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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]

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]

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]

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]

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]

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]

Spleen cell populations producing haemolytic antibody or exhibiting immunocyto-adherence (ICA) to sheep erythrocytes have been studied during ontogeny of the chicken The proportion of ICA cells in spleens of immunized chickens increased steadily from 1 to 12 weeks of age. In immunized adults, as many as 8 per cent of spleen cells exhibited ICA; this was only ten times the number of similarly active cells in unimmunized adults. In immunized chickens, only a comparatively minor population of spleen cells were engaged in producing haemolytic antibody. There was a sudden increase in plaque-forming cells during 8-10 days after hatching but no further increase in the proportion of such cells during the remainder of development. It is suggested that immunological maturation in the normal animal may be due to cumulative antigenic experience during the first few months of development. Immunological maturation is expressed as an increse in the size of the pool of immunologically competent cells. Increases in population of antibody-forming cells from this pool may occur at different stages in ontogeny according to the type of immunity elicited by antigen. [ABSTRACT FROM AUTHOR]

A novel type of immunosuppressive agent, 3-acetyl-5-(4-fluorobenzylidene) 4-hydroxy-2-oxo-2:5-dihydrothiophen (I.C.I. 47,776) inhibits the production of antibodies in rats when given orally. The treatment is still effective when commenced several days after immunization. No effect has been demontrated in mice or in guinea-pigs. [ABSTRACT FROM AUTHOR]

A series of multiple intravenous injections of bovine serum albumin, starting with doses too small to be immunogenic, has been used for the induction of tolerance in adult rabbits. With normal outbred animals, approximately 50 per cent of the rabbits were rendered completely tolerant by an injection schedule with 0·1 mg as the initial dose and a total dose of 9 mg given over a period of 5–7 weeks. The mechanism of this ‘low-dose tolerance’ is discussed. [ABSTRACT FROM AUTHOR]

Attempts were made to immunize F1 hybrid mice to their inbred parental strains by active immunization with either living or dead parental cells and by adoptive transfer of isogenic (F1) lymph node cells to irradiated hybrids that subsequently received parental bone marrow. Cumulative mortality studies and erythrocyte serotyping revealed that many mice survived and retained their parental grafts for long periods of time. In most experiments reported here, 59Fe uptake by erythrocytes and spleens of chimeras 1 week after marrow transplantation was used as a measure of erythropoiesis and thereby of success or failure of the marrow graft. Pre-treatment (‘pre-immunization’)of hybrids with parental spleen cells produced no evidence of specific immunization. However, pre-treatment with spleen cells from parent 1 was detrimental to subse- quently transplanted marrow from parent 2 and vice-versa. Adoptive transfer of viable F1 lymph node cells failed to decrease growth of parental cells in irradiated F1 hybrids. These findings indicate that the poor-growth phenomenon of particular parent-F1 combinations cannot be explained in terms of classical immune rejection of parental cells by F1 hybrids. Instead, growth of parental cells in the F1 environment is inhibited or delayed. [ABSTRACT FROM AUTHOR]

Adult guinea-pigs injected with a small monovalent conjugate of arsanilic acid, from 2 weeks before to the day of immunization, with hapten- conjugate in adjuvant show a profound depression of hapten-specific delayed sensitivity. The same conjugate given 1–2 weeks after immunization is no longer effective. More than 95 per cent of the intravenously administered monovalent conjugate was found to be excreted in 5 days. Passively administered antibody was without effect on the subsequent development of the hapten-specific delayed sensitivity. These observations are consistent with the hypothesis that the non- immunizing intravenous injection of monovalent conjugate is paralysing to precursor cells if given first, but that once these cells are engaged by adjuvant immunization they are no longer repressible but only transiently inhibitable, as long as high concentrations of monovalent conjugate remain in the circulation. [ABSTRACT FROM AUTHOR]

Urine collected and concentrated from three individuals after tetanus toxoid immunization has been studied for antibody activity. Gel-filtration on Sephadex G-200 was used to fractionate the urine concentrates and antibody activity in the eluates was assessed by a passive haemagglutination test. Haemag­glutinating activity was demonstrated by inhibition experiments in the IgG and IgA classes of immunoglobulin but not in the IgM or IgD classes. In no case could antibody activity be ascribed to molecules smaller than intact IgG. [ABSTRACT FROM AUTHOR]

Mice thymectomized within 24 hours of birth were immunized by intraperitoneal injection of sheep erythrocytes at 6 weeks of age and again 60 days later. Serum haemolysin activity was measured at various times following antigen injection. The serum haemolysin was fractionated by sucrose gradient centrifugation into two classes. A time-course of the antibody response was constructed for total, 19S and 7S haemolysins. Both reduction in circulating antibody activity and delay in reaching maximal levels were observed.

