Immune response in the guinea pig to penicillin-autologous carrier proteins.

The spontaneous fixation of penicillin G on guinea pig serum protein was studied. Serum albumin and Ig were shown to fix firmly over 95% of penicillin. Therefore, guinea pigs were immunized with penicillin alone or penicillin-autologous carrier protein conjugates. The production of hemagglutinating antibody, of MIF and the in vitro stimulation of lymphocytes were analyzed as immunological parameters. The results showed that in all cases the strongest immunological response was obtained with animals immunized with the protein-hapten conjugates, whereas no response could be obtained with the carrier protein alone. Finally it was shown that the conjugates significantly stimulated purified T cell population. These results suggest that the antigenic recognition of penicillin involves the participation of lymphocytes.     

Immune response to endotoxin isolated from Bacteroides fragilis in the pregnant guinea pig

The humoral immune response to endotoxin isolated from Bacteroides fragilis was analyzed in the pregnant guinea pig by means of passive hemagglutination, passive hemolysis, a modified Coombs test, and by crossed immunoelectrophoresis. Pregnant animals were immunized with endotoxin on day 30 of gestation, and antibodies were determined on day 61 in maternal and fetal sera, and in amniotic fluid. The IgG and IgM responses in maternal sera were of the same magnitude as in sera of nonpregnant animals. Fetal sera contained IgG and sometimes IgM, and a higher percentage of incomplete antibodies against endotoxin than maternal sera. Low-titer anti-endotoxin antibodies, partially sensitive to dithiothreitol, were found in amniotic fluid. A statistically significant reduction in the growth of fetuses from endotoxin-immunized females was observed.     

Effects of cyclophosphamide on the in vivo response of outbred athymic (nude) mice to a thymus-independent antigen (DNP-AGG-Ficoll).

Both nude mice (nu/nu) and their heterozygous littermates (nu/+) were injected with a single IP dose of 300 mg cyclophosphamide (CY)/kg. CY is a known immunosuppressive agent, which affects primarily B lymphocytes. Immunization with the thymus independent antigen DNP-AGG59-Ficoll after CY treatment disclosed that restoration of the primary direct PFC response occurred more rapidly in nude mice than in nu/+ mice. However in these same experiments, the primary indirect PFC response, recovered earlier in nu/+ mice than in nude mice. After CY treatment, secondary indirect PFC responses were delayed in both nude and nu/+ mice, but the greatest effect was seen in nude mice. The data suggest that the presence of T cells has little if any influence on the recovery capacity of those B cells which are destined to become direct PFC. However the recovery of B cells which are destined to produce indirect PFC responses is facilitated by the presence of T cells.     

Immune response to guinea pig cytomegalovirus polypeptides and cross reactivity with human cytomegalovirus

The immune response to guinea pig cytomegalovirus (gpCMV) was evaluated by immunoblotting. Preinoculation guinea pig plasma did not react with gpCMV antigen, whereas convalescent plasma reacted to at least 18 gpCMV-specific polypeptides. The initial immune response was primarily directed at polypeptides with MWs of 100, 75, and 56 kDa. Over 80% of plasma collected more than 29 days after viral inoculation reacted to these polypeptides and also to those with MW of 54, 52, and 38 kDa. In this report, we also demonstrate cross reactivity between gpCMV and human CMV (HCMV). Human immunoglobulin (IVIG) reacted to at least 20 HCMV polypeptides and cross reacted with six gpCMV polypeptides. GpCMV convalescent plasma also reacted with HCMV polypeptides.     

Immune response of the guinea pig to bovine parathyroid hormone antigen: influence of the booster dose on antisera titer and sensitivity

Thirty guinea pigs were immunized with 40 microgram of bovine parathyroid hormone, bPTH(1-84), and divided into 6 groups with a similar primary response. Each group was boosted twice with a different dose of antigen (0.04 to 40 microgram). The titer (30% binding of tracer) and affinity (% displacement with 320 pg of bPTH(1-84) were studied. The primary response was maximal at 61 days; titers were 1 x 10(-4) or less and affinity was low. The response was maximal 25 days after each booster. After the first, the highest titers, 5.2 +/- 2.6 x 10(-4) (mean +/- S.D.) were seen in the 40 microgram group and a positive correlation was observed between the dose of antigen and the titer (r = .7386, p less than 0.001) for doses greater than 0.6 microgram. This remained true after the second booster although titers were lower in all groups. Affinity was greater after the first booster (35.85 +/- 20.38%, n = 30) than after the second (23.24 +/- 20.73, n = 30, p less than 0.0025), but was similar in all groups. Selected antisera reacted with antigenic determinants in regions 1-34, 53-84 and 35-53 of the bPTH molecule. Cross-reactivity with human PTH(1-84) was maximal in the 53-84 region. In conclusion, antibodies against all regions of bPTH can be raised in the guinea pig. Once a primary response is elicited, best titers are achieved by boosting with high doses of antigen without any detrimental effect on antisera affinity.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Splenic dependence of the antibody response to thymus-independent (TI-2) antigens

