Masao Okuda

A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic immunopotentiation

Abstract A glycoprotein extract (CVS), derived from the unicellular green alga Chlorella vulgaris, strain CK22, exhibited a pronounced antitumor effect against both spontaneous and experimentally induced metastasis in mice. Inhibition of tumor metastasis was enhanced when intratumor administration of CVS was followed by s.c. injection of CVS. Anti-metastatic immunopotentiation was observed in euthymic mice, but not in athymic nude mice. The antitumor activity of CVS was reflected in antigen-specific, T-cell-mediated immunity. Both CD4 and CD8 T cells contributed to the antimetastatic effects, as shown by in vivo depletion experiments with anti-T-cell subset antibodies. Furthermore, CVS caused the recruitment of T cells to the regional lymph nodes and their proliferation in these organs. The CD4-positive population, following CVS injection at the time of tumor rechallenge, displayed a pronounced increase in the proportion of T cells that were CD18 bright, CD44 bright, CD25+, CD54+, CD69+ or CD71+ in the lymph nodes. Thus, CVS induces T cell activation in peripheral lymph nodes in tumor-bearing mice. We conclude that CVS augments antimetastatic immunity through T cell activation in lymphoid organs and enhances recruitment of these cells to the tumor sites. Presurgical treatment with CVS might prevent metastasis or tumor progression.

Effect of hot water extract of Chlorella vulgaris on cytokine expression patterns in mice with murine acquired immunodeficiency syndrome after infection with Listeria monocytogenes

We have previously reported that oral administration of hot water extract of Chlorella vulgaris (CVE) enhances resistance to Listeria monocytogenes through augmentation of Listeria-specific cell-mediated immunity in normal mice and mice with murine acquired immunodeficiency syndrome (MAIDS) caused by murine leukemia virus (MuLV) LP-BM5. To elucidate the mechanisms whereby CVE augments the cell-mediated immunity, we examined the expression patterns of mRNA for cytokines in normal and MAIDS mice given CVE orally after L. monocytogenes infection. The expression levels of IL-1 alpha, IL-12, GM-CSF, MIP and TNF alpha genes were significantly augmented in the peritoneal adherent cells by oral administration of CVE for 2 weeks before Listeria infection. The expression levels of gamma IFN and IL-12 mRNA were significantly higher in the spleen after Listeria infection in CVE-treated mice than in normal mice, while the expression of IL-10 mRNA in the spleen was decreased by CVE administration. In MAIDS mice, oral administration of CVE also augmented the expression of gamma IFN and IL-12 mRNA in the spleen after Listeria infection, while it rather reduced the expression of IL-10 mRNA. These results suggest that CVE may preferentially augment THI responses against Listeria via activation of macrophages to produce IL-12 and enhance host defence against Listeria infection both in normal and MAIDS mice.

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

Abstract An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD50 value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4 – 9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent.

Hot water extracts of Chlorella vulgaris reduce opportunistic infection with Listeria monocytogenes in C57BL/6 mice infected with LP-BM5 murine leukemia viruses

The bacterial elimination after infection with Listeria monocytogenes was impaired in mice with murine acquired immunodeficiency syndrome (MAIDS) by infection with LP-BM5 murine leukemia virus. Oral administration of hot water extracts of Chlorella vulgaris (CVE) restored the capacity of MAIDS mice to eliminate L. monocytogenes in association with improvement of the deteriorated immune response to L. monocytogenes. DTH response to Listeria in CVE-treated MAIDS mice was significantly higher than that of MAIDS mice after Listeria infection in association with increases in number of CD4+CD8- and CD4-CD8+ alpha beta T-cells in the infected sites. CVE might be effective in the treatment of opportunistic infection in retrovirus-induced immunodeficient patients.

Augmentation of the resistance against Listeria monocytogenes by oral administration of a hot water extract of Chlorella vulgaris in mice.

Oral administration of a hot water extract of Chlorella vulgaris(CVE)(20mg/mouse, 10 consecutive days) augmented the resistance against an i.p. infection with Listeria monocytogenes in mice. The numbers of bacteria in a CVE-administered group were significantly lower in the peritoneal cavity or spleen than those in a control group. FCM analysis revealed that gamma delta +Thy1.2+ cells in the nonadherent PEC from CVE-administered mice increased more prominently in number at the early stage on day 3 or on day 5 after infection as compared with those in control mice. The increment of gamma delta +Thy1.2+ T cells was also evident in spleen in CVE-administered mice at this stage after infection. The proportion of TCR alpha beta +Thy1.2+ T cells in the nonadherent PEC of a control group increased from 13% on day 0 to 49% at the late stage on day 10 after infection, whereas the proportion of TCR alpha beta +Thy1.2+ T cells in the nonadherent PEC in CVE-administered mice increased to 64% on this stage after infection in association with augmentation of DTH response to Listeria. These results suggest that CVE-administration effectively augment cell-mediated immunity against Listeria through the increment of gamma delta + T cells in the early phase and the increment of alpha beta + T cells in the late phase after listerial infection.