Adult rats were immunized with human γ-globulin, in heat aggregated form intravenously, and with complete adjuvant intravenously or in the footpads. In all animals an intravenous booster injection was given on day 25. Splenectomy or sham splenectomy was performed 7 days prior to or 3 and 21 days after immunization, and 19S and 7S antibody activities were determined separately in the sera of these animals in the primary and the secondary response. Splenectomized young rats immunized with human γ-globulin in complete adjuvant intravenously or in the footpads were similarly studied. The formation of 19S and 7S antibody was affected separately depending upon the route of antigen administration and time of splenectomy. Furthermore, the inhibitory effect of splenectomy on antibody formation was more pronounced in young than in adult rats.

Immunizing injections of rat liver and kidney mitochondrial fractions were administered to rabbits, and the effects of injecting the antisera into rats were investigated. Rats receiving antiserum to rat liver mitochondria showed impaired clearance of sulphobromphthalein, and developed hepatic fibrosis which in one animal progressed to cirrhosis. There was no increase in albuminuria and the kidneys showed no histological differences from those of control rats receiving normal rabbit serum. Renal changes were virtually confined to rats receiving antiserum to rat kidney mitochondria : these animals excreted increased amounts of protein and showed increased cellularity of the glomeruli, tubular casts and hydropic changes in the tubular epithelium. The significance of these findings is discussed. [ABSTRACT FROM AUTHOR]

The ability of mouse antiserum obtained early after immunization to induce passive cutaneous anaphylaxis (PCA) using a short (4 hours) and a long (72 hours) latent period was studied. It was observed that the ability of an antiserum to induce PCA reactions after a short latent period is not correlated with its ability to induce PCA reactions after a long latent period. Furthermore PCA reactions induced 4 hours after sensitization are not abolished by heating of antisera and are completely prevented by mepyramine whereas PCA reactions induced 72 hours after sensitization are completely abolished by heating but are not prevented by mepyramine. It is suggested that antiserum obtained from mice a few days after a single dose of antigen contains at least two biologically distinct antibodies. [ABSTRACT FROM AUTHOR]

Ten previously untransfused Rh(D)-negative subjects were given an intravenous injection of approximately 0·3 ml of Rh(D)-positive red cells and at about the same time, or 24-48 hours previously, were given an intramuscular injection of 1-1000 μg of anti-D. Following the injection of the red cells there was a variable period before the onset of red cell destruction; when the antibody was injected at the same time as the red cells the delay was due partly to the time taken for the antibody to reach the circulation; when the antibody was injected at least 24 hours before the red cells there was still some delay due to the time required for the antibody to be taken up by the red cells; the delay before the onset of a maximum rate of red cell destruction varied from about 0·2 hours following the injection of 1000 μg of antibody to approximately 100 hours following the injection of 1 μg. The maximum rate of red cell destruction was calculated to be approximately proportional to the square root of the amount of antibody on the cells. After the injection of the smallest amount of antibody (1 μg) the amount on the red cells was calculated to be about 0·03 μg/ml corresponding to about ten molecules of antibody per cell. This dose produced clearance with a T½ of the order of 100 hours. The significance of these observations in relation to protection against Rh-immunization is briefly discussed. [ABSTRACT FROM AUTHOR]

Donor guinea-pigs were immunized with antigen in Freund's complete adjuvant. Three weeks later these donor guinea-pigs showed strong delayed hypersensitivity to the antigen. Peritoneal exudate cells (macrophages) and serum were then transferred intravenously from the immunized donors to normal recipients. Good 24-hour skin reactions were obtained in the recipients which had received cells and serum from donors immunized with bovine serum albumin, y-globulin, ovalbumin and haemocyanin. The reaction transferred by immune cells and serum was greater than the reaction transferred by either singly. This synergy was not found with PPD or blood group substance as antigen. Good transfers of 24-hour skin reactions to PPD were obtained with peritoneal exudate cells both alone and with serum. Good transfers could not be obtained with blood group substance. The synergic action of cells and serum in the passive transfer of 24-hour skin reactions was specific. The ability of peritoneal exudate cells to transfer 24-hour skin reactions was present at 1 week and greater at 3 weeks. In contrast the ability of serum to enhance passive transfers was absent at 1 week and present at 2 and 3 weeks. The histology of reactions passively transferred by spleen, lymph node and peritoneal exudate cells in the presence and absence of serum was studied. Serum alone produced a predominantly polymorphonuclear infiltrate which was maximal at 18-24 hours and almost absent at 48 hours. Cells alone produced only a very slight reaction. The combination of cells and serum produced lesions at 4 and 18 hours resembling those caused by serum alone. At 24 hours however the histiocytes and lymphocytes were more numerous than in the pure serum reaction and by 48 hours the infiltrate consisted of histiocytes and lymphocytes. It was concluded that, with certain antigens, there is a synergic effect of immune cells and serum in the passive transfer of 24-hour skin reactions and reasons are given for considering that these passively transferred 24-hour skin reactions are indeed delayed hypersensitivity reactions. [ABSTRACT FROM AUTHOR]

