Humio Osaki

Trypanosoma gambiense: Immunosuppression and polyclonal B-cell activation in mice

The relationship between the suppression of antibody response and polyclonal B-cell activation was studied in mice treated with a cell homogenate of Trypanosoma gambiense. The cell homogenate injection in mice caused a progressive increase in splenic background plaque-forming cell response to sheep erythrocyte. In the mice with markedly increased background plaque-forming cell response, the different reactivity in the primary antibody response to sheep erythrocytes was observed between the intraperitoneal and intravenous immunization with sheep erythrocytes. The intraperitoneal immunization of mice with sheep erythrocytes strongly suppressed the antibody response, while the intravenous immunization with sheep erythrocytes led to an enhancement of the antibody response. The intraperitoneal injection of silica particles, a toxic agent to macrophages, 30 min before intraperitoneal immunization with sheep erythrocytes abolished the suppression of the antibody response completely. In addition, restoration of the suppressed antibody response was found in mice immunized intraperitoneally with a high dose of sheep erythrocytes. It appears that the suppression of antibody response is not attributable to polyclonal B-cell activation, and is associated with the elevation of the phagocytic activity of peritoneal macrophages.

Toxoplasmacidal activity of Obioactin derived from hydrolyzed Toxoplasma immune bovine serum in heterologous cell cultures.

Serum samples were collected from Toxoplasma-infected cattle after challenge and 24 h after Toxoplasma lysate injection. They were hydrolyzed with proteinase, HCl and NaOH. Then substances 3,000 to 5,000 in molecular weight were obtained from fractionation by chromatography. Native serum was collected from cattle immune only to Toxoplasma was affected in homologous bovine monocytes. Of the hydrolyzed fractions, one termed Obioactin inhibited the growth of Toxoplasma in heterologous cells, such as mouse macrophages and kidney cells, canine monocytes, and human heart and brain cells. Its toxoplasmacidal activity was presumed to be derived from its non-specific cell potentiating effects. In mice immunized to Toxoplasma peritoneal macrophages released about 10 times as much superoxide (O-2) and hydrogen peroxide (H2O2) as glycogen-elicited macrophages in normal mice. The release of O-2 and H2O2 from glycogen-elicited and resident macrophages in normal mice showed a tendency to increase up to 72 h of incubation with 0.5% Obioactin . No changes in the O-2 and H2O2 release were found in kidney cells during the period of incubation with Obioactin . In those immunized mice activated macrophages and kidney cells inhibited the intracellular multiplication of Toxoplasma parasites remarkably. It was suggested that the increase in O-2 and H2O2 generating activity in the macrophages might be associated with the enhancement of antitoxoplasmatic activity. The mechanism of inhibition of Obioactin , however, might be different from that of mouse macrophages on the multiplication of Toxoplasma in mouse kidney cells.

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.

天然免疫調整物質オビオアクチン由来合成ペプチド, Obiopeptide- 1, の生物活性

Tachyzoites of Toxoplasma gondii were killed in mouse macrophage and human somatic cell monolayers by a novel synthetic peptide (Obiopeptide-1) which is a Glycil-penta-Glutaminate (GpG) derivative of native Obioactin. In view of the worldwide prevalence of this protozoan disease and the lack of effective treatments, Obiopeptide-1 may be a new and unique antimicrobial active substance of non-antibiotic chemotherapeutic agents for intracellular parasites, T. gondii and associated nonspecific hypoimmune responses that occur in infected hosts.

Effects of bleomycin on kinetoplast DNA and nuclear DNA in trypanosoma gambiense using in situ microfluorometry technique

The effect of Bleomycin, an anti-tumour antibiotic, on either kinetoplast DNA(K-DNA) or nuclear DNA(N-DNA) in a single Trypanosoma gambiense cell was measured successfully utilizing the in situ microfluorometry technique. This microfluorometric assay clearly revealed that the relative fluorescence intensity (RFI) of K-DNA and N-DNA was greatly reduced by bleomycin, suggesting the occurrence of the single strand breaks in these DNAs. The present results have shown that bleomycin acts on N-DNA stronger than K-DNA at least in Trypanosoma gambiense. Microfluorometry technique can be employed valuably for studying the DNA damages of several anti-protozoan drugs.

