Hidetaka Sasaki

Effect of Glycyrrhizin, an Active Component of Licorice Roots, on HIV Replication in Cultures of Peripheral Blood Mononuclear Cells from HIV-Seropositive Patients

The effect of glycyrrhizin (GR) on HIV replication in cultures of peripheral blood mononuclear cells (PBMC) from HIV-infected patients was investigated. After the depletion of CD8+ T cells, PBMC from HIV+ patients (patient PBMC) and PBMC from healthy donors (healthy PBMC) were cocultured in the presence or absence of GR (100 µg/ml) for 21 days. In cultures of 13 of 42 samples of patient PBMC (13/42, 31%), GR inhibited more than 90% of HIV replication. Among 42 samples of patient PBMC, 20 were identified to be infected with a non-syncytium-inducing variant of HIV (NSI-HIV), 15 with a syncytium-inducing variant of HIV (SI-HIV), and the remaining 7 were classified as cells infected with SI-HIV and/or NSI-HIV. GR inhibited more than 90% of HIV replication in cultures of 12 patient PBMC samples infected with NSI-HIV (12/20, 60%). In patient PBMC infected with SI-HIV, GR inhibited HIV replication in only 1 patient (1/15, 7%). In cultures of patient PBMC, GR induced the production of CC chemokine ligand (CCL)4 and CCL5 in a dose-dependent manner. When the assay was performed in PBMC cultures supplemented with a mixture of monoclonal antibodies for CCL4 and CCL5, no evidence of anti-HIV activity of GR was found. These results indicate that GR has the potential to inhibit NSI-HIV replication in patient PBMC cultures by inducing the production of β-chemokines.

An immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, Z-100, restores the balance of Th1/Th2 cell responses in tumor bearing mice

The regulatory effect of Z-100 on the balance of Th1/Th2 cell responses in BALB/c mice bearing Meth-A fibrosarcoma was investigated. In tumor bearing mice, Th1 cytokine production (IL-2, IFN-gamma) are suppressed and Th2 cytokine production (IL-4, IL-10) are increased, as compared with those of normal mice. The administration of Z-100 (10 mg/kg) to tumor bearing mice restored the balance of Th1/Th2 cell responses from Th2 dominant state to the normal state. This regulatory effect of Z-100 was eliminated by depletion of adherent cells from splenocytes derived from tumor bearing mice, and by the treatment with 2-ClAdo (a macrophage inhibitor). Similarly, this regulatory effect was diminished by the treatment with anti-IL-12 mAb and anti-IFN-gamma mAb. In addition, the IL-12 p40 mRNA expression in splenic adherent cells and IFN-gamma mRNA expression in CD4+ T cells were increased by the administration of Z-100 to tumor bearing mice. These results suggested that Z-100 restored the balance of Th1/Th2 cell responses to the normal one in tumor bearing mice through the activation of macrophages and up-regulation of IL-12 production from macrophages and IFN-gamma production from CD4+ T cells.

Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli, on the pulmonary metastases of Lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor, IL-4

In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p<0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p<0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5×107 cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.

Antiviral effects of recombinant human tumor necrosis factor-alpha in combination with natural interferon-beta in mice infected with herpes simplex virus type 1

The protective effects of combination therapy utilizing recombinant human TNF-alpha (rTNF-alpha) and natural murine interferon-beta (IFN-beta) in mice infected with herpes simplex virus type 1 (HSV-1) was investigated. Mice treated with rTNF-alpha alone at all of the doses tested (a single i.v. administration, 2.3-2,300 micrograms/kg; multiple i.p. administrations 0.4-250 micrograms/kg) as well as mice that received IFN-beta alone at doses of 16 x 10(4) U/kg or less resulted in a 0% survival rate. Combination therapy consisting of a single administration of rTNF-alpha (230 and 23 micrograms/kg) and multiple administrations of IFN-beta (4 x 10(4) U/kg) resulted in a 40% and 60% survival rate. Multiple treatments of infected mice with rTNF-alpha (50 and 10 micrograms/kg) in combination with IFN-beta (4 x 10(4) U/kg) resulted in 50% and 70% survival rates, respectively. These results suggest that the combination therapy of rTNF and natural murine IFN-beta produce synergistic protective effects in mice infected with a lethal amount of HSV-1.

Anti‐Tumor Mechanism of Z‐100, an Immunomodulatory Arabinomannan Extracted from Mycobacterium tuberculosis Strain Aoyama B, on Pulmonary Metastases of B16F10 Melanoma: Restoration of Helper T Cell Responses via Suppression of Glucocorticoid‐Genesis

In the present study, the anti‐tumor mechanism of Z‐100 was investigated with the use of pulmonary metastasis of B16F10 melanoma. In B16F10 mice, Th1 cytokine production (IL‐2, IFN‐γ) was suppressed in comparison with normal mice. On the other hand, Th2 cytokine production (IL‐4, IL‐10) was increased in the B16F10 mice. The administration of Z‐100 to B16F10 mice restored the balance of Th1/Th2 cell responses from the Th2 dominant state to the normal state. Z‐100 significantly suppressed the pulmonary metastasis of B16F10 melanoma in a dose‐dependent manner. These results suggest that Z‐100 restored the breakdown of Th1 cell responses, resulting in the suppression of pulmonary metastasis of B16F10 melanoma. Moreover, Z‐100 decreased the corticosterone levels, which is known to suppress the Th1 cell responses, in both serum specimens and splenic tissue, and the steroidogenic CYP11A1 mRNA expression in CD4+ T cells. These results suggest that a suppression of pulmonary metastasis and restoration of Th1/Th2 cell responses by Z‐100 may be due to the decrease in the corticosterone levels and the steroidogenic CYP11A1 mRNA expression of CD4+ T cells in B16F10 mice. Further, the role of Th1 cytokine, IFN‐γ, on these activities of Z‐100 was examined. The suppressive effects of Z‐100 on pulmonary metastasis and restoration of Th1/Th2 cell responses were eliminated by the administration of anti‐IFN‐γ mAb. Moreover, the suppressive effects of Z‐100 on glucocorticoid‐genesis were eliminated by the administration of anti‐IFN‐γ mAb. These results suggest that Z‐100 restores the balance of Th1/Th2 cell responses via the suppression of glucocorticoid‐genesis by Z‐100‐induced IFN‐γ. IFN‐γ acts as a key cytokine in anti‐tumor activities of Z‐100.

