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Effect of secondary injections of antigen upon the retention in liver of a primary injection

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Abstract

The retention of antigen in rabbit liver tissue, resulting from a primary intravenous injection, is influenced by immunization brought about by subsequent intravenous injections of the same antigen. In rabbits given a single primary intravenous injection of radioactive antigen, the retention of radioactivity in liver tissue, after a period of 21 days, was greater than when the primary injection was followed by secondary injections of the same, but non-radioactive antigen. The results were similar for both S^(35)-azohemocyanin and S^(35)-azo-bovine-serum-albumin, except the hemocyanin was retained to a greater extent than the albumin. There was very little if any correlation between the number of secondary injections and retention of the initial injection. Quantitative antibody nitrogen data, obtained for the serum of each rabbit showed, in general, an inverse relationship between circulating antibody and radioactivity retained, i.e. the higher the circulating antibody titer, the lower the retention of radioactivity in liver tissue. Passively administered homologous antibody did not produce a change in the retention of the primary injection of antigen nor did secondary injections of a heterologous native protein injected according to the same immunization schedule as the homologous azoprotein. From these results it may be concluded that an intracellular antibody-forming activity influences the loss (or retention) of antigen deposited in liver tissue and that the mechanism is immunologically specific.

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Article
The hypothesis presented here embodies the following propositions: 1.(1) γ-Globulins are assembled by combinatorial arrangement of precursor peptides, as proposed by Markert for isozymes.2.(2) The synthesis of each peptide is governed by one gene. Thus many “isoglobulins” can be made with a few genetic loci.3.(3) The antigen selects among these isoglobulins those that best fit its determinant groups and this interaction determines the specific tertiary configuration of the antibody. Thus the number of possible antibodies is vastly greater than that of possible combinatorial arrangements of isoglobulins.4.(4) Each γ-globulin peptide represses its own synthesis.5.(5) Antibody combined with antigen cannot activate the repressor mechanism. Therefore, the formation of an antigen-antibody complex in the cell derepresses the synthesis of antibody globulin.6.(6) Immunologically competent cells, potentially capable of making a variety of γ-globulins, are thus differentiated into specialized antibody forming cells.7.(7) Immunological memory depends on the persistence in the competent cell of antigen or a product originally elicited by antigen, and on the maintenance of an adequate number of such pre-empted cells in the organism.8.(8) The kind of response to antigen—immune or tolerant—will depend at the cellular level on the antigen: antibody ratios in the cell, and at the organism level on the ratio between total amount of antigen and total number of competent cells.Some of these propositions are more speculative than others but no conclusive evidence either for or against any of them is available yet. The detailed mechanisms suggested cannot be summarized without much sacrifice of cogency or precision. No ad hoc mechanism which has no precedent in biological systems or has no precise meaning in molecular terms has been admitted.
Article
Bovine serum albumin conjugated to S/sup 35/-sulfanilic acid was injected intravenously in standard doses into three groups of rabbits: Group A (control): normal rabbits; group B: rabbits injected 18 hours after 450 r of wholebody irradiation (primary antibody response inhibited); and group C: rabbits injected immediately after 450 r of whole-body irradiation (primary antibody response not inhibited). The plasma, liver, spleen, lymph node, kidney, appendix, and lung were studied for radioactivity and protein content at 1, 7, and 21 days after injection. In all groups, the retention of radioactivity in the tissues decreased with time. The overall metabolism of the antigen by lymph node was retarded when compared to other tissues. There were no differences among all three groups in total-organ retention of antigen at all time intervals. When group A was compared to groups B and C, there were certain differences when antigen retention was expressed in terms of tissue weight or protein content. These differences were attributed to organ-weight changes secondary to irradiation. These differences did not appear when group B was compared to group C. The cells responsible for the retention and degradation of the bulk of injected antigen are not inhibited by irradiation under conditions which inhibit the primary antibody response. When large amounts of antigen are injected, only a small fraction may be important in induction of antibody formation. (auth)
Article
When rabbits were fed S(35)-labelled amino acids and simultaneously injected with antigen at the peak of antibody production, the circulating antibody became rapidly labelled within a few hours with the isotope. The specific radioactivity of antibody was measured as antibody was allowed to decline in the absence of antigenic stimulation. At various times, in different animals, antigen was reinjected and circulating antibody was measured for specific radioactivity. The initial antibody which appeared after the antigenic stimulus always had a higher specific activity than antibody circulating just prior to the reinjection. The appearance of antibody of higher specific activity was demonstrated to be a specific response to the antigen which was reinjected. It was concluded that there is a reservoir of antibody which is stabilized in tissue and which is not in equilibrium with that in circulation. A mechanism for this stabilization is suggested and discussed from previous investigations demonstrating the long retention of antigen in liver tissue.
