Localization of 125I-labelled alpha-r-atrial natriuretic peptide in rat tissues by whole-body and microautoradiography

125I-labelled alpha rat atrial natriuretic peptide (28 amino acids: Ser 99-Tyr 126) ([125I]alpha-rANP) was given i.v. to Sprague-Dawley rats and the distribution of radioactivity in the tissues was examined by whole-body and microautoradiography at intervals from 2 min to 4 h after the administration. Inhibition of uptake of the [125I]alpha-rANP by simultaneous injection of an excess of non-labelled alpha-rANP was taken as an indication that highly labelled structures in rats injected with [125I]alpha-rANP alone are due to an abundance of specific receptors for the peptide. In the rats given only the [125I]alpha-rANP a rapid and high radioactivity occurred in the renal glomeruli, the endocardium of the heart ventricles, the endothelium of the processus ciliares of the eyes, the portal vessels and a few larger vessels of the liver, the subcapsular vessels of the adrenal glands and the parenchyma of the lungs. Other tissues showing a distinct, but less prominent, radioactivity were the endocardium of the heart atria, the walls of the great afferent and efferent vessels in the thoracic cavity, the choroid plexuses of the brain ventricles, the pia mater, brown fat, the muscularis layer of the stomach and the intestines, the lamina propria of the villi in the small intestine and the walls of a few small blood vessels in the kidney medulla. The specific labelling was highest at 2 min after injection and then diminished at later intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of 125I-labelled protein A for the detection of humoral immunity to gross murine leukaemia virus

A solid-phase radioimmunoassay utilising bind of 125I-labelled protein A to antibodies bound to virus adsorbed onto microtitre plates was shown to be suitable for detection of humoral immunity to Gross murine leukaemia virus (MuLV). The specificity of the reaction was shown by the fact that only homologous or closely related viruses effectively inhibited binding of antibodies to adsorbed virus. With this method a low level of spontaneous humoral immunity was demonstrated in sera from AKR/Crc mice, a strain with high concentrations of endogenous virus, whereas little or no anti-viral activity was found inCBA/H-T6Crc, a subline that does not appear to express MuLV.     

A microassay for antibody binding to tumor cell surface antigens using 125I-labelled protein a from Staphylococcus aureus.

A sensitive assay for antibodies bound to the surface antigens of adherent tumor cells using protein A from Staphylococcus aureus is described. Cells, as monolayers in microtest wells, are incubated first with immune or control serum, and then with 125I-labelled protein A (IPA), which binds to IgG antibodies bound to cell surface antigens. The IPA assay is shown to be more sensitive than the conventional isotopic antiglobulin (IAG) assay, because IPA binds to a lesser extent to IgG bound non-specifically to cells.     

Localization of 125I-labelled antigen in germinal centres of mouse spleen: effects of competitive injection of specific or non-cross-reacting antigen

Studies were performed on localization of ¹²⁵I-human γ-globulin in spleen lymphatic tissue germinal centres during the primary and secondary immune response as influenced by competitive injections of specific or non-cross-reacting antigens. Isologous mouse 7S serum protein labelled with ¹²⁵I was used as the control. The results of these studies support the following conclusions:

(1) Antigen retention in germinal centres during the primary immune reaction is a dynamic process. For some antigens there may be opsonins available at the the time of injection which promote initial localization in germinal centres. However, the continued localization of antigen over weeks and months is a function of specific antibody production.

(2) For some period of time, germinal centres are specific to the antigen that stimulated their development, and eventually these centres will respond to a different antigen.

(3) Antigen persisting in germinal centres is functional in the development of the secondary immune potential.

     

Phylogenetic studies on the immune response: I. Localization of antigens and immune response in the toad, Bufo marinus

Toads of the species Bufo marinus were injected subcutaneously with ¹²⁵I-labelled flagella from Salmonella adelaide. Observations were made on the ensuing serum antibody response, the antigen localization pattern and sequential cellular changes in lymphatic and other tissues.

The serum antibody findings confirmed the work of previous investigators in showing a good primary response, prolonged synthesis of mercaptoethanol sensitive antibody and little or no evidence of secondary responsiveness.

Antigen became localized in the jugular bodies and spleen where proliferation of pyroninophilic cells could be observed after 5 days. Both the antigen-trapping cells and the first pyroninophilic blasts were scattered randomly throughout the jugular bodies. There was no clear-cut separation into cortex and medulla. Nothing resembling the antigen-trapping web of rat lymph node follicles was observed, nor were there any germinal centres. In the spleen, antigen was trapped in the red pulp and some degree of concentration around the islands of white pulp could be noted 1 day later. However, unlike in the rat, entry of antigen into the white pulp did not occur.

Both focal and diffuse collections of lymphoid and pyroninophilic cells were found in the kidney after antigenic stimulation. It seems likely that the kidney is a major antibody-forming organ in the toad.

