Clifton A. Ogburn

Gel-Precipitation of Streptococcal Culture Supernates with Sera of Patients With Rheumatic Fever and Streptococcal Infection.∗

Summary 1. Precipitin tests in semi-solid agar (gel-precipitin) have been carried out between concentrates of streptococcal culture supernates and sera of various sources. Two technics of diffusion in 1 dimension (tube) were used: the single-diffusion technic of Oudin, and a double-diffusion technic. 2. Sera of rabbits previously injected with streptococcal culture supernates showed increasing numbers of bands as the course of injections progressed. Sera of such rabbits showed as many as 5 bands when tested with such concentrates, indicating that this was the minimum number of antigen-antibody systems present. 3. Sera of patients with acute rheumatic fever and of patients convalescent from an acute streptococcal infection (scarlet fever) were also tested by this technic against concentrates of streptococcal culture supernates. The group with rheumatic fever showed a greater number of bands (a range of 2-7) than did the group with scarlet fever (0-4), when tested by the double-diffusion technic. The corresponding ranges found with the single-diffusion technic were 1-5 and 0-2, respectively. In a sampling of sera from convalescents from scarlet fever the number of bands was found to be no greater 6 weeks after the onset of the infection than 3 weeks after the onset.

Solubilization of h-2 histocompatibility antigens of the mouse by triton x-100 and butanol.

Histocompatibility antigens (HCA) have been solubilized by the use of Triton X-100 and n-butanol from cell membrane fragments of spleens and livers of BALB/c and CBA mice. These soluble preparations could inhibit or neutralize allogeneic antibody (CBA anti-BALB or BALB anti-CBA) in two biological tests, cytotoxicity and the suppression of antibody formation by appropriately stimulated spleen cells of the same strain. The neutralizing activities of the spleen-derived preparations, per milligram, were about 10 times those derived from liver, and preparations extracted by butanol or by papain were of substantially higher specific activity than those extracted by Triton X-100. All preparations contained active molecular species excluded by Sephadex G-200 and others, nonexcluded, in various proportions. Both Sephadex fractions could be adsorbed to sheep red blood cells (SRBC) but only the adsorption of the G-200-excluded material rendered the SRBC agglutinable by CBA anti-BALB antibodies; the non-excluded fraction could compete with the excluded fraction to prevent agglutination of the coated SRBC by such antibody. The soluble antigen could inhibit the allogeneic antibody in two serological tests, the adsorption hemagglutination (AH) reaction referred to, and the standard hemagglutination test of BALB RBC by CBA anti-BALB antibody.

Suppressive antibody in heterologous anti-lymphocytic serum estimated in vitro by the hemolytic antibody plaque test, species-specific and strain-specific antibodies in rabbit-anti-mouse anti-lymphocytic sera.

Summary: Heterologous anti-lyniphocytic sera (ALS) produced by injection of rabbits with spleen cells from CBA or BALB/c mice were found to suppress hemolytic antibody plaque formation by antigenically stimulated spleen cells following prior incubation of such cells with ALS and complement. Cytotoxicity of such antisera for mouse spleen cells was also shown. Both antibody effects were demonstrable in high titer in both anti-CBA and anti-BALB ALS tested against cells of both strains. The bulk of the antibody was species-specific. However, the presence of significant amounts of strain-specific antibody was indicated, first, by higher titers in some classes of sera tested against cells of the immunizing strain than against those of the other strain; second, by absorption of the ALS with cell fragments of the other strain before testing against cells of the immunizing strain; and, third, by sequential absorption of ALS by cell fragments of the other strain and then those of the immunizing strain and elution of antibodies from these absorptions. Hemolytic antibodies appearing in such sera were more preponderantly species-specific. If strainspecific hemolysins were present, they could have constituted at most a substantially smaller fraction. The strain-specific component of an anti-CBA ALS was shown in vivo to cause prolonged retention of BALB skin grafts by CBA mice and reduction in the production of agglutinins to Sltigella by such mice

Relation of the butanol-solubilized rabbit histocompatibility antigens to the suppression of antibody-producing allogeneic lymph node cells.