The murine antibody response to T-independent (TI)-2 antigens [2,4-dinitrophenyl-Lys-Ficoll (DNP-FIC) and DNP-hydroxyethyl starch (HES)] was impaired long after splenectomy, while responses to TI-1 [trinitrophenylated lipopolysaccharide (TNP-LPS)] and thymus-dependent [TNP-keyhole limpet hemocyanin (KLH)] antigens were largely unaffected. The antibody response to these TI-2 antigens was exclusively against the conjugated epitopes [DNP-, fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-Ficoll or -HES]. Fluorescent conjugates of Ficoll and HES localize selectively to the splenic marginal zone macrophages. This localization was not affected by 750 cGy of X-irradiation, but the antibody response to the TI-2 antigens was abrogated for 14 days. Administration of spleen cells restored the antibody response to these TI-2 antigens in otherwise intact irradiated mice but not if they had been splenectomized. Our findings indicate that the antibody response to TI-2 antigens depends upon stimulation of B cells in a splenic environment. This probably involves antigen presentation by marginal zone macrophages.     

Evidence for epitope-specific thymus-independent response against a repeat sequence in a protein antigen.

We have studied the immunogenicity of a recombinant heat-shock protein-related stress protein of 70,000 MW (Pfhsp) of the human malaria parasite Plasmodium falciparum in H-2 congenic and non-congenic strains of inbred and outbred mice. Most mice of different genetic backgrounds produced antibodies to Pfhsp, indicating a lack of any apparent genetic restriction of immune response. A peptide corresponding to the GGMP repeat sequence in the C-terminal region of Pfhsp was recognized by more than 75% of sera from immunized mice. The GGMP repeat epitope-specific antibodies were largely of the IgM isotype, especially in all seven inbred strains of mice tested. The lack of significant boosting of the immune response, a predominantly IgM isotype of antibodies and generation of antibody responses in athymic nude mice suggest a thymus-independent response against the GGMP repeat epitope in the Pfhsp molecule.     

Interactions between the immunological responses of a thymus-independent antigen (Salmonella adelaide O antigen) with a thymus-dependent antigen (sheep erythrocytes) in the adult bird.

The bird's antibody response to a thymus-dependent antigen (sheep erythrocytes) (SRBC) and a thymus-independent antigen (SALMONELLA ADELAIDE O antigen) were characterized: whereas the former proceeded through a brief 19S response to a declining 7S response, the latter failed to switch from 19S TO 7S for several weeks and consisted in repeated excursions of 19S antibodies. When injected intravenously and simultaneously an injection of S. adelaide-killed organisms and SRBC interact, so that the response to the latter fails to switch from 19S TO 7S and consists of repeated excursions of 19S antibodies. The changed character of the SRBC response is interpreted to be due to the relative lack of 7S antibody: passive 7S antibody to S. adelaide O antigen or 7S anti-SRBC produces a negative feedback inhibition of their respective responses, so that only one excursion of 19S antibody is observed. The effect is not, however, symmetrical; the thymus-independent antigen is dominant. Thus, whereas 7S antibody to S. adelaide produces the same negative feedback inhibition on the response to S. adelaide and the response to SRBC (when injected with adlaide), 7S antibody to SRBC inhibits only the response to SRBC and not the response to S. adelaide. These results are discussed relation to current hypotheses of antibody biosynthesis and mechanisms of adjuvant action. They are also discussed in relation to the function of the germinal centres of the spleen which may function to mediate the negative feedback of 7S antibody on the antibody response.     

Immune response to foot-and-mouth disease virus in a murine experimental model: effective thymus-independent primary and secondary reaction.

The immune response against foot-and-mouth disease virus (FMDV) was studied in a murine model. In untreated control mice, the inoculation of 10,000 suckling mouse 50% lethal doses of Ol Campos FMDV i.p. was followed by a burst of viraemia that disappeared in less than 4 days, i.e. when the neutralizing antibodies (NAb) reached titres above one neutralizing unit. In mice treated with cyclophosphamide, the curves of viraemia and NAb were significantly delayed. Nu/nu mice injected with FMDV had curves of viraemia and NAb identical to those of their nu/t littermates. We then studied the secondary (memory) immune reaction in the same model. In order to investigate which preimmunized cells participate in the elimination of actively replicating FMDV, mice were irradiated, then infected with FMDV, and 24 hr later repopulated with cells obtained from either donor mice that had been previously immunized by infection with live virus, or non-infected controls. The transfer of control (non-immunized) lymphoid cells was unable to eliminate the viraemia in recipient animals at times significantly different from those observed with irradiated recipients receiving no cells, while repopulation of recipients with 10(8) immune lymphoid cells (obtained from pooled thymus, blood, peritoneal exudate, spleen and lymph nodes of preinfected donor mice) led to undetectable titres of viraemia at Day 5 post-infection (p.i.). High doses of thymus cells were totally inactive, while a few as 10(7) donor spleen cells were able to abort viraemia at 6 days p.i. When enriched preparations of B or T spleen cells were adoptively transferred, only B cells were able to abort viraemia in irradiated recipients. It is concluded that, in the murine model of FMDV infection, B cells are mainly responsible for primary response and short-term immunological memory. In both cases the protective immune reaction is T-independent.     