Article
The fate of injected S(35)-labeled sulfanilic acid-azoalbumin in the serum, various organs, and liver fractions was compared in the newborn and adult rabbit and in specifically unresponsive and normal adult rabbits. Exponential decay of the injected antigen without the usual immune phase of elimination was observed in newborn and unresponsive adult animals. Comparison of organ distribution of radioactivity in adults and animals injected at birth and 21 days of age showed persistence in the liver at least as long as 3 weeks in all groups (which was the time chosen for observation). The slight differences in spleen and thymus concentrations with age are of undetermined significance. Comparison of the organ distribution of antigen in unresponsive adult rabbits and in normal ones showed slight differences which were similar to those predicted from previous immunization of adults which gave low grade antibody response. There was a slight selective accumulation of antigen in the nuclear fraction of liver homogenates of unresponsive animals, but no other differences were observed.
Article
Soluble BSA-anti-BSA complexes, formed in antigen excess, give immediate skin reactions in normal guinea pigs. The mechanism of the reaction is not that of passive or reversed passive anaphylaxis. The complex itself is toxic. Skin activity of the complex depends on its composition. It has become obvious that the complex composed of two antigen molecules and one antibody molecule, (Ag(2)Ab), does not have the activity, whereas, Ag(3)Ab(2) and more complicated complexes do. The role of complement as well as speculation on the structural changes of antibody-antigen complexes is presented.
1. A method is described for the colorimetric determination of peroxidase with N,N-dimethyl-p-phenylenediamine. The amount of red pigment formed by peroxidase is proportional to the concentration of enzyme and to the time of incubation during the first 40 to 90 seconds. The influence of the concentration of enzyme, N,N-dimethyl-p-phenylenediamine, H2O2, the time of incubation, pH, the temperature, and the possible interference by oxidizing and reducing agents of tissues has been tested. 2. The method has been used to follow the uptake of intravenously injected horseradish peroxidase by 18 different tissues of the rat over a period of 30 hours. The highest concentration of the injected tracer enzyme was found in extracts of kidney, liver, bone marrow, thymus, and spleen. Considerable amounts were taken up by pancreas, prostate, epididymis, and small intestine. Lower concentrations were found in extracts of lung, stomach, heart, and skeletal muscle, aorta, skin, and connective tissue. No uptake was observed by brain and peripheral nerve tissue. 3. Tissue homogenates containing high concentrations of the injected peroxidase, in general also showed high or average activity of acid phosphatase. 4. Six hours after intravenous administration, the liver contained 27 per cent, the kidney 12 per cent, and the spleen, 1.4 per cent of the injected dose. 5. Approximately 20 per cent of the injected peroxidase was excreted in the urine during the first 6 hours, and the concentration of peroxidase in blood serum and urine fell exponentially during this time. After 6 hours, only low concentrations were excreted in the urine but low enzyme activity was still detectable after 30 hours. Approximately 6 per cent of the injected dose was excreted in the feces from 6 to 20 hours after administration. 6. After feeding through a stomach tube, low concentrations of peroxidase were found in blood serum and urine. Considerable variations in the extent of absorption from the gastrointestinal tract were observed in individual rats.