Soluble antigen prepared by Triton and butanol from cell membrane fragments of rabbit lymph node and spleen has been shown to be associated with the suppression or rejection of allogeneic lymph node cells by its abilities (1) to inhibit or absorb the suppressive antibody in rabbit antileucocyte serum, (2) to induce actively in normal rabbits the rejection of transferred lymph node cells, and (3) to stimulate production of antibodies suppressive for allogeneic lymph node cells. This can be demonstrated in cell transfer or, in vitro, in the hemolytic antibody plaque test. Analysis by chromatography and sucrose density gradient ultracentrifugation shows that the rejection-associated antigen can be found within approximately half the total protein of the butanol extract, and suggests nonidentity of the antigen detectable by inhibition of suppressive antibody and that detectable serologically. Further evidence of nonidentity of the antigens and antibodies detected is given by substantial disproportionality of titers determined in suppressive and serological tests in primary and secondary antisera to whole allogeneic cells, respectively, and in secondary and tertiary antisera to the butanol-solubilized antigen.

Serologically detectable rabbit histocompatibility antigens solubilized by several agents.

Three water-soluble preparations have been obtained from cell membrane fragments of rabbit spleen and lymph nodes and give serological evidence of containing rabbit histocompatibility antigens. These materials have been solubilized by the use of Triton X-100, and, from the water-insoluble fraction of the Triton extract, by snake venom phospholipase and by butanol. The evidence for soluble histo-compatibility antigens in these preparations is given by their reaction with antibodies present in rabbit antiserum to rabbit leucocytes, as indicated by their ability to agglutinate tanned sheep red blood cells (RBC) coated with specifically purified antibody from the antileucocyte serum, by their ability to render tanned RBC agglutinable by the antileucocyte serum, and by their inhibition of the agglutination of antigen-coated RBC by antileucocyte serum. The butanol-solubilized material has about 12 times the specific activity of the phospholipase-solubilized material; both yield approximately the same total amount of soluble antigens. These two materials are entirely excluded from Sephadex G-200, whereas the greater part of the Triton-solubilized is not excluded. On diethylaminoethyl cellulose chromatography of the butanol-solubilized preparation, almost all of the serological activity can be found within approximately half the total protein applied to the column.

Accelerated rejection of allogeneic skin grafts in the mouse after injections of histocompatibility antigens solubilized by Triton, butanol, and papain.

BALB/e histocompatibility antigens (HCA), solubilized from cell fragments of spleens and livers by Triton X-100, butanol, and papain, were tested for immunogenicity as transplantation antigens by injection into normal CBA mice and later observation of the time of rejection of BALB skin grafts in these mice. Each type of preparation, injected 3 times in saline or in oil adjuvant, caused accelerated rejection of such grafts. Preparations solubilized by butanol or papain showed higher specific activity than the Triton-solubilized, and preparations from spleen showed higher activity than those from liver. These differences were similar to the differences in specific activity shown by the respective preparations in the inhibition of 2 effects of CBA anti-BALB antibody, cytotoxicity, and the suppression of hemolytic antibody plaque production by BALB spleen cells. CBA antigen solubilized by butanol from liver cell fragments similarly caused accelerated rejection of CBA skin grafts in BALB mice following injection on the same schedule.

Various Effects Of Alloantibody-containing Globulins On The Time Of Retention Of Skin Allografts In The Mouse

A study of the effect of alloantibody-containing ascitic fluid globulin of mice on the retention of skin allografts has been carried out using, as a tentative measure of the relevant antibody, the titer of the antibody in such globulins which can suppress the production of hemolytic antibody plaques by donor strain spleen cells. When globulins with sufficiently high suppressive titer were available in sufficient quantities, pools of such globulins were injected into normal mice which were given skin allografts in the same strain combination as that used for production of the alloantibodies. Among globulins, all of which were above a given threshold of suppressive titer and all within a given range, four kinds of effect were found on the time of retention of the allograft: accelerated rejection in almost all of the grafted mice (relative to control mice injected with normal ascitic globulin), or accelerated rejection in some of the grafted mice, or no apparent effect, or, finally, prolonged retention of the graft. There was no relation between the suppressive titer of the globulin pool and its effect on the time of rejection of the skin graft. The fact that anti-H-2 antibody-containing globulin pools could cause, in given instances, these opposite effects of accelerated rejection and prolonged retention of the allogeneic skin grafts suggested the participation of at least two alloantibodies with the same specificity but with differing biological effect, the fate of the graft depending on the relative concentrations of the two. The possible roles of two such competing antibodies are being explored.