T. P. M. Schetters

Protective and pathological activity in serum of mice developing resistance to Plasmodium berghei infection

Summary The serum from mice developing resistance against Plasmodium berghei infection using chemotherapeutic treatment has been analysed in vivo and in vitro. During the immunization period pathological as well as protective activities which could be transferred by serum were generated. The pathological activity, which was defined as destruction of erythrocytes in normal recipient mice, was generated early in the immunization procedure, peaked at day 21, and decreased to undetectable levels by day 35. After reinfection of the donor mice the pathological activity reappeared in the serum, and was maintained for at least 56 days. Analysis of the transferred serum samples showed the presence of anti‐erythrocyte antibodies (ELISA), but no correlation with the in‐vivo anti‐erythrocyte effect could be found. The anti‐erythrocyte effect of the serum samples indirectly increased the parasitaemia in the recipient mice through the induction of reticulocytosis. The protective effect of the serum samples could only be detected in samples taken from animals beyond day 61 of the immunization procedure. This net protective effect was reflected in a decreased parasitaemia at 7 days after challenge of the recipient mice with P. berghei infected erythrocytes. The protective activity of the serum was correlated with high titres of anti‐erythrocyte antibodies. Anti‐erythrocyte antibody titres were strongly correlated with titres against heterologous red blood cells as well as total immunoglobulin content of the serum samples, indicative of polyclonal activation of lymphocytes. Except for IgGl, all (sub‐) classes were elevated during the immunization procedure, of which IgG3 was abundant. After immunity was obtained these immunoglobulin levels remained high, and the relative amount of IgGl in the serum was restored.

Plasmodium berghei: Reduction of the mouse's specific lymphoproliferative response in relation to corticosterone and pregnancy

Spleen cells from mice immune to Plasmodium berghei exhibited a significantly increased in vitro proliferative response to parasitized reticulocytes compared to spleen cells from normal mice. The specific response to malaria antigen was decreased in spleen cells from pregnant immune mice in contrast to the nonspecific response to the mitogen phytohemagglutinin. Addition of mouse serum to spleen cell cultures of immune mice depressed both the phytohemagglutinin and the specific proliferative response, whereas serum of pregnant mice exerted an even stronger inhibition than serum of nonpregnant mice. Charcoal adsorption of mouse sera for the elimination of steroid hormones removed the serum dependent immunosuppression from normal as well as pregnant serum. Corticosterone added to the spleen cell cultures depressed also the proliferative response. These findings demonstrate that the response to malaria antigen is decreased in immune mice during pregnancy. The possible effect of serum corticosterone on the depression of the immune response is discussed.

Plasmodium berghei: Relative immunogenicity of infected reticulocytes and infected oxyphilic red blood cells

Hyperbleeding of mice 1 day before and 1 day after infection with Plasmodium berghei resulted in a more aggravated infection. Parasitemia rose significantly faster, but the mean survival time of these mice was not significantly different from control mice. At Day 5 of infection, parasites were almost exclusively in reticulocytes in contrast to control infections in which parasites were found in oxyphilic erythrocytes at Day 5 after infection. Purified parasitized reticulocytes taken from hyperbled mice at Day 5 after infection contained more young developmental parasite stages than purified parasitized oxyphilic erythrocytes taken from normal mice at Day 5 to 7 after infection. Parasitized reticulocytes were more readily opsonized by antibodies from immune serum when compared to parasitized oxyphilic red blood cells and when used to stimulate immune spleen cells the former were better stimulator cells than the latter. Results suggest either that parasitized reticulocytes are more immunogenic then parasitized oxyphilic red blood cells or that suspensions of parasitized reticulocytes contain more immunogenic parasite stages than suspensions of parasitized oxyphilic red blood cells.