Article
The following investigation was divided into two parts. The first was concerned with the rate and amount of excretion of soluble ^(35)S-labeled hemocyanin (KLH) and bovine serum albumin (BSA) following a single intravenous injection into normal rabbits and also with the properties of the excreted antigen material and its possible association with ribonucleic acid (RNA) or nucleotides. The second involved the release and excretion of ^(35)S-labeled material which remained in tissues, or extravascular spaces, at 7 days following the primary injection. This release was accomplished by injection of the unlabeled native protein antigen as described previously by Garvey and Campbell (1). In a recent report by Garvey and Campbell (2) the loss of antigen material from hepatic tissue following a secondary injection of the native protein carrier was clearly demonstrated by radioautography. Although much of the released material was taken up by spleen and lymph nodes, some of it escaped through the kidneys. It should be pointed out that the radioautographic studies did not bear out the speculation presented by Campbell and Garvey (3, 4) that release of primary antigen resulted in uptake by adjacent hepatic cells and thus production of a "clone." However it is possible that clones of cells might be formed in lymphoid tissues by such a mechanism of release and transfer of antigen. The level may be too low for detection but still significant with respect to antibody formation. It was expected that the present study of excreted material would give further information as to the nature of retained antigen and also provide material for future investigation of the biological properties of the released material.
Article
The distribution of 14C-DNP-BSA and 14C-DNP-PGM in the tissues of the rabbit is reported. The data show that after the advent of antibody production, antigen is retained primarily in the liver, spleen and lymph nodes. Fractionation by phenol of liver homogenates from immunized rabbits indicates that the retained form of the antigen is qualitatively different from injected antigen. Sedimentation in sucrose gradients of the ‘retained antigen’ fraction gave an average S value greater than that of the starting antigen which suggested that ‘retained fragment’ was associated with macromolecular material. Sedimentation analysis in CsCl gradient confirmed that all of the antigen present in the water phase and precipitable by alcohol after extraction with phenol was bound to RNA. Partition studies in a phenol-detergent-water system at different temperatures suggests that the RNA of the ‘Ag fragment-RNA’ complex has solubility properties characteristics of the messenger type of RNA. The relationship of fragmentation of antigen to the theory of subcomplementarity in antigen-antibody interactrions is reviewed and a new theory for the biosynthesis of polypeptide chains in γ-globulin molecules involving ‘antigen fragment-RNA’ complex and a sub-cistronic m-RNA from lymphocytes is proposed.
Article
CBA mice sensitised to A-strain antigens by ip injection of spleen cells or by skin grafting develop a form of immunity detected by their treatment of 51Cr-labelled (A × CBA) F1 lymph node cells 8 days later. The responses are a reduction in the amount of radioactivity recovered in the lymph nodes and spleens and an increased amount in the livers of sensitised mice, as compared to normal mice, 24 hr after the injection of cells. Elimination of cells takes place in sensitised lymph nodes and spleens, and the trapping of cells takes place in the livers and spleens. Trapping is mediated by immune serum, but elimination seems to involve synergic action between sensitised cells and serum.In mice grafted with A-strain skin, the responses appear at the same time as the graft is being rejected. However, the duration of the response after administration of (A × CBA) F1 spleen cells ip differs from that of homograft immunity: It is extremely persistent. In the non-H-2 system C3H → CBA ip spleen cells elicit a response, but skin grafts fail to do so.
Chapter
The purpose of some early studies from this laboratory was to isolate, by mild chemical procedures, antigen material† that had been retained in the tissues of an immunized rabbit. Liver tissue was chosen for the technical reason that the quantity of retained antigen was greater in this tissue than in any other tissue. When liver brei was fractionated with sucrose, antigen material was concentrated in the soluble fraction. Further fractionation to remove non-antigen material was effected by absorption on Dowex-2 resin and subsequent elution with sodium salicylate. Ribonucleic acid was present with the concentrated antigen, and when further fractionation was carried out with 50% ammonium sulfate, the nucleic acid and antigen remained in the soluble fraction with an Sw value of 3.6 to 4.3 (Garvey and Campbell, 1957). The fraction that contained antigen and RNA was at least 200-fold more active for in vitro anaphylactic sensitization, i.e., in the Schultz-Dale reaction than the original antigen and was tentatively regarded as a complex since neither component was dialyzable unless denatured by heat or alkali. Studies carried out over the past dozen years have amplified and extended these initial findings but determining their significance in the immune reaction still requires much work.
