely related times, of' movement to sites of inflammation and non-specific cytotoxicity as well as the capacity to passively transfer cellular immunity. [ABSTRACT FROM AUTHOR]

Thirty-six guinea pigs were injected with a suspension of pooled guinea-pig adrenals incorporated into Freund adjuvants; twenty-two rabbits were immunized in a similar manner with rabbit adrenal suspensions. Sera of twenty-six guinea pigs and twelve rabbits reacted in complement fixation and tanned cell haemagglutination tests with adrenal extracts but not with extracts of other organs. Potent antisera also produced one line reaction in double diffusion gel precipitation test with adrenal extracts. The adrenal-specific antigen was thermolabile, possibly of protein character. Reactions of guinea-pig antisera were limited to guinea-pig adrenal; rabbit antisera reacted with adrenal of all tested mammalian species. The serological reactions exhibited by antigens prepared from the antibody producer's own adrenal were as strong as those produced by adrenals of other animals of the same species. Additional evidence for auto-specificity of the antibody under investigation was obtained from the experiment in which a guinea pig injected with its own gland removed by left adrenalectomy formed adrenal antibodies. Some but not all animals developed histological lesions in the adrenal gland. The interpretation of this observation was complicated by occasional positive histological findings in the adrenals of control animals. [ABSTRACT FROM AUTHOR]

A state of protein-overloading paralysis has been induced to bovine γ globulin (BGG) in adult CBA mice. Paralysis was demonstrated by a lack of immune elimination of 131I labelled BGG after prior challenge with Freund's adjuvant containing BGG. The degree of paralysis was found to diminish when the time between the paralysing injection and challenge injection was increased. This loss of the paralysed state could be prevented by injections of antigen whilst the mice were still paralysed. The superficial nature of the differences between paralysis (unresponsiveness induced in adults) and tolerance (unresponsiveness induced in neonatals) to BGG in CBA mice is discussed. The possibility that suppression of antibody production in previously immunized animals given a massive dose of antigen, is a distinct phenomenon from the induction of paralysis/tolerance in animals not previously immunized, is briefly discussed. [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]

A complete evaluation of the immunogenicity of oligo-L-lysines and their mono-ε-DNP derivatives requires a determination of their capacity to prepare animals for secondary responses to a highly immunogenic analogue and to elicit secondary responses in animals prepared with such an analogue. It is shown here that 1-ε-DNP-tetra-L-lysine is not effective, or only marginally effective, in preparing PLL+ guinea-pigs for secondary anti-DNP responses to DNP-poly-L-lysine whereas 1-ε-DNP-nona-L-lysine is active in such preparation. Similarly, in animals primed to DNP-ovalbumin, supplemental immunization with tetra-L-lysine is not effective in preparing strain 2 guinea-pigs for a secondary anti-DNP response to DNP-poly-L-lysine whereas both nona-L-lysine and poly-L-lysine are effective, the latter to a considerably greater degree. In animals primed with DNP-poly-Llysine or with DNP-ovalbumin and poly-L-lysine, DNP-poly-L-lysine elicits secondary anti-DNP responses whereas neither 1-ε-DNP-nona-L-lysine or 1-εDNP-tetra-L-lysine do. The data indicate that a hierarchy in the degree of immunogenicity exists within the lysyl peptides series with poly-L-lysine more immunogenic than nona-L-lysine and this, in turn, more immunogenic than tetra-L-lysine which is either nonimmunogenic or marginally immunogenic. Further, the determination of immunogenicity is a feature either of the 'helper' cell population or of a step in the response which is prior to the activation of 'helper' cells. [ABSTRACT FROM AUTHOR]

Lymph node cells from Black Hooded (BH) rats immunized with Freund's complete adjuvant (CFA) are only poorly cytotoxic to target cells treated with target cell specific antibody. Normal spleen cells are highly cytotoxic to antibody coated target cells. When 106 lymph node cells, from rats immunized with Chang cells in CFA, are mixed with 107 spleen cells from unimmunized rats, the mixture is 8.6 times more cytotoxic than the sum of the cytotoxicities of the two lymphoid cell populations alone. Immune lymph node cells were no more cytotoxic in the presence of 5 per cent fresh guinea-pig serum than in the presence of heat inactivated guinea-pig serum. Immune spleen cells are prevented from damaging target cells by antagonists of protein synthesis such as Puromycin. Puromycin, however, does not alter the cytotoxic effect of either immune or nonimmune spleen cells on antibody coated target cells. From these experiments it is tentatively suggested that lymphoid cells from immunized animals mediate target cell damage by a reaction involving firstly immunologically specific responder cells which synthesise antibody and secondly non-specific effector cells which damage the antibody coated target cells. Evidence is presented which suggests that stable soluble factors may not be responsible for mediating target cell damage in this system. [ABSTRACT FROM AUTHOR]

