Takusaburo Ebina

Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis

Abstract We have isolated a novel type of natural tumoricidal product from the basidiomycete strain, Agaricus blazei Murill. Using the double-grafted tumor system in Balb/c mice, treatment of the primary tumor with an acid-treated fraction (ATF) obtained from the fruit bodies resulted in infiltration of the distant tumor by natural killer (NK) cells with marked tumoricidal activity. As shown by electrophoresis and DNA fragmentation assay, the ATF also directly inhibited tumor cell growth in vitro by inducing apoptotic processing; this apoptotic effect was also demonstrated by increased expression of the Apo2.7 antigen on the mitochondrial membranes of tumor cells, as shown by flow-cytometric analysis. The ATF had no effect on normal mouse splenic or interleukin-2-treated splenic mononuclear cells, indicating that it is selectively cytotoxic for the tumor cells. Cell-cycle analysis demonstrated that ATF induced the loss of S phase in MethA tumor cells, but did not affect normal splenic mononuclear cells, which were mainly in the G0G1 phase. Various chromatofocussing purification steps and NMR analysis showed the tumoricidal activity to be chiefly present in fractions containing (1→4)-α-D-glucan and (1→6)-β-D-glucan, present in a ratio of approximately 1:2 in the ATF (molecular mass 170 kDa), while the final purified fraction, HM3-G (molecular mass 380 kDa), with the highest tumoricidal activity, consisted of more than 90% glucose, the main component being (1→4)-α-D-glucan with (1→6)-β branching, in the ratio of approximately 4:1.

Interferon Induction by Glycyrrhizin and Glycyrrhetinic Acid in Mice

Licorice extract is an herbal drug which has long been used as a demulcent and elixir in Chinese medicine. One of the main active components of licorice extract is glycyrrhizin (GL), a kind of saponin. The biological effects of GL and its aglycone, glycyrrhetinic acid (GA), have been extensively studied. Their anti-inflammatory (5), anti-ulcerous (3), and antiviral (14) effects have been reported from 1948 (15) to 1979 (14). Moreover, a preparation of GL combined with glycine and cysteine (SNMC), has been widely and successfully used in Japan as an antihepatitis drug (6, II, 19), although its mechanism of pharmacological action remains unclear. Recently, interferon (IFN) with or without adenine arabinoside (Ara-A) has been used to treat hepatitis B patients (4, 7). Studies show that IFN consistently decreases the level of either DNA polymerase or hepatitis B surface antigen in hepatitis patients. There are also many reports which suggest that some irnmunopotentiators induce IFN (13) and explain their antiviral activity in vivo as interferon mediated (8, 16, 18). Therefore, in the light of GLs clinical effect on hepatitis patients and of the fact that its structure resembles that of hydrocortisone, the possibility that GL induces IFN was proposed. In this study we investigated the ability of both GL and GA to induce IFN in mice. Sixto 8-week-old male DDI mice obtained from the Institute for Experimental Animals, Tohoku University School of Medicine, were used in this experiment. Six-week-old male C3H, ddY, CDF-l, C57BL, BALB/c, and athymic nude mice (nujnu) of BALB/c background were obtained from the Funabashi Farm Co., Ltd., and were used to study the effect of the different mouse strains on interferon induction. GL and GA were supplied by Minophagen Pharmaceutical Co. Drugs were dissolved in 0.01 M phosphate buffered saline (PBS) and adjusted to pH 7.2 with I N sodium hydroxide. Pooled sera obtained from three mice were tested for anti-viral activity, which was determined by the 50% plaque reduction method on L-929 monolayer cell cultures with vesicular stomatitis virus (VSV) and was expressed in international reference units based on NIH reference mouse IFN (Catalog No. 002-904-511) (12).

Induction of interferon-γ in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes

Abstract A bacterial antitumor and immunopotentiating agent, OK-432, induced Interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 × 10 7 spleen cells were cultured in the presence of 5 μg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN-γ of molecular weight approximately 40,000, but also contained IFN-α and IFN-β.

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/ c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (106 cells) and left flank (2 × 105 cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibitition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 × 107 cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

Prevention of rotavirus infection by oral administration of cow colostrum containing antihumanrotavirus antibody

After immunizing 8-month pregnant Holstein cows with human rotavirus, Wa strain, cow colostrum containing neutralizing antibody to human rotavirus, designated as Rota colostrum, was obtained. After randomly grouping 13 infants from a single orphanage, 6 infants received 20 ml of Rota colostrum every morning and 7 control infants received 20 ml of market milk. One month later, rotavirus associated diarrhea was observed in 6 of the 7 infants given milk and 1 out of the 6 infants given Rota colostrum. Orally administered Rota colostrum significantly protected infants from diarrhea caused by rotavirus (P < 0.05). Two out of 5 Rota colostrum recipients who were free from diarrhea showed rises in complement fixation (CF) antibody titer after the rotavirus infection epidemic. Thus, Rota colostrum prevented the outbreak of diarrhea but did not prevent immunological responses to natural rotavirus infection. In the therapeutic trial Rota colostrum had no effect on duration of diarrhea, bowel movements or virus shedding in stool. However, there were no side-effects of Rota colostrum.

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge‐132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat‐ or acid‐treatment, suggesting that the induced IFN is of ‐nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge‐132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge‐132, neither induction of IFN nor augmentation of NK activity occurred, and X‐ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge‐132 are mediated by the induced IFN.