Article
Newborn rabbits were injected intraperitoneally with a tolerance-inducing dose of labeled radioactive bovine serum albumin. At varying times, 12 hr to 6 wk after injection, tissues were obtained for radioautography. Cellular uptake of antigen appeared confined to the liver, occurring as follows: (1) It became attached initially to erythrocytes in the intravascular system of the liver. (2) Within 1-2 days after injection, it adhered to the cells in extravascular sites where erythropoiesis persisted. (3) Still later. it was associated with hepatocytes. Erythrocytes with adherent antigen were also found in the peripheral blood throughout the period of investigation. The latter finding led to studies of the peripheral erythrocytes of the neonate; membrane properties of these were found unique compared with the adult. It is inferred that erythropoiesis in the liver represents a distinct stage in cellular development. Studies of nonforeign protein, i.e., rabbit serum albumin in neonates, strongly suggested that the erythrocyte-antigen-hepatocyte interaction in the neonate involved a pattern of cellular recognition of foreign protein. Both hematopoiesis and maturation of the liver are discussed as principal factors modulating antigen handling, antibody production, and/or its detection in the neonatal environment that led to the condition commonly defined as "tolerance."
Article
Anamnestic antihapten responses were obtained to trinitrophenyl (TNP) when rabbits sensitized to trinitrophenyl-hemocyanin (TNP-KLH) were challenged with TNP-heterologous protein conjugates. Hapten-heterologous carrier conjugates elicited antihapten titers similar in magnitude to those elicited by the homologous carrier conjugate. Hapten-heterologous carrier recall of antihapten was successful as early as 37 days and as late as 11 months after sensitization. There was no correlation between anti-TNP-precipitating antibody titer after sensitization and the ability to respond to challenge by hapten-heterologous carrier. The results are discussed in terms of immunogenicity of sensitization, suppressive effects of persisting postsensitization antibody, and submolecular haptenic environment as factors possibly affecting the heterologous recall process.
Article
This review is about the involvement of antigen, and more specifically that of antigen-RNA complexes, in the inductive phase of an immune response. Early studies are reviewed that were begun by the senior author more than twenty years ago to probe the question of the role of antigen in antibody production in rabbits. It was shown that the quantity of antigen declined with time after injection but persisted at significant levels for very long intervals of time after injection, suggesting that all the antigen may never be lost. Since the liver yielded more detectable antigen than any other tissue it was the main source of in vivo antigen for biological and chemical characterization. As more extensive experiments were undertaken these showed stability of the radioactive hapten labels used to detect antigen and also the dynamic involvement of the liver in handling antigen. A body of evidence was thus accumulating that implied, at least indirectly, an immunological role for the liver-retained antigen. As for the antigen findings, one of the earliest was that antigen persisted, not in the original form of the injected protein, but as antigen fragments associated with ribonucleic acid, from whence originated the term antigen-RNA complexes. In later studies that utilized deproteinizing techniques, aimed at obtaining pure ribonucleic acid, antigen peptide was found to persist as a moiety of the isolated nucleic acid, thus providing convincing evidence for the in vivo origin of these complexes. The antigen-RNA complexes were immunologically active by both in vivo and in vitro tests. Recent experiments, using rats injected with aniline-azo-BSA have extended the earlier findings and it is these studies upon which attention will be focused in the experimental section. Liver polysomes and isolates of pure hepatocytes have been found to contain antigen material associated with RNA, and studies are in progress using these sources of material for elucidation of antigen-RNA function. It seems timely to suggest that an equally important application may be the use of these products of in vivo metabolism as probes to study the many inductive phenomena in an immune response.