Anti-haemagglutinin titres and measurements of the changes in the number of antibody molecules per millilitre (A), and in their average avidities (K) were carried out by equilibrium filtration on antisera prepared in rabbits to SW, MEL and LEE influenza viruses. These studies showed: (1) Immunization of rabbits with influenza virus by the intravenous route induced sufficient levels of antibodies by the 5th day after vaccination for testing by equilibrium filtration, immunization by the intraperitoneal or subcutaneous routes did not induce sufficient antibody levels for testing by the 5th day. (2) The avidity of the antibodies present at 5 days after vaccination was higher than at 10 days after vaccination. (3) The avidity of antibodies increased during the initial response period (from the low point at 10 days) and was independent of the routes or schedules of vaccination tested. (4) Secondary vaccination did not cause a pronounced increase in the avidity of antibodies, although a few groups of rabbits showed some further increase; tertiary vaccination caused no further increase. (5) The number of antibody molecules produced in rabbits after vaccination with influenza virus depended on the route and schedule of inoculation; the routes could be ordered intravenous, intraperitoneal and subcutaneous in decreasing order of efficiency; multiple doses increased the number of antibody molecules by all routes. (6) Increases in the antihaemagglutinin levels after secondary stimulation were due mainly to increases in the number of antibody molecules. [ABSTRACT FROM AUTHOR]

Precipitating antisera against the four subclasses of human IgG were prepared by immunizing different species of animals (monkeys, rabbits, chickens, guinea-pigs and sheep) with whole IgG or Fc fragments. Freund's complete and incomplete adjuvant and Al(OH)3 were used to enhance antibody formation. In most of the experiments with rabbits, immunization against a particular subclass was accompanied by induction of unresponsiveness to the other subclasses. Different animal species showed distinct preferences for production of certain IgG subclass antisera. Anti-IgG3 could quite easily be produced in each of the animal species tested (monkey, rabbit and guinea-pig). Anti-IgG2 and anti-IgG4 could be raised in monkeys and, with greater efforts, in rabbits. Anti-IgG1 could easily be raised in guinea-pigs and with difficulty in rabbits. In our hands monkeys did not react to IgG1, possibly because only Gm(f+) paraproteins were used; experiments with rabbits and guinea-pigs showed that antibody formation against IgG1 was promoted by the presence of the Gm(a) marker. The Gm(n) marker was found to influence anti-IgG2 formation to an even greater extent: no subclass-specific antibodies were obtained when Gm(n-) IgG2 paraproteins were used as antigen. Apart from IgG subclass-specific antibodies the following additional antibodies were often found: antibodies directed to a combination of a certain heavy chain and a kappa light chain in monkeys; antibodies specific for one or more antigenic determinants common to IgG2 and IgG3 in monkeys and rabbits immunized with IgG2; antibodies specific for one or more antigenic determinants common to IgG1 and IgG2 in guinea-pigs and rabbits immunized with Gm(f+) IgG1. [ABSTRACT FROM AUTHOR]

Immunization leads to an increase in serum immunoglobulin levels, but only a fraction of this increase is attributable to specific antibody. This report presents data showing that this increase in serum immunoglobulin levels can be correlated with an increase in the number of immunoglobulin-secreting cells. An absolute increase in the number of immunoglobulin-secreting cells occurred in every organ tested but was greatest in spleen and peripheral blood. Peyer's patches contained very few such cells. A comparison of cells secreting immunoglobulin with those containing surface immunoglobulin receptors indicated that there is a differential anatomical distribution of the two cells following antigenic challenge. [ABSTRACT FROM AUTHOR]

HgA-negative rabbits were given an intravenous injection of HgA-positive red cells followed within 30 minutes by an intravenous injection of IgG anti-HgA. Several weeks later a second injection of HgA-positive red cells, without antibody, was given; the survival of these red cells was compared with the survival of red cells given to animals which had received no passive antibody. As judged by a relatively good survival of the second injection of red cells, primary immunization to HgA was at least partially suppressed when the dose of anti-HgA given with the first injection of red cells was such that 260-750 μg antibody was bound per ml red cells. In three groups of animals given HgA-positive cells and anti-HgA in doses such that 20-40 μg antibody was bound per ml red cells there was apparently no suppression of the antibody response. It is concluded that the amount of specific IgG antibody required for the suppression of primary immunization is at least ten times greater, in terms of μg antibody per ml red cells, for the rabbit antigen HgA than for the human antigen Rh; the difference may be due partly to the greater number of HgA antigen sites on the rabbit red cell compared with the number of Rh(D) sites on the human red cell. [ABSTRACT FROM AUTHOR]

We studied the effect of passively administered anti-carrier arid anti- hapten antibodies on the primary anti-hapten antibody response to hapten-carrier conjugates in mice. Bacterial α-amylase (BαA), Taka-amylase A (TAA) and key-hole limpet haemocyanin (KLH) were used as carrier molecules, and 2,4-dinitro-phenyl (DNP) group was used as a haptenic determinant. Three groups of mice were injected intravenously with anti-carrier antiserum, anti-hapten antiserum and normal serum as the control, respectively, immediately after the immunization with the hapten-carrier conjugate. The primary anti-carrier antibody response was markedly suppressed by the passively administered anti-carrier antibody but not anti-hapten antibody. However, the primary anti-hapten antibody response was suppressed not only by passively administered anti-hapten antibody but also by the injection of anti- carrier antibody in an early period after the immunization. When anti-carrier antibody was given twice 0 and 7 days after immunization, the primary anti-hapten antibody response was markedly suppressed and the suppressive effect was observed even at a later period. In contrast, anti-hapten antibody given by the same schedule as above suppressed only the primary anti- hapten antibody response, but not the anti-carrier antibody response. Passively administered anti-carrier antibody did not suppress carrier-specific helper cell development, but still suppressed the development of B memory cells and antibody formation against carrier determinants. The antigen dose required for the development of B-cell memory was much higher than that necessary for the stimulation of' T cells. Passively administered anti-carrier antibody clearly inhibited the cellular cooperation between carrier-committed helper cells and hapten-specific B cells and the augmented primary anti-hapten antibody response induced by carrier-primed T cells was clearly abolished. Furthermore, mice preimmunized with carrier showed significantly lower anti-hapten antibody response following the hapten- carrier challenge. Moreover, development of hapten-specific memory cells was also suppressed. Thus, even in the non-specific antibody-induced suppression by anti-carrier antibody, negative feedback effect of a B-cell product with anti-carrier specificity exclusively regulates the B-cell line development and differentiation. [ABSTRACT FROM AUTHOR]