Studies on the activity of obioactin with acetylspiramycin on mice infected with toxoplasma gondii

In acutely infected mice, acetylspiramycin (ASPM) administered in combination with Obioactin , or sulfamethoxypyrazine ( SMPZ ), prevented Toxoplasma organisms from encysting in the brain and heart tissues. When administered alone, ASPM prevented these organisms from encysting in the heart but not in the brain. All the regimens used failed to reduce the bulk of parasites in the brain and heart tissues of chronically infected mice. Treatment with an ASPM- Obioactin combination, however, brought about the most remarkable reduction, or 52.4%, of the cyst count in the brain of all the methods of treatment applied. Light and electron microscopy of the mice with chronic Toxoplasma infection revealed no changes in the brain or heart tissue of the ASPM- Obioactin treated mice and degenerative changes in the cyst wall and bradyzoites and a remarkable increase of microglias with cysts in the brain in some of these mice.

Antimicrobial activity of Obioactin.

A newly developed enzyme-hydrolyzed toxoplasma-hyperimmune bovine serum preparation, Obioactin , was examined for its anti-microbial and related actions against heterologous protozoa, bacteria, and viruses both in-vivo and in-vitro. An appreciable efficacy of Obioactin in a combination with nifurtimox, Lampit was observed in experimental Trypanosoma cruzi infections in mice. But prophylactic applications of the agent alone or joint with Lampit were in vain in mice. In-vitro digestion of certain bacteria in mouse peritoneal macrophages was accelerated in the presence of the agent being three to ten times stronger than that in untreated macrophages. Inhibitory effect of Obioactin on in-vitro infectivity of viruses was suggested.

In situ Microfluorometry of Whole Kinetoplast and Nuclear DNA in a Single Cell of Trypanosoma gambiense and Trypanosoma cruzi

In situ microfluorometry of whole kinetoplast DNA (K-DNA) and nuclear DNA (N-DNA) from a single trypomastigote cell of Trypanosoma gambiense (strain Wellcome) and Trypanosoma cruzi (strain Tulahuen) was attempted by using the microfluorometer combined with a photon counter. The results obtained were summarized as follows; 1. The K-DNA value of 2K 1N (2 kinetoplasts and 1 nucleus) T. gambiense was about twofold as much as that of 1K 1N on. 2. The N-DNA value of 2K 1N T. gambiense was a little less than two times as much as that of 1K 1N T. gambiense, showing that the N-DNA had already bee increased nearly twofold before the nuclear division was initiated. 3. The K-DNA value of 1K 1N T. cruzi was about fivefold as much as that of 1K 1N T. gambiense. The N-DNA value was contrarily lower than that of T. gambiense, being approximately 70% of the latter. 4. Percent K-DNA/N-DNA value was, in average, about 7 in either 1K 1N or 2K 1N T. gambiense and about 50 in T. cruzi. 5. Percent K-DNA/N-DNA value in individual trypomastigote cell was variable, showing that the increase of K-DNA and N-DNA was not synchronized at least in the two species of trypanosomes used here. 6. The fact that K-DNA and N-DNA values varied from cell to cell although they do not divide, suggested the possibility of DNA increase in the trypomastigote of T. cruzi.

Agglutination Antibody Responses to Trypanosoma gambiense Homogenate in Mice Treated with Dextran Sulfate 500 and Carrageenan

Dextran sulfate 500 and carrageenan, sulfated polysaccharides, have been considered as macrophage-toxic giving profound effects on the immune response. This work deals with agglutination antibody responses to T. gambiense homogenate in mice treated with dextran sulfate 500 and carrageenan. Antibody responses to the first immunization were suppressed in mice treated with the agents before the first immunization but reverse was the case with the response to the second immunization. These suppression and enhancement appeared to be dependent on the timing of treatment in regard to the time of the first immunization. The enhanced antibody response was abolished by either treating the mice with anti-thymocyte serum or transferring of spleen cells treated with anti-thymocyte serum into mice. Thus, the enhancement of antibody response is seemingly attributable for the most part to potent memory T cells induced by treatment with the agents.

In situ microfluorometry of kinetoplast and nuclear DNAs in Trypanosoma gambiense unusual repairment of DNA after treatment with bleomycin

The blood stream form of Trypanosoma gambiense was smeared on a nonfluorescent slide glass with 1 microgram/ml of Hoechst 33258 in 1 mM Tris-HCl buffer (pH 7.2) containing 1% 2-mercaptoethanol and subjected to the in situ microfluorometry. Effects of bleomycin (BL) on the kinetoplast (K)-DNA and nuclear (N)-DNA of T. gambiense were examined in the time course to 6 h after injection of BL into the infected mice. An enhancement of fluorescence occurred 30 min after the injection and then slowed down. This enhancement was due to DNA synthesis both in the K-DNA and N-DNA. This suggests that the strong repairment occurs in both DNAs after treatment with BL.