The hypothesis is advanced that the absence of immunological memory may be due to the absence of the follicular antigen-trapping web and of resultant germinal centres.

     

Antigens in immunity. VII. Analysis of immunological memory

Rats were given primary and secondary injections in saline solution without adjuvants, of Salmonella flagellar antigens, the dose varying from 10 pg (10^-11g) to 1 mg. Rats were bled at various times after injection, and levels of both total (7S+19S) and mercaptoethanol resistant (7S) anti-H antibody were determined, using a serial two-fold dilution assay. The data from several thousand such titrations was entered on IBM punched cards and a series of programs were written to allow computer analysis thereof.

The chief findings which emerged from the study were:

(1) Important differences exist in the kinetics of primary and secondary responses.

(2) The excess antibody formation characteristic of the secondary response is essentially a short-lived phenomenon.

(3) Conditions may be defined for the demonstration of excellent secondary responses where the additional antibody formed is solely 19S.

(4) Secondary responses are evoked only when the second dose of antigen equals, or preferably exceeds, the first dose.

(5) Peak antibody titre: antigen dose curves differed for primary and secondary responses.

(6) Memory can be quantitated in a variety of ways. The hypothesis that memory stems from a series of cellular events induced by antigen but independent of actual antibody formation is discussed.

(7) Optimal conditions for the study of the role of antigen in the secondary response were established.

     

Localization of 125I-labelled α-r-atrial natriuretic peptide in rat tissues by whole-body and microautoradiography

¹²⁵I-labelled α rat atrial natriuretic peptide (28 amino acids: Ser 99–Tyr 126) ([¹²⁵I]α-rANP) was given i.v. to Sprague—Dawley rats and the distribution of radioactivity in the tissues was examined by whole-body and microautoradiography at intervals from 2 min to 4 h after the administration. Inhibition of uptake of the [¹²⁵I]α-rANP by simultaneous injection of an excess of non-labelled α-rANP was taken as an indication that highly labelled structures in rats injected with [¹²⁵I]α-rANP alone are due to an abundance of specific receptors for the peptide. In the rats given only the [¹²⁵I]α-rANP a rapid and high radioactivity occurred in the renal glomeruli, the endocardium of the heart ventricles, the endothelium of the processus ciliares of the eyes, the portal vessels and a few larger vessels of the liver, the subcapsular vessels of the adrenal glands and the parenchyma of the lungs. Other tissues showing a distinct, but less prominent, radioactivity were the endocardium of the heart atria, the walls of the great afferent and efferent vessels in the thoracic cavity, the choroid plexuses of the brain ventricles, the pia mater, brown fat, the muscularis layer of the stomach and the intestines, the lamina propria of the villi in the small intestine and the walls of a few small blood vessels in the kidney medulla. The specific labelling was highest at 2 min after injection and then diminished at later intervals. Several of the labelled structures are localized in tissues involved in the regulation of blood pressure, and fluid and electrolyte homeostasis, processes in which the atrial peptides are considered to play a role. It is suggested that high concentrations of receptors are present at the sites at which the [¹²⁵I]α-rANP was strongly localized and that biological effects of the atrial peptides are exerted via these structures.     

Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response.

An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.     

Dynamics of 125I-labelled horse serum albumin presentation in vivo in guinea pigs.

Low antibody production in guinea-pig sera was determined by passive hemagglutination after 125I-labelled horse serum albumin (HoSA) injection. The appearance of radiolabelled HoSA on T cells, B cells and monocytes/macrophages (Mo) of guinea pigs was detected and followed as a function of time. The radioactivity peaks appeared first on B cells and Mo, later on the T cells. The circulating T lymphocytes contained labelled antigen 7 days after the injection while T and B lymphocytes in the spleen preserved their radioactivity 15 and 20 days later. Helper, suppressor and effector T cells were able to fix 125I-HoSA, this was shown by autoradiography using monoclonal antibodies 4 days following the antigen injection.     

Uptake of 125I-labelled C3a by cultured human endothelial cells.

The interactions of C3a anaphylatoxin with vascular endothelium were studied in vitro using human endothelial cells in culture and 125I-labelled human C3a. Cultured endothelial cells took up 125I-C3a in a time- and concentration-dependent manner and inactivated it. Uptake was not associated with binding to specific receptors since the amount of radioactivity accumulated by the cells was not influenced by treatment with excess unlabelled peptide, metabolic inhibitors or by low temperature. Further, we observed that uptake was not saturated during 90 min of incubation or within the concentration range of C3a tested (10(-9)--10(-6) M). C3a was taken up more rapidly than other labelled, less basic compounds, including Tyr5-bradykinin, lysozyme and albumin. Examination of the cells by autoradiographic electron microscopy revealed labelled material within the cell cysoplasm but not within specific intracellular structures, such as vesicles or vacuoles. C3a was partially inactivated after incubation with endothelial cells for 15 min, but some spasmogenic activity was retained even after 90 min incubation. Since the peptide is readily inactivated by the cells, the radioactivity in the cytoplasm may be inactive C3a and possibly C3a fragments. The combination of uptake and inactivation of C3a by endothelial cells may be an effective means of removing the peptide from circulation.     