The effect of the time and dose of heterologous antithymocyte serum (ATS) on reaginic antibody formation was studied in the rat. Animals were treated with a single intravenous injection of ATS at various times before or after the primary immunization with dinitrophenylated Ascaris suum extract (DNP-As) and Bordetella pertussis vaccine. If animals were treated with a large lymphopenic dose of ATS shortly before or at the time of immunization, the production of reaginic as well as IgM and IgG antibodies was greatly suppressed, whereas the same treatment if given shortly after the immunization was started significantly enhanced and pro- longed the reagin production. On the other hand, smaller doses of ATS even if given at the time of immunization only delayed the reaginic antibody response which also showed a marked prolongation. Furthermore, an unusual sequential production of IgM and IgG antibodies was observed in some of the ATS-treated animals. These results suggest that ATS can inhibit either inductive or regulatory function of the thymus-derived lymphocytes (T cells) depending on the time when it is administered and on the dose of ATS. Some other supporting data indicating that ATS inhibits the T cells specialized in the regulation of antibody formation are also presented. [ABSTRACT FROM AUTHOR]

The role of Peyer's patches in primary and secondary immune response was studied with Vibrio cholerae as the antigen. V. cholerae E1 Tor ME 7 is a typical enteric antigen which was shown to be suitable for the demonstration of antibody forming cells by localized lysis in agar. No plaque-forming cells (PFC) against this antigen were found in the organs of non-immunized mice. Consequently a primary immune response could be studied. In vitro an immune response against this antigen could only be obtained with spleen cells when the donor animal had been primed. After primary intraperitoneal or enteral immunization many PFC were found in the spleen but none in Peyer's patches. this cannot be explained by insufficient penetration of the antigen into Peyer's patches as secondary responses were obtained with both routes of immunization. After local injection of antigen directly into Peyer's patches, PFC only appeared after 5 days. This is the indication that an essential cell type for the unable to restore the immune response in lethally irradiated mice, unless they were injected together with thymus cells or cells from peripheral lymph nodes. This suggests the absence of active T cells and indicates the presence of B cells in Peyer's patches. The combination of thymus cells and bone marrow cells was inactive in restoring the immune response. This is an indication that the B cells in Peyer's patches are immunologically more mature than bone marrow cells. [ABSTRACT FROM AUTHOR]

When nuclei from the lymphocytes of mice immunized with rabbit erythrocytes are injected into unprimed animals the number of cells forming antibody to rabbit erythrocytes increases to a level approaching that of the immunized donor animals. The specificity of the response induced by nuclei is equally as good as that induced by native antigen. When ‘T’ and ‘B’ mice are immunized with rabbit erythrocytes a poor response is observed but when the nuclei from primed ‘T’ animals are used to immunize ‘B’ mice the number of antibody-producing cells is very much increased. In view of current theories about cellular co-operation it is pointed out that this co-operative effect of ‘T’ lymphocyte nuclei is independent of both cell membrane and additional antigen. [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]

CBA mice were immunized by painting the skin with the contact sensitizing agent 'oxazolone'. The draining lymph nodes were taken at 4 days and mixed with 51Cr-labelled mastocytoma tumour cells. Cytotoxic killing was assessed by the release of 51Cr. Little killing occurred in the absence of phytohaemagglutinin. However, immunized but not normal lymph node cells killed the mastocytoma cells providing phytohaemagglutinin (PHA) was present during the killing reaction. Analysis by the `one hit' hypothesis suggested that up to 14 per cent of the lymphocytes in immunized lymph nodes were effector or killer cells. The cytotoxicity was non-specific. The evidence for this was that DBA/2 lymph nodes immunized with oxazolone killed DBA/2 mastocytoma cells. The killing reaction did not require macrophages but was due to large cells. These were shown to be θ-positive in carefully controlled experiments. The non-specific cytotoxicity was greatest 4 days after immunization with oxazolone. Reimmunization of the mice on day 10 did not cause a classical secondary response, as assessed by non- specific cytotoxic killing. Instead the time course resembled the primary while the magnitude of killing was slightly reduced. There were several similarities between non-specific cytotoxic cells and the large pyroninophilic cells and the cells which move to sites of inflammation which are found in lymph nodes immunized with contact sensitizing agents. These similarities included θ-positivity, large size and peak occurrence 4 days after immunization. It is postulated that non-specific cytotoxicity and movement to sites of inflammation are due to blasts or their immediate descendants which originated from T lymphocytes. [ABSTRACT FROM AUTHOR]

