Repeated Injections Of Antigen Research Articles

Progression of articular destruction and the production of tumour necrosis factor-alpha in antigen-induced arthritis in rabbits.

We examined the progression of articular destruction and the production of tumour necrosis factor-alpha (TNF-alpha) in antigen-induced arthritis (AIA) in rabbits, i.e. flare-ups of inflammation induced by repeated intra-articular injections (single, twice and three times) of antigen. A marked progression of articular destruction and an infiltration of inflammatory cells in the synovium were observed with the increase in the number of antigen injections. An immunohistochemical analysis of the synovial lesions following three injections of antigen revealed that the lymphoid follicles consisted mainly of CD4+ T cells and IgG/IgM+ B cells. There were marked infiltrations of IgG+ plasma cells around the lymphoid follicles. In contrast, the production of TNF-alpha in the synovial fluid and the erythrocyte sedimentation rate (ESR), which is a marker of systemic inflammatory activity in rheumatoid arthritis, peaked at 6 h 24 h, respectively, following the last injection of antigen. These values were also greater following the repeated injections of antigen compared with the single injection. The TNF-alpha was produced markedly in the joints at the onset of the flare-ups of arthritis following the repeated injections of antigen, and the elevation of the ESR and an acceleration of the inflammatory response in the synovium were observed with a concomitant progression of severe articular destruction, suggesting that the marked production of TNF-alpha at the time of flare-ups may be involved in the exacerbation of AIA in rabbits.

Generating antibodies against secreted proteins using vascular smooth muscle cells transduced with replication-defective retrovirus.

The traditional method of antibody (Ab) generation requires repeated injections of antigen (Ag). We have developed an alternative method that allows an investigator to generate a polyclonal antiserum with only a cDNA in hand. We cloned a cDNA encoding the coding frame for baboon tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Fischer rat arterial smooth muscle cells (SMC) transduced with the baboon TIMP-1 using a replication-defective retrovirus were propagated in culture. TIMP-1 overexpressing rat SMC were seeded into de-endothelialized rat carotid arteries. Three weeks after cell seeding in the rat, the presence of Ab to the baboon TIMP-1 was detected by dot blot and enzyme-linked immunosorbent assay in 5 of 6 of the animals. The major portion of the Ab generated against baboon TIMP-1 during the 12-month monitoring period after the cell seeding was identified as belonging to the IgG1 subtype. More interestingly, the titer of the Ab kept rising throughout an 8-month monitoring period. Among the salient features of this Ab are its capacity to block TIMP-1 activity and its utility for detecting TIMP-1 by immunohistochemistry. These results demonstrate that Ab against a secreted protein can be obtained in response to continuous expression of the cDNA by vascular SMC. Purified Ag is not required.

Identification and characterization of the 35-kDa beta subunit of guanine-nucleotide-binding proteins by an antiserum raised against transducin.

Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification.

Decline and Restoration of Antibody-Forming Capacity in Aging Beagle Dogs

Abstract The production of antibody to coliphage was studied in young (1 1/2 years) and old (7 to 11 years) beagles. In five old dogs given a single injection of T2 coliphage three of them showed a severe deficiency of antibody-forming capacity as compared to five young controls. When three injections of T2 or T4 coliphage were given in 5 days, 15 old dogs produced the same amount of antibody as did 15 young control dogs. Thus, antibody-forming capacity was restored to aging dogs by giving multiple injections of antigens. The relative serum titer to the cross-reacting antigen, T4 coliphage, was equal in young and old dogs given T2 coliphage. Our results are interpreted in accord with the Antigen Selection Hypothesis (33). They showed that one of the age-associated losses in antibody-forming capacity results from a decline in the proliferative activity of antigen-stimulated cells because the proliferative state can be maintained by repeated injections of antigen. We also concluded that the precursor cells suffer no loss with age in sensitivity to antigenic stimulation because there is no relative increase in the fraction of cross-reacting antibody.

The antibody response of pigs to dinitrophenylated bovine IgG administered with a novel type of adjuvant.

AbstractHigh and reproducible antibody response to miligram quantities of 2,4‐dinitrophenyl (DNP)‐conjugated bovine IgG was elicited in pigs with the aid of a novel adjuvant composed of aluminium hydroxide, paraffin oil and Sorbitan trioleate (Al‐Span‐Oil). Repeated injections of antigen produced antibody levels up to 8 mg per ml of serum. The antibody level reached the maximum around the 12th day after first antigen injection, but around the 7th day after subsequent antigen injection. Isolated anti‐DNP antibody was found to be chemically heterogeneous. It bound ϵ‐DNP‐lysine with a mean association constant of about 106 M−1.

In-vitro detection of reaginic antibody.