A radioimmunoassay for virus antibody using binding of 125I-labelled protein A.

An assay for virus antibodies using protein A from Staphylococcus aureus is described. Type B and type C RNA tumour viruses adsorbed on to polystryrene microtitre plate wells were incubated with antiserum and then with 125I-labelled protein A (I-pA) and bound radioactivity was determined. Technical details such as labelling, antigen concentration, storage of I-pA are reported. The specificity of the reaction was investigated in detail by competition experiments with purified unbound homologous viruses. This assay also proved to be sensitive for demonstration of autogenous immunity to both type B and type C RNA tumour viruses. A study using antisera against purified core and envelope virus proteins of mammary tumour and leukaemis viruses suggested that the reaction mainly involves surface antigens of the intact virions.     

Localization of brucella antigens that elicit a humoral immune response in Brucella abortus-infected cells.

Localization of brucella antigens, to which brucella-infected cattle make antibodies, and the surface characteristics of Brucella abortus smooth strain 19 and rough strain 45/20 were studied by the use of monospecific antisera in absorption tests, electron microscopy, and electrophoretic mobility of organisms in microelectrophoresis. Antigenic determinants of electrophoretically defined antigen A5 were present on the surface of B. abortus rough strain 45/20 organisms, and protein moieties were most probably exposed on the surface of this strain in contrast with smooth strain 19 organisms. Several antigens distinct from the smooth lipopolysaccharide complex, to which brucella-infected cattle make antibodies, were not detected on the surface of smooth organisms. Agglutinating antibodies present in anti-B. abortus strain 19 serum did not bind to all areas on the surface of the smooth cells, suggesting the presence of different antigenic moieties on their surface. It is also postulated that the surface of B. abortus rough strain 45/20 displays receptors able to strongly bind immunoglobulin molecules, as well as other serum components.     

Studies on the control of antibody synthesis. VIII. Selection for high affinity antibody production in the secondary response.

Priming with haptenic determinants can augment the antibody response to a new haptenic determinant presented on a different carrier to which the original haptens are also coupled. This augmentation may be accompanied by a slight increase in the affinity of the antibody formed. However, only if the test hapten is present on the original priming antigen is there a selection for high affinity antibody-forming cells and a marked increase in the affinity of the antihapten antibody produced upon boosting is seen. The data are consistent with the assumption that antibody affinity is primarily controlled by the selection of B lymphocytes by antigen, while additional factors may be involved in controlling the magnitude of the response.     

Role of plasmid-encoded antigens of Yersinia enterocolitica in humoral immunity against secondary Y. enterocolitica infection in mice

In the present study the role of Yersinia enterocolitica antigens in humoral immunity against secondary Y. enterocolitica infection has been investigated. For this purpose, BALB/c mice, 4 weeks after immunization by primary infection with a sublethal dose of various Y. enterocolitica strains and mutant strains, were challenged with a lethal dose of highly virulent Y. enterocolitica strains of serotype O:8. As evident from the survival rate and protection against bacterial growth in the spleens of mice, only immunization with wild-type or attenuated strains harbouring an intact virulence plasmid mediated protection against a lethal challenge. Protection by immunization with plasmid-harbouring strains correlated with persistence of the bacteria in spleens for at least 7 days after immunization and high serum titres of Yersinia-specific antibodies directed against both chromosomal and plasmid-encoded determinants. Furthermore, the adoptive transfer of the purified IgG fraction of a rabbit serum specific for the adhesin YadA encoded by virulence plasmid pO8 from Y. enterocolitica O:8 mediated protection against a lethal challenge with the serotype O:8 strain harbouring the virulence plasmid pO8 but did not mediate protection when this strain harboured the virulence plasmid pO9 from serotype O:9. In summary, the results demonstrate that in our experimental mouse infection model: (i) the presence of the intact virulence plasmid in an immunizing Y. enterocolitica strain is essential for induction of protection against a lethal challenge with highly virulent Y. enterocolitica and (ii) that antibodies against plasmid-encoded determinants of Y. enterocolitica, especially YadA, are of major importance for achievement of protective immunity in mice.     

Measurement of the clearance function of macrophages with 125I-labelled polyvinyl pyrrolidone

The rate constant of the exponential decay in blood radioactivity between 18 and 48 hr after intravenous injection of 30–80 μg of ¹²⁵I-labelled polyvinyl pyrrolidone (PVP) in mice has the characteristics of a test of the clearance function of macrophages. It is reduced by carbon, PVP or hydrocortisone, and increased by oestrogen. Clearance is independent of dose over this range, and probably measures resting unstimulated phagocytic function; this is perhaps different from the function measured by existing techniques. Preliminary evidence suggests that it is applicable to man.