The antibody response of A strain mice to immunization with C57BL mouse leukemia, EL4, is confined to the 7S γ-globulin. A rapid chromatographic method has been developed for the isolation of mouse 7S γ-globulin in a high state of purity as judged by immunological and electro-phoretic techniques. The apparent molecular weight (MSD) of the immune γ-globulin preparation did not differ significantly from that of the normal γ-globulin preparation. However, weight average (Mw) and Z average (Mz) molecular weights determined from sedimentation equilibrium experiments showed differences in the two preparations. The Mz/Mw ratios showed that the molecular polydispersity in the immune sample was significantly greater than that in the normal sample. [ABSTRACT FROM AUTHOR]

A study of the secondary response of regional lymph nodes to immunization with diphtheria toxoid adsorbed to aluminium phosphate has been made using a combined immunofluorescent and autoradiographic technique. No difference was detected in the kinetic pattern of the specific antibody-containing cells compared to the general population of those containing γ-globulin. The morphology of the replicating antibody-containing cells and their progeny are described. A population of large basophilic, DNA-synthesizing cells which did not contain γ-globulin was found to be present in the nodes. Rates of cell division were examined in rats immunized with HSA. The blast cells were found to have a rapid turnover and their progeny, the plasma cells, a short life span. [ABSTRACT FROM AUTHOR]

Rats, injected with crude bovine heart extracts, showed dissociation of their immunological response. Complement-fixing and capillary precipitating anti-bodies were produced, active against particulate antigens, while tanned-cell haemagglutinating and gel-diffusing antibodies were active against soluble antigens. Particulate antigens were centrifuged down from tissue extracts as 60,000 g but not at 12,000 g. The kinetics of production and specificity differed in the two groups of antibody. When rats were injected with bovine tissues from birth, to produce immuno-tolerance, they did not produce complement-fixing or capillary precipitating antibodies on later challenge with bovine heart but did produce haemagglutinating and gel-diffusing antibodies. Even before challenge, pretrated rats showed haemagglutinating antibodies, while the response by this test to the further immunization was partially suppressed. [ABSTRACT FROM AUTHOR]

Dissociation rate measurements were determined on antisera following single and multiple intravenous injections of antigen and after injection of antigen incorporated into adjuvant. It was found that regardless of the amount, route, or number of injections the dissociation characteristics of all sera were similar, reaching the same maximum binding strength within the same time period following immunization. The relative production of 19S and 7S antibody was studied and it was found: (a) insufficient 19S antibody is formed to account for the change in dissociation characteristics that occur following immunization; (b) at the same stage of immunization, 19S and 7S antibody have similar rates of dissociation from antigen, but 19S antibody combines more slowly with antigen than 7S antibody; (c) the haemagglutination technique show's no correlation with binding or precipitin methods when estimates as to the relative amounts of 19S and 7S antibody present in sera are made. [ABSTRACT FROM AUTHOR]

The sera of rabbits injected with rat liver, kidney, heart, muscle, spleen and brain in Freund's complete adjuvant fixed complement with rabbit tissue. This complement-fixing activity was attributed to autoantibodies which were able to fix complement in vitro with the tissue of the rabbit in which they occurred. Absorption, gel diffusion and antibody and antigen titrations indicated that some of the anti-liver, anti-kidney, anti-heart, anti-muscle and anti-brain sera contained organ-specific autoantibody. The sera also contained autoantibody reacting with widely distributed antigen(s), which was relatively labile at 65°. The anti-kidney and anti-brain sera reacted with distinct antigens which were extracted from rabbit kidney and brain with a mixture of chloroform and methanol. The natural autoantibody of Kidd and Friedewald was usually labile at 65° and behaved like a macroglobulin on sucrose gradient centrifugation. Sera taken 1 week after immunization with rat tissue contained heat-labile macroglobulin antibody. However, sera taken I month after immunization also contained small molecular weight antibody which was stable at 65°. [ABSTRACT FROM AUTHOR]

Describes a method for the partial purification of the opsonins in pig serum which are directed against a strain of Salmonella typhimurium. Factors which occur in the beta-globulin fraction; Occurrence of antibodies produced by specific immunization of the pig in the gamma globulin fraction.

Two or three graded infections with oocysts of Eimeria acervulina, E. tenella, E. necatrix and E. maxima produced a resistance to further infection with the immunizing species. The oocyst output after the second infection, in each case, was lower than that after the initial dose indicating the substantial immunizing effect of the initial infection. The species could be placed in a descending order of immunizing activity as follows: E. maxima, E. acervulina, E. tenella and E. necatrix. A solid immunity to the immunizing species in no way prevented the development of an additional infection, here referred to as ‘cross-infection’, with any of the species studied. Serum precipitins were produced in infections with all four species, the response to infection with E. necatrix being less marked than to the other species. A first challenge of immune fowls with the immunizing species produced some increase in precipitation in agar whereas a second challenge had no such effect; the significance of this lack of response is discussed. Usually, fowls immunized against one species and then infected with an additional one, produced serum precipitins which reacted only with the antigen of the additional species. But E. tenella immunized fowls, when given an additional infection with E. necatrix, produced precipitins that reacted with antigens of both species. The same was also true when E. necatrix immunized fowls were infected with E. tenella. [ABSTRACT FROM AUTHOR]