ACTH‐dependent modulation of malaria immunity in mice

Summary Tetracosactrin, a synthetic adrenocorticotrophic hormone (ACTH) analogue delivered by osmotic minipumps implanted s.c. in mice induced a dose‐dependent increase of plasma corticosterone levels. In mice with an established immunity to Plasmodium berghei the increase of the plasma corticosterone level due to tetracosactrin treatment correlated with loss of immunity against this malaria parasite. The observed plasma corticosterone levels associated with loss of malaria immunity were of the same order as those in mice that lost their immunity during pregnancy. Adrenalectomy before administration of the ACTH analogue prevented both the increase of plasma corticosterone and loss of malaria immunity. Adrenalectomized mice still lost their malaria immunity when treated with the synthetic corticoid dexamethasone. The effector function of malaria immunity is sensitive to corticoids, and. at least during pregnancy, the naturally occurring serum corticosterone level appears to be an important regulator of malaria immunity.

Stage-specific proteins ofPlasmodium berghei-infected red blood cells detected by antibodies of immune mouse serum

The asynchronously developing malaria parasitePlasmodium berghei was synchronized using in vitro cultivation techniques (Mons et al. 1985). After the infection of naive mice, preparations of parasitized erythrocytes with a high level of synchronism could be obtained. Immunofluorscence and immunoblotting techniques using serum from immunized mice were applied to determine stage-specific immunogenic molecules in the parasitized erythrocyte preparations. These techniques allowed the detection of not only parasitederived but also altered-self molecules. Membrane fluorescence of infected erythrocytes was detected only in preparations containing late trophozoites and schizonts. The appearance of this fluorescence pattern coincided with the presence of immunogenic polypeptides of mol. wt.>200 kD, 86 kD, and 56 kD especially, among some other polypeptides. Preliminary experiments using lactoperoxidase-catalyzed radioiodination suggested that the >200 kD and 56 kD molecules were present at the erythrocyte surface. One molecule with mol. wt. 153 kD was associated with the presence of ringinfected erythrocytes. However, membrane fluorescence of ring-stage-infected erythrocytes was not found. Noninfected erythrocytes sometimes showed membrane fluorescence.

Selective depletion of immunoglobulin isotypes after decomplementation of mouse sera by heat treatment.

Inactivation of mouse serum by heat treatment (30 min at 56 degrees C) decreased antibody reactivity as shown by haemagglutination assays. This appeared to be due to loss of antibody since Ouchterlony analysis showed disappearance of antibody after heat treatment. By both isotype-specific ELISA and Ouchterlony analysis it was shown that mouse IgM, IgG2b and IgG3 are sensitive to heat treatment whereas IgG2a is relatively resistant. IgG1 is not sensitive to heat treatment. To avoid complement-dependent lysis in haemagglutination assays EDTA should be added to the serum samples.

Impaired immune responsiveness in Plasmodium berghei immune mice.

Summary Mice immunized against Plasmodium berghei parasites by drug–controlled infection exhibited decreased immunoresponsiveness against rabbit red blood cells (RRBC). Increasing RRBC antigen dose increased responsiveness, but agglutinating anti–RRBC antibodies of the IgG class remained undetectable. Clearance of colloidal carbon from the bloodstream of malaria–immunized mice was not different from controls. Removal of all the persistent parasites from immune mice did not restore responsiveness until 140 days after treatment, suggesting that the parasite per se did not influence responsiveness directly. Because of this, and because of the fact that priming of mice with RRBC before P. berghei immunization was not more effective than priming after immunization, it was concluded that antigen uptake and subsequent presentation were not impaired in P. berghei immune mice, in contrast to infected mice. Anti–RRBC antibodies were detected in serum of P. berghei immune mice, but regulation of responsiveness to RRBC by transfer of such immune mouse serum was not found. Immunoglobulin levels, especially of the IgG2 and IgG3 subclass were elevated in sera of P. berghei immune mice, which indicated an LPS–like polyclonal activation. The results also suggest that during drug–controlled infection, which leads to immunity against infection, a state of B–cell tolerance is induced.