The parameters of a system for testing both actively acquired and passive immunity to the cattle lungworm in the guinea-pig are described. It was found that the passive transfer of guinea-pig immunoglobulins conferred a strong immunity. The globulins responsible for immunity were present in a serum fraction containing electrophoretically fast moving globulins and anaphylactic activity. Guinea-pigs were not protected by artificial immunization with adult worm homogenate even though precipitating and anaphylactic antibodies were produced. Artificial immunization may have failed because &gamma1 and &gamma2-antibodies were produced against irrelevant antigens or because blocking &gamma2-antibody was produced in excess.

Immunization with 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-protein conjugates, and assay of antisera by binding of N131IP-ε-amino-n-caproic acid (N131IP-aminocap), is used to investigate the response of CBA mice. Primary immunization is obtained more efficiently with alum-precipitated conjugates mixed with pertussis, than with conjugates alone or in Freund's complete adjuvant, and is most efficient with conjugates of chicken serum globulin (NIP-CG) or edestin. The mice invariably respond to efficient immunization. Secondary responses have been obtained by stimulation in vitro or in vivo of spleen cells transferred from primed donors. Cells taken from donors primed with NIP-ovalbumin respond detectably to as little as ∼0·001 μg NIP-ovalbumin/mouse, but are less sensitive to stimulation with NIP-bovine serum albumin (NIP-BSA). Secondary stimulation by NIP-conjugate is subject to competitive inhibition by NIP-aminocap. Prolonged administration of NIP-BSA or NIP-BGG renders mice partially unresponsive to immunization with NIP-CG, but the degree of immunological paralysis obtained is less than that obtained towards the carrier protein. [ABSTRACT FROM AUTHOR]

Immunization of rats with rat liver resulted in the production of antibodies which reacted with autologous liver, as determined by haemagglutination. The antisera were also capable of reacting with heterologous and homologous liver. The specificity of the reaction with autologous liver was confirmed by fluorescence microscopy. Rat anti-rabbit liver antisera showed the same specificity for rat liver when tested with other rat organs. These antisera were also capable of reacting with autologous rat livers. [ABSTRACT FROM AUTHOR]

Rats killed 24 hours after a single injection of 30–100 mg/kg Isoproterenol (N-iso-propyl noradrenaline), show focal myocardial necrosis. Animals previously immunized against homologous (rat) heart homogenate showed a marked increase in severity and extent of the lesions. Immunization against rabbit heart caused a lesser degree of enhancement; rat kidney, rabbit serum and kidney and saline-adjuvant emulsion as antigens, gave results similar to the controls. There was no correlation between the level of circulating anti-heart antibodies, as measured by passive haemagglutination, and the severity of the myocardial necrosis. Within each treatment group, however, animals with higher titres tended to show greater myocardial damage. It is suggested that circulating anti-heart antibodies, normally non-toxic in vivo, become cytopathogenic after the myofibre cell barrier is broken down by Isoproterenol. [ABSTRACT FROM AUTHOR]

The antigen-elimination test has been calibrated by means of passive immunization experiments. The effect of altering the amounts of antigen or antibody used in this test has been demonstrated. Experiments have been carried out to investigate the possibility of inducing immunological paralysis in mice immune to BGG. It was found necessary to start with an injection of 300 mg BGG, and follow this by a course of twice-weekly injections of exponentially (×2) decreasing amounts. X-irradiation increased the ability of antigen to paralyse. The difference between non-immunogenic antigens (BGG in mice) and immunogenic antigens (BSA, etc.) is discussed. The possible role of intracellular antibody in determining the amount of antigen necessary to induce immunological paralysis is also discussed. [ABSTRACT FROM AUTHOR]