A fair degree of correlation has been found between the in vivo antitoxin content of sera from horses immunized with crude Corynebacterium diphtheriae culture filtrates and the direct agglutinin titre of the sera when tested with sheep cells sensitized with diphtheria toxoid. Haemagglutination inhibition tests at the LA level of test with the same sera showed some rather large discrepancies from the in vivo values and further tests with special agglutinin inhibiting toxins suggested that specific antitoxin free from other accessory, antibodies might be non-agglutinating, and therefore not titratable by haemagglutination inhibition. The phosphate-stable, pepsin-stable and trypsin­stable antigens isolated from culture filtrates of C. diphtheriae were found to contain extremely small quantities of specific toxoid, and cross titration of each of the three antigen preparations showed that there was very little contamination by other antigens within the group. Absorption of diphtheria antiserum with red cells sensitized with each of the three accessory antigens individually, showed that the antibodies were highly specific and distinct. Absorption of diphtheria antiserum with a mixture of red cells sensitized with the three different antigens removed all demonstrable accessory antibodies, and the absorbed serum would no longer agglutinate cells sensitized with complete diphtheria toxoid. The absorbed serum, however, retained a large proportion of its neutralizing capacity for diphtheria toxin, when titrated in vivo. Titration of each of the accessory antibodies in a number of horse sera by haemagglutination inhibition demonstrated a correlation between the values for the accessory antibodies to the phosphate-stable and pepsin-stable antigens, but no correlation with the values for the antibody to the trypsin­stable antigen, when compared with results of the flocculation test. The relative proportions of diphtheria toxin and of the phosphate-stable and pepsin-stable antigens remained constant at all stages of purification of a filtrate including several recrystallization procedures. However, the concentration of the trypsin­stable antigen declined steadily during the process. The relative proportions of toxin, phosphate-stable and pepsin-stable antigens in several crude culture filtrates were constant under reasonable conditions of storage and very varied where deterioration had occurred. These three antigens also maintained a constant ratio in growing culture, the trypsin­stable antigen did not. [ABSTRACT FROM AUTHOR]

Human chorionic gonadotrophin (HCG) is antigenic in rabbits when injected with Freund's adjuvant. The HCG used for immunization showed the presence of five antigens in the Ouchterlony plates against the homologous antiserum. Three of these antigens were common to human serum and one of these three to normal human urine. Rabbit antiserum to HCG absorbed with human serum did not form any antigen-antibody precipitin line with either normal human serum or normal human urine. It formed two precipitin lines with HCG. One ml. of the rabbit antiserum to HCG could inhibit even 150 I.U. of HCG as tested by the Aschheim-Zondek test in mice and ovarian hyperaemia test in rats. The absorbed antiserum could inhibit hormonal activity of HCG even when the antiserum and HCG were injected simultaneously at separate sites. [ABSTRACT FROM AUTHOR]

The elimination of 131I­labelled BGG from the circulation of the mouse has been used as a measure of the immune response to BGG. It has been shown that intravenous injection of BGG solution alone is an unreliable method of immunization. Successful immunization was obtained after subcutaneous injection of Freund's adjuvant containing BGG. BSA and HSA have short biological half-lives in adult CBA mice. They are therefore unsuitable for use with the antigen elimination technique. [ABSTRACT FROM AUTHOR]

An in vitro assay procedure for evaluating the antibody response to glomerular basement membranes, lung basement membranes and tendon fibrils indicated a sharing of identical or similar antigenic components. At least 70 per cent of the glomerular basement membrane-binding antibodies are absorbable by tendon fibrils, and eluates from such tendon preparations are not nephrotoxic. Within the species employed (man, rabbit and dog) for glomerular basement membrane sources, there appears to be no species specificity as regards the presence of the nephrotoxic component. Immunization of sheep with either canine or rabbit glomerular basement membrane leads to the elaboration of a haemagglutinin for the respective species. [ABSTRACT FROM AUTHOR]

Normal adult spleen cells were antigenically stimulated by incubation with isolated flagella from Salmonella bacteria, and after neutralization of free antigen and washing, were transferred to normal and sublethally X-irradiated mice and rats of various ages by intraperitoneal injection. Neonatal rats and Hall Institute mice could not produce antibody following such injections, but more mature rats and Hall Institute mice, and neonatal C3H mice, could produce antibody. Cells from immunized spleens were secondarily stimulated in vitro and transferred to neonatal and more mature recipients. Regardless of the age of the recipients, antibody production typical of a secondary response ensued. Cells from neonatal rat spleens were stimulated in vitro and transferred to more mature recipient rats. Some antibody production resulted. The theoretical implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

A method developed for localizing antibody in imprints of lymphoid organs of immunized animals involves treating the fixed imprints with radio-iodinated homologous antigen, washing to remove uncombined antigen and then making an autoradiograph. Finally the imprints are stained with the autoradiographic film in situ. A number of artifacts interfere with this procedure, and steps have been taken to overcome them. Antibody is localized by this method in plasma cells. The number of grains over an individual cell is proportional to the amount of radioactive material it has fixed, so that the method is potentially capable of yielding quantitative results. [ABSTRACT FROM AUTHOR]