Article
A broad spectrum of literature has been reviewed dealing with diverse cellular and subcellular reactions to antigen and their subsequent effect upon immunological phenomena.A working hypothesis or model has been proposed which suggests that antigenic material may have multiple effects upon cells capable of globulin synthesis. The antigen may complex with surface globulin receptors thereby inducing an increase in the rate of globulin synthesis. Adaptive enzyme synthesis for catabolizing the complexes is initiated. In addition, fragments of the antigen may react with cellular tRNA and repress globulin synthesis by forming two specific complexes-one between the antigenic determinant and the nascent peptide; and the second between the tRNA and the ribosome bound mRNA. Derepression occurs when other nearly identical antigen-peptidyl-tRNA complexes displace the repressor molecule at one of its combining sites.These activities may be carried out in more than one cell. The induction of an increased rate of globulin synthesis occurs in the specific antigen-sensitive cell. The repressed polysomal complexes are carried in antigen-activated macrophages and lymphocytes which act as memory cells. Finally the derepressed polysomes accumulate in antibody producing plasma cells and become incorporated in the globulin synthetic mechanism of the host cell. In this manner the antigen-sensitive cell could contribute the information or code for the variable portion of the H chain in the immunoglobulin molecule. The code for the constant portion of the H chain and all of the L chain would be contributed by the plasma cell.This concept of induction, repression and derepression of subcellular polysomal units by peptidyl-tRNA complexes can be broadly applied to the regulation of protein synthesis in fields other than those of immunity and tolerance.The proposed model appears to be in agreement with many experimental observations and may be of value in designing and interpreting new experiments.
Article
Two soluble antigens, BSA and KLH labeled with sulfanilate-³⁵S, when injected intravenously into normal animals, were excreted in the urine to over 70% in 24 hr. Over the next 6 days, 25% more was excreted after which time only a trace could be detected. Much of the antigen remaining from the primary injection appeared in the urine following a secondary injection of the unlabeled protein carrier at 7 days after primary injection. The antigen material found in the urine was quite heterogeneous with respect to physical properties and much of it was associated with RNA material as shown by chromatographic analyses. The main difference between the labeled material released following the primary and secondary injection was the higher degree of association of antigen material with nucleotide material after secondary injection as compared with primary injection. Further study is needed to distinguish qualitative from quantitative changes of the components, antigen and nucleic acid, and also the nature of their association. Possible similarities were found for the RNA-antigen material released from tissue after secondary injection of unlabeled antigen, and the material that was isolated previously from liver.
Article
Soluble BSA-anti-BSA complexes, formed in antigen excess, give immediate skin reactions in normal guinea pigs. The mechanism of the reaction is not that of passive or reversed passive anaphylaxis. The complex itself is toxic. Skin activity of the complex depends on its composition. It has become obvious that the complex composed of two antigen molecules and one antibody molecule, (Ag2Ab), does not have the activity, whereas, Ag3Ab2 and more complicated complexes do. The role of complement as well as speculation on the structural changes of antibody-antigen complexes is presented.
Chapter
The chapter discusses the persistence of foreign antigenic material in tissues, its chemical and biological properties, and speculations as to its role in immune mechanisms. A native antigen molecule soon changes after entering an intracellular environment. The persistence or retention of “antigen material” is important and not the original native substance that was used for immunization. Retention of antigen such as localization of antigen in normal and immune animals, retention of antigen in normal and immune animals, and retention of antigen in unresponsive animals is also provided in this chapter. It will also be seen that the retained antigen is not only a fragment of the immunizing material but that it is combined with normal tissue components such as ribonucleic acid (RNA) and under certain conditions with its specific antibody. Information related to the retention of foreign antigen material should provide a challenge to investigators of immune mechanisms. Future studies will probably revolve around investigations of chemically modified RNA in antibody-forming cells and studies of synthetic polypeptides or copolymers of various types with regard to their antigenic behavior. Characterization of retained antigen (physical properties and biological properties) and retained antigen and antibody formation is also discussed in this chapter.
Article
A sensitive biological test has been used to detect the persistence of minute traces of a foreign protein, bovine γ-globulin, in the blood and livers of rabbits intravenously injected with it, as an antigen. At various intervals after injecting these rabbits (donors) serum or liver tissue was transferred from them to the peritoneal cavities of normal or unilaterally adrenalectomized mice (recipients) with the aim of rendering the latter hypersensitive to the antigen that might be persisting in the transferred materials; a state of affairs detectable, 2 days later, by the appearance of signs of reversed passive anaphylaxis when the recipient mice were intravenously challenged with a strong anti-bovine γ-globulin rabbit serum. The protein persisted in the blood of the donor rabbits, in readily demonstrable amounts for 1 month, and in the blood of one animal, in minute traces, or as long as 6 weeks. It was detectable in the livers for 8 weeks. The persistence of bovine γ-globulin in rabbits, which form circulating antibodies to it well, is not as long as that in mice, which form antibodies to it poorly, since in previous work with the mouse the antigen was found (1) in the blood after 8 weeks and in the liver for 14 weeks. Nevertheless the antigen persists in the rabbit much longer than is generally supposed. Indeed it can be found in the liver all through the period in which circulating antibody is demonstrable in the blood. Explanations for the phenomenon have been suggested. Its significance in relation to the mechanisms of antibody formation is obvious.