Gastroenteritis in suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a protein-bound polysaccharide preparation (PSK)

Oral inoculation of human rotavirus MO strain (serotype 3) into 5‐day‐old BALB/c mice cause gastroenteritis characterized by diarrhea. Clinical symptoms, histopathological changes in the small intestine, and the detection of rotavirus antigen in enterocytes were all characteristic of rotavirus‐induced gastroenteritis. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of immunoglobulin (IgY) from the yolks of eggs of rotavirus‐immunized hens. When IgY against a rotavirus strain homotypic to the challenge virus (MO strain) was administered in the mice, complete protection against rotavirus infection was achieved. On the other hand, with oral administration of IgY against a heterotypic strain (serotype 1, Wa strain), a lower protective effect was nevertheless obtained. The four different strains of human rotavirus (Wa, KUN, MO, and ST3) were inactivated in vitro by treatment with PSK, a protein‐bound polysaccharide preparation, in a dose‐dependent manner. Oral administration of 2.5 mg of PSK caused a therapeutic effect on experimentally MO‐infected suckling mice. The antiviral effect of PSK was indicated by the reduction of the duration of diarrhea.

In vitro production of immune interferon (IFNγ) by murine spleen cells when different sensitizing antigens are used in vivo and in vitro

OK-432, a killed preparation of Streptococcus pyogenes, as well as bacillus Calmette-Guérin (BCG) and Corynebacterium parvum are all known to induce immune interferon (IFN gamma) in mice. To examine the mechanisms of IFN gamma induction by OK-432, DDI mice were sensitized with various doses of OK-432, either by a single injection of a 1-mg dose or repeated injections of 0.1-mg doses given intraperitoneally. Spleen cells removed from the mice 7-9 days after the last injection produced high-titered IFN gamma (600-800 IU/ml) in vitro in the presence of 5 micrograms/ml of OK-432. In the absence of OK-432, however, in vitro IFN gamma production of sensitized spleen cells was quite limited. Moreover, when inducers of different antigenic entities such as serologically unrelated Streptococcus faecalis, Listeria monocytogenes, or Con A were added in vitro, instead of OK-432, to the OK-432-sensitized spleen cells, high-titered IFN gamma production also occurred. This may indicate that the signal required by T cells to produce IFN gamma in vitro need not necessarily be the same as that required to sensitize mouse macrophage in vivo. Once sensitized with OK-432, mice spleen cells continued to produce high-titered IFN gamma for more than 3 but less than 5 weeks.

Clinical effects of MND-19 (Inosiplex) on subacute sclerosing panencephalitis: — A multi-institutional collaborative study—

A total of 151 cases of subacute sclerosing panencephalitis (SSPE), comprising 89 cases treated with MND-19 (Inosiplex) and 62 untreated cases, were retrospectively investigated as to background characteristics, survival rate and clinical course in order to compare the findings in the 2 groups of cases. The survival rate for the cases treated with MND-19 (MND-19-treated group) was significantly higher than that for the untreated cases (control group), which was also true on stratified analysis or on smoothing of the background factors by means of Coxs multiple regression model. Investigation of the clinical course revealed that progression through the disease stages was significantly slow in the MND-19-treated group, compared with in the control group. Global rating of the clinical course showed that a prolonged remission was obtained in more MND-19-treated cases than control cases. The measles virus antibody titer was in no way affected in the former group. Side effects of MND-19 were observed in 17 of the 89 treated cases (19.1%).

Two Distinct Electrophoretic Migration Patterns of RNA Segments of Human Rotaviruses Prevalent in Japan in Relation to Their Serotypes

Polyacrylamide gel electrophoretic analysis of viral nucleic acids has revealed II segments of double-stranded RNA patterns in rotaviruses (8). On the basis of differences in the migration of RNA segments on gels, Espejo et al have demonstrated the presence of two distinct RNA patterns among human rotaviruses obtained from different epidemics (1-5). Most recently, the existence of a correlation between the RNA patterns of human rotaviruses and their serotypes (subgroups) has been demonstrated (6). This communication describes the existence of two distinct RNA patterns among rotaviruses obtained from infants hospitalized with acute diarrhea between 1975 and 1980 in Sendai and Yamagata. Eighteen rotavirus strains were isolated and examined in this study. Extraction and purification of the rotaviruses were carried out by the method developed by Espejo et al (I). As a standard strain, the Wa strain of human rotavirus type 2, which was kindly provided by Dr. R.C. Wyatt (National Institutes of Health, U.S.A.) was used. The Wa strain was grown in MAI04 cells. The purified virus was disrupted at 100 C for 2 min in 0.0625 M Tris-HCl, pH 6.8, containing 1% (w/v) sodium dodecyl sulfate, 4 M urea and 5% (v/v) 2-mercaptoethanol. For electrophoresis of the RNA, 10% (w/v) polyacrylamide-0.27% (w/v) bisacrylamide slab gels were prepared. Appropriate amounts of samples were applied to the gels and electrophoresis was carried out at the constant current of 12 mAjslab gel for 8 hr. The gels were subsequently stained for 15 min with ethidium bromide solution (I !,g/ml in 0.02 M Tris-HCl, pH 7.4) and photographed. Figure 1 shows the RNA gel electrophoretic profiles of rotavirus strains obtained in Sendai (80S1, 80SR002; 80S2, 80SR004) and in Yamagata (80Yl, 80Y003; 80Y2, 80YOOI) in 1980 and the Wa strain. When compared with the RNA pattern of the Wa strain, the most striking difference in mobility was found in RNA segments 10 and II of the 80S1 and 80S2 strains, which moved significantly more slowly than those of the Wa strain. In contrast, as far as RNA segments 10 and 11 are concerned, no difference was found between the Wa strain and 80Yl or 80Y2. The RNA pattern with slower moving 10th and 11th seg-