The migration of peritoneal exudate cells (PEC) from rats with delayed skin reactivity to diphtheria toxoid (DT) and tuberculin (PPD) was inhibited in the presence of antigen. Three days after an intravenous injection of 2.5 mg of DT there was no inhibition with DT. Seven and 28 days later migration was inhibited but less than in sensitized controls. In 6 weeks the difference disappeared. Three days after the DT challenge, the migration inhibition with PPD was weaker than in the sensitized controls. From day 7 on, non-specific reduction of migration inhibition was not detected. The 24-hour skin reactivity to DT and PPD closely paralleled the in vitro phenomena. The mechanism of desensitization appears to be the lack or a functional defect of the specific cells or a blocking factor unremovable by washing or produced during the migration assay. [ABSTRACT FROM AUTHOR]

About 50 per cent of guinea-pigs immunized with egg albumin possess strong antibody activity in an immunoglobulin class with antigenic properties different from those of the well-known guinea-pig immunoglobulins. This immunoglobulin is present in normal guinea-pig serum in very small amounts and its concentration rises during immunization with egg albumin. Immunization with bovine insulin or bovine γ-globulin does not stimulate increased synthesis of this immunoglobulin class. Owing to the special conditions that are needed for its stimulation it is provisionally called IgS. It was found to be a heat stable 6·5S immunoglobulin and is not involved in PCA reactions. [ABSTRACT FROM AUTHOR]

Normal mice were immunized with picryl chloride. 51Crk-labelled normal lymph node cells were injected 1-10 days afterwards and the net arrival of radioactivity at the draining lymph nodes estimated. The net arrival at normal unimmunized lymph nodes was 5.3 per cent of the injected dose. This rose to 13.4 per cent 1 day after immunization and then gradually declined. This increased arrival caused by picryl chloride was reduced in mice rendered unresponsive by the repeated injection of picryl sulphonic acid or given a single injection of picryl sulphonic acid 1-6 days before immunization. Blast cells labelled with 125IUDR from lymph nodes immunized with an unrelated agent also showed an increased arrival at draining lymph nodes. This increased arrival was reduced in unresponsive mice painted with picryl chloride. A single intravenous injection of picryl sulphonic acid increased the arrival of normal lymph node cells at lymph nodes 1 day later and this effect was also diminished in mice rendered unresponsive by repeated injections of picryl sulphonic acid. The arrival of normal lymph node cells at the spleen was reduced by painting with picryl chloride or by a single injection of picryl sulphonic acid given a few days beforehand. Painting the ears of unimmunized mice with picryl chloride increased the arrival of normal lymph node cells and blast cells at the ears. However, the arrival was unaffected by pretreatment with picryl sulphonic acid. It was concluded that the increased arrival of cells in lymph nodes caused by picryl chloride and picryl sulphonic acid had both an immunological and a chemical inflammatory component. In contrast the arrival at painted ears lacked an immunological component. This provided evidence for antibody, or antigen-sensitive cells in apparently unimmunized mice which rapidly release pharmacologically active agents on exposure to antigen. [ABSTRACT FROM AUTHOR]

After intradermal injection of xenogeneic cells, antibody producing cells were demonstrated in the regional lymph nodes and peritoneal fluid as early as 5 days post-immunization. By 19 days antibody-producing cells were also found in the bone marrow, spleen and peripheral blood. They were not found in significant numbers in the thymus or non-regional lymph nodes. Antibody was demonstrable in the blood as early as 4 days post-immunization and reached maximum titre at 19 days. Both IgG and IgM were recovered from the thoracic duct lymph after intravenous injection. The significance of these findings is discussed in relation to the rejection of solid tumours and grafts. [ABSTRACT FROM AUTHOR]

This study has investigated the charge of IgG involved in antibody dependent lysis of cells by lymphocytes. The following conclusions are drawn: 1. That under certain conditions the physical alteration of IgG can result in molecules which compete with antibody bound to target cell antigens for receptors on cytotoxic lymphocytes. This phenomenon is used in a cytotoxicity inhibition test to determine the charge distribution of antibodies, which are capable of interacting with cytotoxic lymphocytes. This test is independent of the antigenic specificity of the inhibiting antibody. 2. The charge distribution of rat antibodies capable of reacting with cytotoxic lymphocytes, as assessed by cytotoxicity inhibition is similar to that of specific rat antibody capable of inducing cytotoxic activity against a xenogeneic target cell. By both criteria the isoelectric points ranged between pH 5 and 10. 3. Spontaneously occurring human antibody against a human line of target cells was found to have a highly restricted range of isoelectric points separating between pH 8 and 9.5. On the other hand assay of these sera by cytotoxicity inhibition showed a normal distribution of total antibody capable of reacting with cytotoxic lymphocytes. It is suggested that a likely explanation of these findings is that the human antibody with specificity for an allogeneic strain of target cells is cross reacting with a single antigen on the target cells and that the antibody from specifically immunized rats is likely to be against multiple antigens. [ABSTRACT FROM AUTHOR]

Five immunoglobulin classes designated γ2a, γ2b, γ2c, γ1, and γA were identified in the middle peak of Sephadex G-200 gel filtrate of mink sera. γ1 globulin and γA globulin appeared to share: an antigenic determinant which was not present on the γ2 globulins. Radioimmunoelectrophoresis revealed weak antibody activity of γ2 globulins but strong antibody activity of γ1, and γA globulins in immune sera of two mink immunized with bovine serum albumin. [ABSTRACT FROM AUTHOR]