Article
1. The foregoing experiments show that significant differences in tissue localization of antigen occurred in rabbits when the titer of circulating antibody was greater than 0.1 mg N. 2. The increased uptake of antigen by the lungs of immune animals may be largely attributed to hypersensitive reaction of this tissue whereas the greater localization in the liver represents complexing of antigen with circulating antibody. 3. A method of acid-salt dissociation was found useful in isolating the antigen and antibody from the immune complexes obtained as insoluble material in a perfusate from liver tissue. 4. There was no apparent redistribution of antigen when the usual 6 hour test period for localization was extended to 72 hours.
Article
A sensitive biological test has been used to detect the persistence of minute traces of a foreign protein, bovine gamma-globulin, in the blood and livers of rabbits intravenously injected with it, as an antigen. At various intervals after injecting these rabbits (donors) serum or liver tissue was transferred from them to the peritoneal cavities of normal or unilaterally adrenalectomized mice (recipients) with the aim of rendering the latter hypersensitive to the antigen that might be persisting in the transferred materials; a state of affairs detectable, 2 days later, by the appearance of signs of reversed passive anaphylaxis when the recipient mice were intravenously challenged with a strong anti-bovine gamma-globulin rabbit serum. The protein persisted in the blood of the donor rabbits, in readily demonstrable amounts for 1 month, and in the blood of one animal, in minute traces, or as long as 6 weeks. It was detectable in the livers for 8 weeks. The persistence of bovine gamma-globulin in rabbits, which form circulating antibodies to it well, is not as long as that in mice, which form antibodies to it poorly, since in previous work with the mouse the antigen was found (1) in the blood after 8 weeks and in the liver for 14 weeks. Nevertheless the antigen persists in the rabbit much longer than is generally supposed. Indeed it can be found in the liver all through the period in which circulating antibody is demonstrable in the blood. Explanations for the phenomenon have been suggested. Its significance in relation to the mechanisms of antibody formation is obvious.
Article
The S^(35)-label of S^(35)-BSA was detected in the liver tissue of rabbits to the extent of 0.02 per cent (10 µg or ≃ 10^(14) molecules) of the injected material at 140 days after injection. The rate of loss of antigen at the termination of the experiment was of such an order that significant amounts would be expected to persist for at least several years. Data are reported which extend the retention data previously reported on S^(35)-labelled hemocyanin. They indicate that amounts of the order of 0.05 per cent (25 µg.) of antigen material persist at 330 days after injection. All of the radioactivity of material retained in the liver tissue 6 weeks after injection was immunologically related to the original S^(35)-BSA antigen. Preliminary studies are reported which indicate that the retained antigen is bound to ribonucleic acid. A new method is described for the isolation of p-azophenylsulfonate bovine serum albumin from tissue extracts by means of a Dowex 2 adsorbent.
Article
1. The catabolism of I∗BGG in immune rabbits is much more rapid than in non-immune rabbits, especially within the first 6 hours. 2. Only specific antibody is necessary for the rapid immune catabolism, as indicated by the identical rates of I∗BGG catabolism in actively and passively immunized rabbits.
Catabolism of I131-1abeled bovine gamma globulin in immune and non-immune rabbits The persistence of bovine v-globulin injected as an antigen into rabbits, f
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  • D W Talmage
  • P D Mcmaster
  • It Kruse
  • E Sturm
  • J L Edwards
Dixon, F. J. and Talmage, D. W., Catabolism of I131-1abeled bovine gamma globulin in immune and non-immune rabbits, Proc. Soc. Exp. Biol. and Med., 1951, 78, 123. 8. McMaster, P. D., Kruse, It., Sturm, E., and Edwards, J. L., The persistence of bovine v-globulin injected as an antigen into rabbits, f. Exp. Med., 1954, 100,