Immune response to hepatitis B surface antigen.

A total of 69 persons were investigated for assessment of cell-mediated and humoral immunity to hepatitis B surface antigen (HBsAg). Three groups, each consisting of 20 normal persons, 20HBsAg carriers, and 20 convalescent hepatitis B patients, were studied for HBsAg, anti-HBs, and leukocyte migration inhibition with purified HBsAg. Sequential sampling if an additional group of nine acute hepatitis B patients defined the cellular and humoral immune response to HBsAg. The antigen was eliminated rapidly by mounting of cell-mediated immune response detectable for a limited period, followed by antibody response in relatively few patients moore than 3 months after clearance of circulating HBsAg.     

Impaired antibody response of C57BL/6 beige mutant mice to a thymus-independent type 2 antigen

The murine equivalent of the human Chediak-Higashi Syndrome is the beige (bg) mutant in the C57BL/6 (B6) background. Besides the well-known lack of natural killer (NK) activity in bg-homozygous mice, functional abnormalities of T cells, macrophages and various granulocytes have been reported. With the exception of one study indicating a decreased in vitro response to lipopolysaccharide, there is no report concerning the B cell compartment of the beige mutant. The in vivo anti-trinitrophenyl antibody response to a TI-2 antigen (TNP-Ficoll) was found here to be significantly lower in B6 beige than in B6 wild mice, although both strains responded similarly to an analogous TD antigen (TNP-ovalbumin). Since the marginal zone macrophages of the spleen were previously shown to be essential for the initiation of antibody responses to TI-2 antigens, they might be another target of the beige mutation.     

Conditioned suppression of a thymus-independent antibody response

An illness-induced taste aversion was conditioned in mice by pairing cyclophosphamide, an immunosuppressive drug, with the consumption of saccharin, a novel drinking solution. Two weeks after conditioning, animals were injected with the hapten trinitrophenyl (TNP) coupled to the thymus-independent carrier, lipopolysaccharide. Serum antibodies to TNP were titered 6 days later by passive hemagglutination. Relative to control groups, conditioned animals provided with saccharin at the time of antigenic stimulation and, again, 3 days later showed a significant attenuation of their anti-TNP antibody response. In a second experiment, the conditioned stimulus (CS) consisted of the novel saccharin drinking solution plus the noxious internal effects of an injection of LiCl. Conditioned animals reexposed to the CS again showed the lowest antibody titers, but differed significantly from only one of the control groups. Taken together, the results of these experiments confirm previous reports of conditioned immunosuppression and suggest that the effects of conditioning on a primary humoral antibody response can be observed in response to a T-cell independent antigen in the mouse.     

T-lymphocyte response in a guinea pig model of tuberculous pleuritis.

The ability to induce tuberculous pleuritis in Mycobacterium bovis BCG-vaccinated guinea pigs was investigated as a model of human disease. A pleural effusion of 5 to 10 ml was obtained 6 to 7 days after the bilateral pleural injection of a suspension of heat-killed M. tuberculosis cells. Histological lesions were indicative of granulomatous pleuritis. Comparative studies of T lymphocytes obtained from pleural fluid and peripheral blood revealed increased antigen-driven lymphoproliferation and E rosette formation in pleural effusion lymphocytes. The CD2+ T-lymphocyte population appeared to be expanded or concentrated in pleural fluid, suggesting a compartmentalization of antigen-reactive T lymphocytes. These data demonstrate that experimental tuberculous pleuritis with effusion, closely resembling the human disease, can be produced in BCG-vaccinated guinea pigs.     

Antibody formation in mouse bone marrow. V. The response to the thymus-independent antigen Ecsherichia coli lipopolysaccharide.

The occurrence of plaque-forming cells (PFC) in mouse bone marrow was studied during primary and secondary responses to the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Anti-LPS responses were induced by various doses of LPS. During the primary response, doses of 1 and 10 mug LPS intravenously (i.v.) were found to evoke a distinct PFC response in both spleen and bone marrow. The spleen contained the majority of PFC until about 5 days after immunization. During the course of the reaction the number of PFC in the bone marrow rose to a level which equalled or surpassed the level in the spleen. LPS doses of 0-001, 0-01 and 0-1 mug i.v. only induced a PFC response in the spleen. Apparently there is a minimal threshold dose of LPS of about 1 mug for PFC to appear in the bone marrow. The secondary response was studied in mice primed with 1 mug LPS i.v. and boosted with either 0-001, 0-1 or 10 mug LPS i.v. 3 months later. After each dose tested the PFC activity in the spleen was several times higher than during the primary response. As was observed in the primary response doses of 0-001 and 0-1 mug LPS i.v. did not evoke a PFC response in the bone marrow. After boosting with 10 mug of LPS i.v. a significant PFC response was found in spleen, bone marrow, thymus, lymph nodes, Peyer's patches and blood. From about 5 days after the booster injection the number of PFC in the bone marrow exceeded the total number found in all other lymphoid organs. The results are discussed in relation to the bone marrow PFC response to the thymus-dependent antigen sheep red blood cells. To this antigen a clear PFC response in the bone marrow is found only during the secondary response.