The arrival of cells from normal and immunized lymph node cells at sites of inflammation was studied. Mice were immunized with ‘oxazolone’ or picryl chloride and 3–4 days later the draining lymph node cells were dissociated, labelled in vitro with 51Cr and injected intravenously into recipients. Sites of inflammation were produced in the recipients by painting the ear with chemically reactive contact sensitizing agents or croton oil. The net arrival of of normal lymph node cells at sites of inflammation was 0.1 per cent. In contrast the arrival of cells from immunized lymph nodes was 4–8 times greater. The peak number of cells that moved to sites of inflammation occurred 4 days after immunization with ‘oxazolone’. An increase in the percentage of cells that moved to sites of inflammation occurred in lymph nodes after immunization with a number of agents including contact sensitizing agents, skin grafts and Freund's complete adjuvant. There was little or no increase in mice rendered unresponsive to picryl chloride and then immunized with picryl chloride or in mice injected with aluminium hydroxide, alum precipitated mouse serum or pneumococcal polysaccharide. This suggested that immunization and possibly the induction of delayed hypersensitivity was necessary for the generation of these cells. The arrival of the cells did not depend on antigenic similarity between the agent used to immunize the lymph node and the agent used to produce inflammation. For example increased arrival of cells from immunized lymph nodes occurred at sites of inflammation produced in the car by croton oil or in the peritoneal cavity by paraffin oil. [ABSTRACT FROM AUTHOR]

The primary antibody response of the IgM type against sheep red blood cells, by spleen cells obtained from mice immunized in vivo has been studied at the cellular level. The modified plaque assay technique which was used allows the quantitative recording of plaque formation and growth in a carboxymethylcellulose gel. The distribution of plaque size at different times of incubation indicates that the secretion rate and the final amount of antibody released differ considerably between individual PFC of the same splenic population. However, the mean sizes of plaques from splenic populations at the same time after immunization are similar from mouse to mouse. During in vitro incubation, the kinetics of plaque growth display characteristic features, thus differentiating PFC populations from mice examined at different times after in vivo immunization. Most notably, there is a decrease in the mean size of the plaques between day 4 and day 5 following immunization. These results are discussed in terms of antibody secretion by PFC populations during the primary response. [ABSTRACT FROM AUTHOR]

Antigenic competition between the Fc and Fab fragments of rabbit IgG in mice could be abolished by passive immunization with antiserum against the dominant antigen (Fc). The induction of tolerance to Fc also eliminated antigenic competition. Anti-Fd production, generally very poor in response to rabbit lgG, was considerably enhanced by these procedures, indicating that it is also subject to antigenic competition with Fc. A model is proposed to explain antigenic competition which proposes that it is the 'co-operative antibody' produced to the dominant antigen which acts as an inhibitor to antibody production to the suppressed antigen by competing for sites —presumably on macrophage membranes—where cooperation occurs. The merits and difficulties of such an explanation are discussed. [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]

The synthesis of antibodies and non-specific immunoglobulins by the rabbit spleen perfused with culture medium in vitro has been studied using the incorporation of [14C]glycine. Separate perfusion of the two halves of the same spleen provided an opportunity to determine the influence of several factors on the synthesis of these proteins.It was shown that changes in the rate of synthesis of antibodies and non-specific immunoglobulins in the perfused spleen are in some cases similar to the changes observed in the whole organism, and in some cases they are not. Thus, the synthesis of antibodies in the spleen withdrawn 2 days after secondary immunization increased during the 2 days of perfusion and in the spleen taken 4 days after immunization it decreased in the same way as in the whole organism. However, in the spleen taken 3 days after secondary immunization, the synthesis of antibodies during the perfusion period remained unchanged. Another distinction from the processes occurring in the living body was that the increased rate of antibody synthesis in the perfused spleen was not associated with an increase in nonspecific immunoglobulins synthesis. [ABSTRACT FROM AUTHOR]

Ox red cell antigens F, V, Ε1, J, L, R', S' and T' have been recovered in soluble membrane protein obtained by a butanol extraction method. F, V and Ε1 have been studied in more detail and are found in the sialic acid rich fraction of the membrane protein. Isoimmunization with protein containing F and V antigens produced excellent specific antisera for these antigens. Antigens lost in butanol extraction were not destroyed by sonication, and it has not been possible to regenerate activity for these lost antigens. [ABSTRACT FROM AUTHOR]

The transmission of antibodies across the gut of suckling pouch-young was investigated in three species of rnarsupials (Setonix brachyurus, Macropus eugenii and Trichosurus vulpecula) from Australia. Mother Setonix, immunized against Salmonella adelaide flagella and Bacteriophage φ × 174, transmitted the antibodies in milk to their young. In sucrose density gradient runs, the antibody activity in milk whey and in serum of pouchyoung, of Setonix and Macropus was found to be in the 7S region only; antibody in the 11S and 19S regions was not detected. Chromatographic preparations of IgM antibodies were fed to pouch-young Setonix which were later bled and their serum titrated for anti-S. adelaide agglutinins and antiphage φ × 174 activity, The IgM antibodies were not transmitted across the gut in detectable amounts. Antibodies were present in the blood of pouch-young Setonix within 15-60 minutes of gavage (feeding by stomach tube) of immune serum. In Setonix the capacity to absorb antibodies in the intestine was lost at an age between 170 and 200 days and in Trichosurus it was lost at an age between 98 and 145 days. At these ages the pouch-young were able to leave the marsupium for varying lengths of time. Antibodies did not traverse the rumen wall in a young Setonix whose rumen was isolated from the intestine with ligatures before immune serum was gavaged. [ABSTRACT FROM AUTHOR]