Atopic disease in man is distinguished by the presence of a special type of antibody, variously termed reaginic, skin sensitizing, or Prausnitz-Kustner antibody. While universal agreement niay not exist on the exact relationship of the atopic reagin to symptoms in atopic disease, it is nonetheless the most reliable indicator of the naturally occurring sensitized state that is peculiar to atopy in man, and appears unrelated to levels of blocking, hemagglutinating, or precipitating antibodies, which at times may be detected in the sera of atopic individuals. It is also known that the level of reaginic antibody is influenced by repeated injections of antigen, being initially increased, and later dirninishing to the point of disappearance following continuous, long term hyposcnsit1zation.l This influence on reaginic antibody appears to bear a closer relationship to improvement in the clinical state than do the increascs in blocking and hemagglutinating antibodies developed during hyposensitization. The basic method for the detection and study of the rcaginic antibody for more than 40 years has been the passive local sensitization of normal human skin by atopic serum, the Prausnitz-Kustner phcnomenon.2 Since the first recognition of reagin, investigators have atteinptcd to characterize the antibody by methods which would eliminate thc technical and biologic problems involved in using human skin as an indicator of its presence. Among the earliest characteristics of reagin recognized were its inability to form precipitins, fix complement, or passively sensitize the guinea pig to While the usual laboratory animals are also not susceptible to passive sensitization of skin with human sera, the typical P-K phenomenon can be demonstrated in the skin of the macaca irus monkey when intravenous injection of Evans blue dyc is used to stain the lesions as in passive cutaneous anaphylaxis. Intravenous injection of antigen simultaneously with the dye is more successful in visualizing lesions, and is recommended over direct intradermal injection of antigen into the tcst site.4 Other properties of reagin which have been helpful in defining its character and in separating it from other antibodies are its therniolability anti inability to pass the placental barrier.j Since it is destroyed by heating at 56 for periods of approximately 4 hours, it may be readily separated from blocking antibody.6 Its failure to cross the placental harrier has been useful in relating it to certain of the immunoglobulins.7 Among the in-vitro tests for the demonstration of antibodies in allergic sera,

The Immunoglobulins of Mice

Summary Serum levels of 7 S γ2-, 7 S γ1-, γ1A- and γ1M- globulins and of hemagglutinating antibody were determined in 10 strains of mice immunized with hemocyanin and ferritin. The effect of five factors on the response to immunization were investigated: a) difference between single and repeated injections of antigen, b) differences between antigen administration in saline and in adjuvant, c) effect of different time intervals between first and second administration of antigen, d) effect of adjuvant alone and e) mouse strain differences. A single injection of antigens in saline produced no appreciable change in the serum immunoglobulin levels. After a single injection of antigen emulsified in Freund's adjuvant, the levels of the 7 S γ1-globulins showed a two- to five-fold increase. After a second injection of the antigens in adjuvant, 7 S γ1-globulins showed a 10- to 30-fold increase. The absolute and relative changes in the other three immunoglobulins (7 S γ2, γ1A(IgA) and γ1M(IgM)-globulins) following primary and secondary challenge were not as marked as the increase in 7 S γ1-globulins. Repeated injections of antigen in saline produced a greater increase of γ1M(IgM)-globulins than did several injections of antigen in Freund's adjuvant. The response of specific immunoglobulins to antigenic challenge is strain dependent. The most notable strains were a) the C57BL mice, which showed marked increase of γ1M(IgM) and γ1A (IgA)-globulins and b) the white Swiss (GP) mice, which showed very little increase in γ1M-(IgM)- and γ1A(IgA)-globulins while developing the highest levels of 7 S γ1-globulin. Injection of adjuvant without antigen had no notable effect except in C57 strains of mice, where marked increases of γ1M(IgM)-globulins occurred.

Roles of the Liver and Appendix in the Serum Hemolysin Response in Rabbits

Summary The roles of the liver and appendix in the serum hemolysin response in rabbits immunized with one or more injections of sheep erythrocyte stromata were studied. The effects of extirpation of these organs were observed both in the presence and absence of the spleen. Antibody titers were determined photometrically in 50% hemolysin units, and the serum hemolysin response was analyzed with respect to the lengths of the latent period and rise to peak titer, day and magnitude of peak titer, and rates of hemolysin rise and decline. Using this approach, no antibody contribution by the appendix or liver to the serum hemolysin response was detected. These organs apparently were inactive even after repeated injections of antigen which overrode the depressive effect of splenectomy by inducing increased nonsplenic activity. Changes in the hemolysin response observed following immunization via the portal vein and in partially hepatectomized animals injected via the ear vein could be attributed to the liver's capacity to remove significant amounts of antigen from the circulation. Increased disappearance rates of passively transferred hemolytic antiserum in partially hepatectomized and liver-irradiated rabbits suggest that the regenerating or recovering liver catabolizes antibody faster.

THE MECHANISM OF TOLERANCE PRODUCED IN RATS TO SHEEP ERYTHROCYTES. I. PLAQUE-FORMING CELL AND ANTIBODY RESPONSE TO SINGLE AND MULTIPLE INJECTIONS OF ANTIGEN.