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

SummaryThe effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.

Oral Administration of Chlorella Vulgaris Augments Concomitant Antitumor Immunity

Chlorella vulgaris, an unicellular green algae, or its acetone-extract (Ac-Ex) were administered orally to Meth A tumor bearing BALB/c or (BALB/c x DBA/2)F1 (CDF1) mice. When CDF1 mice were fed daily with 10% dried powder of Chlorella vulgaris (CVP) containing diet before and after Meth A tumor inoculation, the growth of rechallenged Meth A tumor was significantly suppressed in an antigen-specific manner. Augmentation of antitumor resistance was exhibited also by Winn assay using lymph node cells of tumor-bearing mice orally administered with CVP or Ac-Ex. Antigen-specific concomitant immunity in these mice were mediated by cytostatic T cells but not by cytotoxic T cells. Natural killer cells seemed not to contribute in antitumor resistance in this system.

Accelerated restoration of the leukocyte number and augmented resistance against Eschericia coli in cyclophosphamide-treated rats orally administered with a hot water extract of Chlorella vulgaris

The effects of oral administration of a hot water extract of Chlorella vulgaris (CVE) on the restoration of the leukocyte number and on the resistance against Escherichia coli infection were examined in cyclophosphamide (CY)-treated rats. Male Fischer rats (F344/DuCrj) were administered orally 1000 mg/kg of CVE for 14 days and injected intraperitoneally with a single dose of CY (50 mg/kg) (day 0) one day after the 14th CVE administration. CVE was further administered continuously after CY treatment until the rats were sacrificed for analysis. The number of bone marrow cells in the CY + CVE group was significantly higher on day 7 after CY treatment than that in the CY-treated group. The number of spleen cells in the CY + CVE group became significantly higher on day 11 than that in the CY-treated group. In the peripheral blood, the number of PMN recovered efficiently in the CY + CVE group in comparison with the CY-treated group on day 7. When E. coli was injected i.p. into normal, CY-treated, and CY + CVE-treated rats on day 6, the difference in number of bacteria among these three groups was most prominent before 6 h, that is, the number in the CY + CVE group was remarkably lower than those in the CY-treated group, and even in the control group, among all organs so far tested.

Augmentation of the resistance against Escherichia coli by oral administration of a hot water extract of Chlorella vulgaris in rats.

In previous studies, we demonstrated that a hot water extract of Chlorella vulgaris (CVE) augmented the resistance against an intraperitoneal infection with Escherichia coli by its intraperitoneal, intravenous or subcutaneous administration. The augmented resistance appeared to be attributable to the enhanced activity of polymorphonuclear leukocytes (PMN). In this study, the effect of oral administration of CVE against Escherichia coli infection was examined. Male Fisher rats (F344/DuCrj) were administered 1000 mg/kg of CVE orally for 14 days and challenged with 2.7 x 10(8) Escherichia coli intraperitoneally. The numbers of living bacteria in the peritoneal cavity, blood, spleen and liver at 1, 6, and 24 h after the inoculation were counted. The bacterial numbers increased during 1-6 h and reached the peak at 6 h in both control and CVE-administered groups. The bacterial numbers decreased to an undetectable level at 24 h in both groups. In a CVE-administered group, the numbers of viable bacteria in each organ were remarkably lower than those in a control group in all organs so far tested. Whereas, the leukocyte numbers, especially PMN numbers, in the peritoneal cavity and peripheral blood maintained higher levels in the CVE-administered group at 6 h after E. coli inoculation. Chemiluminescent responses of peritoneal exudate cells induced by casein or E. coli were higher in a CVE-administered group. These results form the basis for the judgment that the degree of effectiveness of bacteria clearance from the peritoneal cavity shown by oral CVE administration may be strong enough to warrant developing this material as a new type of biological response modifier.

A new type of biological response modifier from Chlorella vulgaris which needs protein moiety to show an antitumour activity

An immunopotentiator obtained from Chlorella vulgaris strain CK22, showed antitumour effects against various lines of syngeneic tumours, especially by intratumour administration. The immunopotentiator exhibited far greater antitumour activity against a rechallenged tumour than against the primary‐inoculated tumour in Meth A and BALB/c or CDF1 mouse systems. The antitumour effect was at least comparable to that of a streptococcal preparation, OK‐432, which has been widely used for clinical immunotherapy.