An evaluation of the specificity of antigen binding receptors possessed by cells involved in cellular immune responses and by cells in the antibody synthesizing line was made in order to determine to what extent each cell type could distinguish between closely related mono-ε-DNP-oligo-L-lysines. The antigen binding receptors of cells involved in cellular immune responses were studied by the elicitation of delayed hypersensitivity reactions in vivo and by the stimulation of DNA synthesis by lymph node cells in vitro using 1-ε-DNP-nona-L-lysine and 9-ε-DNP-nona-L-lysine. These experiments demonstrated that closely related compounds could be distinguished by this cell population. On the other hand, serum antibody from animals immunized with 1-ε-DNP-nona-L-lysine did not regularly distinguish between various mono-ε-DNP-oligo-L-lysines of differing immunogenicity or between 1-ε-DNP-nona-L-lysine and 9-ε-DNP-nona-L-lysine. Serum antibody specificity is assumed to reflect the specificity of the receptors possessed by the precursors of antibody producing cells. Thus, it appears likely that the cells involved in cellular immune responses or functioning as 'helper' cells in the stimulation of antibody synthesis by other cells are capable of determining the immunogenicity of a compound. On the other hand, precursors of antibody producing cells, at least in primed animals and in the mono-ε-DNP-oligo-L-lysine response, do not appear to make distinctions between peptides of differing immunogenicity. [ABSTRACT FROM AUTHOR]

A detailed evaluation of the capacity of mono-ε-DNP-oligo-L-lysines to initiate anti-DNP antibody synthesis and a state of delayed hypersensitivity in guinea-pigs is presented. Peptides containing as few as two lysine residues elicit the production of significant amounts of anti-DNP antibody when they are administered as Freund's complete adjuvant emulsions. Under these immunization conditions, the serum concentration of anti-DNP antibody is dependent on the chain length of the peptide, and on the amount and kind of mycobacteria in the adjuvant; guinea-pigs lacking the PLL gene produce amounts of anti-DNP antibody indistinguishable from that produced by guinea-pigs possessing this gene. On the other hand, when guinea-pigs are immunized with either 1-ε-DNP-tetra-L-lysine or 1-ε-DNP-nona-L-lysine without the use of mycobacterial adjuvant, anti-DNP antibody is produced only by guinea-pigs receiving the nona-L-lysine and only by those animals possessing the PLL gene. Delayed hypersensitivity results front immunizing PLL+ guinea-pigs with mono-ε-DNP-octa and nona-lysines but not from immunization with mono-εDNP-hexa-lysine; PLL- animals do not exhibit delayed hypersensitivity to any of these compounds. The data suggest that antibody synthesis to positively charged compounds may proceed as a result of formation of charge complexes with mycobacterial proteins; under such immunization conditions the intrinsic immunogenicity of a compound is more reliably revealed by the induction or elicitation of cellular immune responses. On this basis, a definite discontinuity in the degree of immunogenicity of the mono-ε-DNP-oligo-L-lysines occurs as the peptides are lengthened from six to eight residues. [ABSTRACT FROM AUTHOR]

Uranium acetate has been shown to be an effective positive stain for examining thin sections of ferritin-rabbit anti-ferritin complexes in the electron microscope. Although this procedure does not reveal the fine detail of the reactants it does appear to be a useful staining method. Stained sections of equivalence complex revealed some rather large antigen-free areas which are reduced in size or completely removed by exposure to acid. The stained material in these areas appears to consist of auto-antiglobulins which are produced in rabbits experiencing long term immunization with ferritin. [ABSTRACT FROM AUTHOR]

By means of rabbit anti-human thrombocyte antibodies, two organ specific antigens of human thrombocytes were detected. Both antigens were found to be specific for human thrombocytes and did not cross-react with thrombocyte antigens of other species. Separation and purification of the antigens were achieved by ion exchange chromatography and polyacrylamide gel electrophoresis. Production of monospecific antisera against each antigen separately was obtained by using purified antigen preparations for immunization. Physicochemical studies showed that both antigens are thrombin resistant, trypsin sensitive, and denatured at temperatures of 100° but not at 56°. One antigen is a glycoprotein with an αglobulin electrophoretic mobility and a molecular weight of approximately 1.071.17 × 105, while the other is a protein with faster mobility in the post-albumin region and a molecular weight of 2, 8-3, 0 × 104. Both antigens are present in soluble form in the cell sap as well as insoluble components in the cell membranes but only one antigen, the glycoprotein, could be solubilized from the platelet membranes by sodium dodecyl sulphate and pyridine treatments. [ABSTRACT FROM AUTHOR]

The primary and secondary immune responses of toads (Bufo marinus) were studied using the bacteriophage f2 as antigen. Antibodies were identified in the serum 2 weeks after primary immunization and peak antibody levels were reached at 6 weeks. Although both IgM and IgG antibodies can be found in toad serum, most of the antibody activity in our animals persisted in the IgM fraction until 8 weeks after immunization. When a second injection of antigen was given 4 weeks after primary immunization, there was a marked increase in the total serum antibody activity and IgG antibodies were found as early as 4 weeks after the second injection of antigen. These observations suggest that the toad, B. marinus, is capable of a true secondary response to the bacteriophage f2. [ABSTRACT FROM AUTHOR]