Strong 24 hour skin reactions occur in guinea-pigs immunized with antigen in Freund's complete adjuvant. These reactions were reduced by the injection of 1 mg of the same antigen, in either the soluble or alum precipitated form, 14 days before immunization with antigen in Freund's complete adjuvant. There was also a reduction in the corneal reaction, which has been regarded as an index of delayed hypersensitivity. This phenomenon was called immune deviation. The phenomenon was demonstrated for bovine γ-globulin, human serum albumin, bovine serum albumin, egg albumin, diptheria toxoid, hacmocyanin, purified protein derivative (PPD) and dinitrophenylated proteins. Ten mg of soluble bovine γ-globulin caused considerable reduction of the circulating antibody level and of the 24 hour skin reaction. Alum precipitated bovine γ-globulin and smaller doses of soluble antigen reduced the skin reaction but had less effect on the antibody level. Similar results were obtained with human serum albumin. This suggested that the conditions for eliciting immune paralysis and immune deviation were different. Immune deviation was obtained with either footpad or intravenous injections and with as little as 100 µg of antigen. Alum precipitated bovine γ-globulin caused immune deviation when given 14, 7 or 1 day before or 1 day after immunization with antigen in Freund's complete adjuvant. It was inactive when given 6 days after immunization. Splenectomy had no effect on the production or deviation of 24 hour skin reactions. Alum precipitated antigen had a variable and usually slight effect on the level of antibody following immunization with the same antigen in Freund's complete adjuvant. There was, however, a qualitative alteration in the antibody. The sera of guinea-pigs, which had been deviated by a prior injection of bovine serum albumin or bovine γ-globulin, showed only a gamma;1 line of antibody on immunoelectrophoresis, while the sera of control guinea-pigs also showed the γ2 line characteristically seen in guinea-pigs immunized with antigen in Freund's complete adjuvant. It was concluded that immune deviation was distinct from classical immune paralysis and that the immunologically specific reduction of the 24 hour skin reactions might be due, at least in part, to a selective loss of delayed hypersensitivity. [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.

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]

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]

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 lipid-A component of lipopolysaccharides, when presented in a suitable form, acts as an immunogen in rabbits, giving rise to the production of circulating antibodies. Highest anti-lipid-A titers were obtained on immunization with acid-treated bacterial cells coated with lipid A. Anti-lipid-A activity of the immune sera could be assayed by passive hemolysis of lipid-A-sensitized erythrocytes in the presence of complement. On filtration of the immune globulins on Sephadex G-200, antibodies against lipid A were detected in the IgM and IgG fractions. Lipid A preparations derived from various Salmonella and one Escherichia coli strain exhibited strong serological cross reactions with anti-lipid-A antisera. Furthermore, lipid-A antiserum also cross-reacted with all lipopolysaccharides tested, although in some cases to a low degree. On the other hand, only 8 out of 13 antibacterial antisera interacted with lipid-A-coated red cells. Finally, it was found that anti-lipid-A antibodies are effective in sensitizing E. coli O111 bacteria for intraperitoneal phagocytosis. [ABSTRACT FROM AUTHOR]

Antibodies directed against mucopeptide were obtained by immunization of rabbits with Bacillus licheniformis N. C. T. C. 6346 ceil walls extracted chemically to remove the teichoic acids. The teichuronic acid component present in whole walls or alkali extracted walk of the organism was not significantly immunogenic in rabbits. The mucopeptide antisera reacted strongly with sonicates of Bacillus licheniformis and Bacillus subtilis mucopeptides and the homologous reaction was inhibited by soluble fragments of low molecular weight derived from mucopeptide. The best inhibition of the homologous reaction was obtained with fragments containing peptide and hexosamine material isolated from the soluble products of lysozyme treatment of Bacillus licheniformis mucopeptide. Immune sera obtained from rabbits immunized with intact Bacillus licheniformis N. C. T. C. 6346 walls containing mucopeptide, teichuronic acid and teichoic acids contained antibodies directed mainly against the teichoic acids. The immune response to the mucopeptide component was greatly reduced compared to that obtained when wall preparations lacking teichoic acids were used as immunizing antigens. [ABSTRACT FROM AUTHOR]

Antibodies from a rabbit immunized with uridinediphosphate galactose 4-epimerase were shown to inhibit the specific activity of the enzyme and to precipitate [14C]UMP-Iabelled epimerase. It comprised about 0.33% of the immunoglobins and its Fab fragments had a binding constant of 0.2 × 109 M-1 at 25° and 1.1 ×109 M-1 at 4°. Despite the high energy of interaction the antibody did not liberate [14C]UMP from reduced [14C]UMP-epimerase. It gave two precipitation lanes both with the native and with the reduced epimerase in the immunoelectrophoresis. However, the same pattern was obtained with antibody from a UMP-epimerase immunized rabbit that could not precipitate [14C]UMP-epimerase or inhibit the enzymatic activity. It is not clear whether any of the lines represent the epimerase protein unit sensu strictu. [ABSTRACT FROM AUTHOR]