Carl J. Wust

Effect of antigen dose on lymphatic tissue germinal center changes.

Summary Investigations were performed on the effect of various doses of sheep erythrocyte antigen on spleen germinal center changes. These changes were correlated with the production of specific serum antibody during the first 30 days after primary antigen stimulation. The results indicate a dose-response relationship between antigen dose and germinal center changes. A correlation was observed between the germinal center changes and specific serum antibody production.

Actinomycin D: Effect on the Immune Response

Actinomycin D injected simultaneously with sheep erythrocytes in female rats caused a delay in the immune response but had no effect on the rate or maximum amount of hemagglutinin produced. The delay was roughly proportional to the concentration of the antibiotic administered, and was up to 2 days for 75 �g in a 200-gram female rat (sublethal dose for females). The dose effect in the delay in response is consistent with the time when actinomycin would no longer be available to bind with newly synthesized DNA and when messenger-RNA production could occur. Similar results were obtained with another antigen, the enzyme β-galactosidase, in male rats during the secondary response.

Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum.

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibody-dependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given γ globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LD50 of SF, a virus which is in a different subgroup than SIN. All mice survived if the γ globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus. Cobra venom factor treatment of normal C3H mice challenged with SF did not reduce the protection, suggesting that complement was not involved. Methyl palmitate (40 mg/mouse) given before γ globulin and virus challenge suppressed macrophage activity and reduced the level of protection 23% in females and 70% in males. Silica treatment (3 mg/mouse) reduced the protection equally in both males and females by 92%. In vitro experiments were done to test if it were possible that cross-antibody-dependent cellular cytotoxicity (ADCC) could accout for the passive cross-protection observed in this system. Cross-ADCC could be demonstrated in vitro at high dilutions of antiserum (1:25,600). On the basis of the in vitro and in vivo results presented, we suggest that cross-ADCC against SF-infected target cells is one of the likely mechanisms to explain the passive cross-protection observed.

Cell-free amino acid incorporation by rat spleen ribosomes

An energy-dependent, amino acid incorporating system is described for rat spleen ribosomes supplemented with soluble components of rat liver. Ribosomes prepared by deoxycholate treatment of microsomes prior to centrifugation could be stored in liquid nitrogen for 5 days before use in incorporation experiments. During storage, latent RNase was activated at a rate of 0.045 μg. per milligram ribosomal protein per day. After 5 days of storage, the half-life of the ribosomes for incorporation was 3.6 days. The loss of ability to incorporate was attributable to the accumulated activated RNase. Soluble components from rat liver were obtained by treating the 105,000 g supernatant fluids of homogenates with streptomycin to yield amino acid acceptor RNA, followed by (NH4)2SO4 precipitation at 0.75 satn. to yield an enzyme protein concentrate. Maximum incorporation was obtained with 1 mg. of spleen ribosomes, 75 μg. of RNA, and 3.5 mg. of protein concentrate. Other characteristics of this system are presented.

TIME RELATIONSHIP OF INJECTION OF ACTINOMYCIN D AND ANTIGEN TO THE IMMUNE RESPONSE.

Summary Large basophilic cells in the lymphoid nodule of spleen are damaged when actinomycin D is given before, with, or after injection of sheep erythrocytes. The severity of cytotoxicity is dependent upon the time the drug is given in relation to injection of antigen. Consonant with the relative degree of damage is delay in appearance of circulating hemagglutinin. The time for injection of animals for maximal delay in appearance of hemagglutinin and coincident maximal cytotoxicity is 4 to 8 hours before injection of antigen.

Binding of 125I-labeled anti-L chain to polysomes of rat spleen in vitro

Abstract The light (L) chain of rat gamma globulin was isolated and injected into rabbits as antigen. Fractionated immune gamma globulin from the rabbits was labeled with 125I and used to indicate the presence of nascent L chains on ribosomes of rat spleen. After centrifugation in a sucrose gradient, the label was found on ribosomes containing 4 to 9 monomeric units, but was not coincident with the optical density profile (OD). The highest specific activity was found between the hexamer and the septamer or between the septamer and octamer. During the first 24 hours of the immune response to sheep erythrocytes, the amount of 125I anti-L chain bound to ribosomes decreased to a minimal value 12 hours after injection of antigen and increased between 12 and 24 hours to a value comparable to that in nonimmunized animals.

Studies on ribosomes of rat spleen during the immune response

Abstract Microsomes and ribosomes (i.e., microsomes treated with sodium deoxycholate) have been prepared from homogenates of spleens of nonimmunized rats and rats that were immunized to sheep erythrocytes. The method used to prepare microsomes allows the release of ribosomes from endoplasmic reticulum without the aid of detergent. Particles thus obtained were sedimented by zonal centrifugation through a sucrose gradient. Six peaks of optical density of 254 mμ were observed that represented 65S, 81S, 105S, 120S, 154S, and 175S. The predominant peak was 120S. Similar optical density profiles were found with both microsomes and ribosomes from spleens of all rats, immunized and nonimmunized. Labeling in vivo with 32 P of RNA associated with ribosomes from immunized rats showed that the relative specific activity increased to a maximum of about twofold at 72 hours after injection of antigen over that found in nonimmunized animals. The relative amount of amino acid incorporated in a cell-free system by microsomes or ribosomes increased with immunization. With ribosomes, this amount reached a maximal twofold at 3 days after injection of antigen; with microsomes, it was a maximal 1.3-fold at 2 days after immunization. Zonal centrifugation of incubation mixtures showed loss in the 120S region, and larger polysomes with concurrent increase in 81S with time.

Cross-protection correlates with delayed antibody formation to challenge virus after immunization with Sindbis virus.

Mice immunized with Sindbis virus, strains HR (heat-resistant) or AR339, by a dual injection given intracerebrally (i.c.) and intraperitoneally (i.p.) at day zero, were cross-protected from challenge at day 10 with Semliki Forest virus (SFV). Neutralizing and haemagglutination-inhibition antibodies to Sindbis are detected in high titre by day 6 after immunization but no cross-reacting antibodies to SFV were found up to day 42. Immunized mice that were challenged with SFV showed an 8 to 10 day delay in the appearance of antibody to SFV compared to mice that were sham-inoculated. Thus, cross-protection in Sindbis-immunized animals was correlated with a temporary suppression of antibody formation to the challenge virus (SFV), while the appearance of high titre antibody to SFV in sham-immunized mice after challenge with SFV did not protect.