Tadatoshi Miyagami

Microbicidal Activity of Toxoplasma Immune Beagle Plasma and Lymphokines to Toxoplasma Multipliction in Host Cells

In beagles infected with Toxoplasma tachyzoites, IgM and IgG humoral antibodies appeared on the early period of infection as the expression of the humoral antibody response. IgM antibody was no longer observed after 16 days, however, IgG antibody persisted maintaining high titers up to the last day of the experiment. When challenged on the 13th day postinfection, IgG antibody levels were boosted, however, IgM levels did not show any change at all. Lymphokines obtained from spleen cells collected 10, 30, 100 and 300 days postinfection showed a gradual increase in the ability to inhibit Toxoplasma multiplication in normal canine monocytes accordingly compared to the control which showed no inhibition at all. Toxoplasma lysate antigen was injected intravenously to hyperimmuned dog 2 wk after rechallenge. Plasma collected 24 hr after injection inhibited markedly Toxoplasma multiplication. No change in the microbicidal activity was found when plasma was absorbed with anti-canine IgG compared to the unabsorbed one. The results therefore indicated that the substance inhibiting Toxoplasma multiplication in canine monocytes existing in plasma is not derived from antibodies specific for Toxoplasma but as a Toxoplasma growth inhibitory factor (Toxo-GIF) or a similar substance. The plasma obtained also showed antiviral activity of interferon type II and was found to increase in the circulation 6 hr after Toxoplasma lysate antigen injection.

Plasmodium berghei: Long lasting immunity induced by a permanent attenuated mutant*

A long-lasting immunity against challenge with highly virulent Plasmodium berghei (NK65) was observed in Balb/c mice immunized with a permanently attenuated parasite (XAT), a derivative (XAT) of the NK65 strain. Mice infected with living XAT parasites showed an extremely low self-resolving type of parasitaemia followed by a strong immunity against a challenge with the lethal parent NK65 strain. This immunity lasted for nearly one year. Cross immunity was also observed in the immune mice after challenges with P. berghei ANKA, P. yoelii 17XL, P. vinckei, and P. chabaudi. In addition stage specific immunity was found after P. yoelii nigeriensis sporozoites were inoculated into immune mice. Late radical treatment of immunized mice had little effect on the immunity. Both IgG and IgM anti-Plasmodium titers did not correlate with the degree of immunity. Immune suppression was not observed in the test mice as far as responsiveness of spleen cells to several mitogens is concerned.

An alternative approach to malaria vaccine with a permanent attenuated mutant from a high virulence Plasmodium berghei strain.

An alternative approach to malaria vaccine with the use of Plasmodium berghei NK65XAT (XAT) is reviewed. XAT is a permanent low virulence strain derived from high virulence P. berghei NK65 (NK65) by irradiation. Although one organism of parent NK65 could kill one mouse, as many as 10(7) XAT parasites caused modest self limiting parasitaemia in immuno-competent mice. In the mice recovered from XAT infection, long lasting immunity to challenge not only by parent NK65, but also by ANKA so far as different species of rodent Plasmodia was seen. The XAT parasites invaded selectively into immature erythrocytes. Because of this feature, the attenuated parasite might induce potent and long-lasting immunity presumably with the background of MHC antigen expression on infected cells. Immunopathologic reactions in mice infected with XAT were modest comparing to those seen in mice with parent NK65 infection. Attenuation was also tested using P. yoelii nigeriensis with which cyclical transmission with A. stephensi was established. Although similar attenuation occurred by X-ray irradiation, produced parasites eventually reverted to virulence after several animal passages. Irradiation was also attempted to induce attenuated P. falciparum mutant and a parasite of a slow multiplication feature was obtained in an experiment. We would propose an alternative approach in the study of malaria vaccine using attenuated live organisms which confers potent and long lasting immunity to the host.

Modulator effect of Toxoplasma lysate antigen in mice experimentally infected with Plasmodium berghei

Normal mice were pretreated twice at an interval of 2 weeks with an emulsion of TLA (Toxoplasma lysate antigen), PLA (Plasmodium lysate antigen) or both in LMO (light mineral oil) or with a combination of the emulsion and Obioactin or Tp-LKs (Toxoplasma lymphokines) as an immunopotentiator. They were then given Obioactin or Tp-LKs 3 and 25 days after the first treatment and were further given parasitized erythrocytes with 1 X 10(2)-10(4) P. berghei 2 weeks after the second treatment. Thirty (3/10, number of survival/number of examined) per cent of mice treated with TLA, 50 (5/10)% of those treated with a combination of TLA and Tp-LKs and 60 (6/10)% of those treated with a combination of TLA and Obioactin survived as long as 20 days postinfection while none of untreated controls survived more than 15 days postinfection. Only 18.2 (2/11)% of mice treated with PLA or TLA + PLA survived and 20 (2/10), 18.2 (2/11) and 60 (6/10)% of those treated with TLA + Obioactin, PLA + Obioactin or TLA + PLA + Obioactin survived throughout the experiment, respectively while none of controls survived more than 13 days postinfection. Five mice of each group were killed right before infection, and 5, 10 and 15 days postinfection. In mice treated with TLA + Obioactin, more macrophage phagocytosis and macrophage migration inhibition induced by sensitized T-cells were observed than in those treated otherwise. No appreciable differences were noted according to the method of treatment in blood examination values. Cross immunities between Toxoplasma and Plasmodium antigens were tested by counter-immunoelectrophoresis and indirect fluorescent antibody technique. By using counter-immunoelectrophoresis, a specific precipitin line was observed between TLA and anti-PLA which was absorbed by mouse erythrocytes, leucocytes and liver powder. By the indirect fluorescent antibody technique, anti-Plasmodium IgM and IgG titers were detected in sera from mice treated with TLA or TLA-Obioactin before infection.

Correlation between release of reactive oxygen intermediates and inhibition of toxoplasma multiplication in mouse peritoneal and alveolar macrophages and kidney cells after in vitro incubation with obioactin, lonomycin a, muramyl dipeptide, lipopolysaccharide or toxoplasma lysate antigen

The inhibition of Toxoplasma multiplication inside cells does not correlate with an enhanced release of oxygen intermediates except in the case of peritoneal macrophages treated with Obioactin. The inhibition observed in alveolar macrophages treated with Obioactin, in kidney cells treated with Obioactin or lonomycin A and in peritoneal macrophages treated with lonomycin A was not accompanied by an increment of release of oxygen intermediates. Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) enhanced the release of toxic oxygen intermediates in peritoneal macrophages, but did not have any toxoplasmacidal effect. Adenosine triphosphate (ATP) content increased during Obioactin, MDP or Toxoplasma lysate antigen (TLA) treatment. The actual oxygen consumption of the peritoneal macrophages treated with Obioactin increased dose dependently, but that of TLA-, lonomycin A- or MDP-treated cells did not change. These results suggest that the relationship between the intracellular killing of Toxoplasma protozoa and the release of oxygen intermediates differs according to the cells and/or the stimuli, and that the cellular mechanism of Toxoplasma killing in the peritoneal macrophages treated with Obioactin involves an energy-dependent mechanism.