Previous studies suggested that an active immune response was partially responsible for maintaining immunological unresponsiveness to sheep erythrocytes. Measurement of the plaque-forming (antibody-releasing) cell response proved to be a sensitive indicator of an immune response to sheep erythrocytes in the absence of detectable circulating antibody to the antigen. The present studies were undertaken to determine whether an active immune process, measured by the plaque-forming cell response, was partially responsible for induction and maintenance of tolerance. Rats injected intraperitoneally with large doses of sheep erythrocytes beginning at the day of birth develop tolerance to the antigen. In this paper, the plaque-forming cell and antibody response to sheep erythrocytes was characterized for rats receiving a single antigen injection at various ages and for rats which received repeated antigen injections as adults. The dose of antigen was the same as that used to produce tolerance; the injection schedule for repeated immunizations was also the same as that used to produce tolerance. Rats receiving a single antigen injection on the day of birth or at age 7 days had no measurable response to the antigen. Rats receiving a single antigen injection at age 17 days and sacrificed 4 days later had an unequivocal response to the antigen. The spleens had about one-tenth as many plaque-forming cells as spleens of adult animals immunized similarly, but the antibody titers were as high as titers for adult animals. Presumably the high titers of these young animals resulted from the high ratio of plaque-forming cells to body weight and blood volume. Adult animals receiving a single antigen injection had a peak or near peak plaque-forming cell response 4 days after immunization; at this time, sera contained high titers of 19S antibody and the numbers of plaque-forming cells in spleens correlated reasonably well with circulating antibody titers. 7S antibody appeared in serum 5 or 6 days after immunization. The numbers of plaque-forming cells declined progressively 2 and 3 weeks after immunization. Repeated twice weekly, injections of the antigen in adult rats produced a marked decline and then stabilization of numbers of plaque-forming cells in spleens. Although the numbers of plaque-forming cells were fewer, titers of 19S and 7S antibody stabilized at high levels. A progressive recovery of the plaque-forming cell response and a rise in antibody titer occurred when the interval between the last 2 injections was increased from 3 to 10, 17, or 32 days. These findings suggested that repeated closely spaced antigen injections interfered with either cell division or maturation of antibody-forming cells. As the interval between injections was increased, additional antibody-forming cells matured or were formed through cell division. Thus, relatively constant antigenic stimulation provided a mechanism for controlling or limiting the response of antibody-forming cells. In the following paper, it will be shown that this mechanism operating together with a homeostatic mechanism which prevents induction of new antibody-forming cells serves to regulate the immune response to repeated injections of antigen.

Immunological unresponsiveness in rabbits produced by neonatal injection of defined antigens.

The phenomenon of immunological unresponsiveness induced in the neonatal rabbit by a single injection of a defined protein antigen, has been characterized semiquantitatively, and studies bearing upon the mechanism of such unresponsiveness have been presented. A single intraperitoneal injection at birth of 10 to 100 mg. BSA, HGG, ovalbumin, or a human macroglobulin, or an oral feeding of 100 mg. BSA, induced a state of unresponsiveness lasting at least 90 to 120 days. 100 mg. BSA given from birth to 17 days of age, but not later, produced unresponsiveness of 90 to 120 days' duration. Data are presented which show that the duration of unresponsiveness is finite and related to the amount of antigen given at birth, and that it may be indefinitely prolonged by repeated injections of antigen. Disappearance of injected antigen in the unresponsive animal was exponential with time, with no accelerated or immune phase. Administration of the antigen in adjuvants resulted in significant shortening of the duration of unresponsiveness. The transfer of immune cells to the unresponsive host while resulting in vicarious antibody formation, did not affect the underlying unresponsive state. Negative results of attempts to produce unresponsiveness to a variety of bacterial antigens are presented. The implications of the data are discussed, particularly in reference to the other experimental models of immunological tolerance, and to the various theories of acquired immunity. It is clear that any satisfactory theoretical explanation of acquired immunity will have to account simultaneously for the phenomena of immune tolerance.

The role of the spleen and the dynamics of hemolysin production in homologous anamnesis.

Our previous studies on the dynamics of hemolysin production in rabbits after single (1950) and multiple (1951) intravenous injections of sheep red cells have been extended to include a study of the homologous anamnestic response following single and multiple reinjections with the same antigen into intact rabbits and into rabbits splenectomized either at the time of the initial injection or of the reinjection. After an induction period, hemolysin begins to be produced rapidly at one continuous rate of accumulation (cf. fig. 8B) or at two (cf. fig. 12) or more rates to peak titer and thereafter generally declines at several varying rates. Our results indicate that peak antibody titer after both the initial rise and anamnestic rise is due chiefly to the activity of the spleen after a single injection of antigen and is due, relatively, more to the activity of nonsplenic sources after repeated injections of antigen. A review of the literature and a dis-