Cryptococcal encephalitis in thermally injured mice is accelerated by type 2 T-cell responses.

Objective To explore the pathogenic role of burn-associated type 2 T-cell responses on the development of cryptococcal encephalitis in mice with severe thermal injuries. Design Experimental Cryptococcus neoformans infection in normal mice was compared with that in thermally injured mice (TI mice), normal mice treated with a mixture of interleukin (IL)-4 and IL-10, or normal mice inoculated with burn-associated type 2 T cells. Setting University research laboratory. Subjects Male BALB/c mice, 8 to 10 wks of age. Interventions We prepared four groups of mice as follows: a) normal mice, b) TI mice, c) normal mice treated with the IL-4/IL-10 mixture, and d) normal mice inoculated with burn-associated type 2 T cells. These groups of mice were anesthetized and exposed to 1 × 10 cells/mouse of C. neoformans intratracheally. Cryptococcal growth in brains and lungs in normal mice were compared with those of the other three groups. Also, cytokine-producing profiles of T lymphocytes from brains of both normal mice and TI mice were determined. Measurements and Main Results Compared with normal mice, TI mice were susceptible to C. neoformans infection. At the maximum (15 days after infection), numbers of C. neoformans organisms in brains of TI mice were 10 times higher than those of the pathogen in brains of normal mice. After stimulation with anti-CD3 monoclonal antibody, IL-4 (but not interferon gamma) was produced in cultures of T lymphocytes from brains of TI mice 15 days after the infection, whereas the same cell preparation from normal mice produced interferon gamma (but not IL-4). TI mice and mice that were treated with a IL-4/IL-10 mixture or inoculated with burn-associated type 2 T cells were equally susceptible to the cryptococcal infection. Conclusions Burn-associated type 2 T cells or their cytokine products play a key role in the severity of cryptococcal encephalitis that develops in TI mice.

Z-100, a polysaccharide-rich preparation extracted from the human type Mycobacterium tuberculosis, improves the resistance of Meth-A tumor-bearing mice to endogenous septic infection

The effect of Z-100, an immunomodulatory arabinomannan extracted fromMycobacterium tuberculosis, on cecal ligation and puncture (CLP)-induced sepsis in mice bearing Meth-A fibrosarcoma was investigated. When normal BALB/c mice were subjected to the CLP procedure, their mortality rate was 17%. On the other hand, an increased mortality was observed in tumor-bearing mice subjected to CLP 10 days after tumor inoculation, and then all mice died when tumor- bearing mice were subjected to CLP 20 days after tumor inoculation. However, the increased percent mortality was decreased by 50% when these mice were injected intraperitoneally with a 10 mg/kg dose of Z-100. When splenocytes (5 × 107 cells), obtained from Meth-A tumor-bearing mice 20 days after tumor inoculation, were transferred intravenously to normal mice (recipient mice), mortality of these recipient mice were increased by 62% as compared with that of the control (22%). However, no increased mortality (25%) was observed in recipient mice which were transferred with splenocytes from tumor-bearing mice injected intraperitoneally with Z-100 (10 mg/kg). In addition, suppressor cell activity was demonstrated in splenocytes from Meth-A tumor-bearing mice at 20 days after tumor inoculation using one-way mixed lymphocyte reaction. However, the suppressor cell activity was significantly decreased by the intraperitoneal administration of a 10 mg/kg dose of Z-100 (p<0.01). The increase of mortality in recipient mice by adoptive transfer of mononuclear cells (MNCs) from tumor-bearing mice was not detected when these MNCs were treated with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb, but an increase was seen with anti-Lyt 1.2 mAb or anti-immunoglobulin antiserum treated MNCs.These results suggest that the suppressor cells affect the mortality of CLP-induced sepsis and Z-100 may have a therapeutic activity against opportunistic infections in immunocompromised hosts through the regulation of suppressor T-cells.

Effects of Z-100, a Mycobacterium-tuberculosis-Derived Arabinomannan, on the LP-BM5 Murine Leukemia Virus Infection in Mice

Objective: To investigate the effects of Z-100, an arabinomannan extracted from Mycobacterium tuberculosis, on the LP-BM5 murine leukemia virus (LP-BM5 MuLV) infection in mice. Methods: C57BL/6 mice infected intraperitoneally with 4.5 × 102 PFU/mouse of LP-BM5 MuLV (MAIDS mice) were treated intraperitoneally with a 10-mg/kg dose of Z-100 every other day beginning 1 day after the viral infection. MAIDS mice treated with Z-100 were compared with control mice (MAIDS mice treated with saline) for their survival and splenomegaly after LP-BM5 infection. Cytokine-producing profiles of splenic T cells from these two groups of mice were also compared. Results: When MAIDS mice treated with Z-100 were compared with those of control mice, a decrease in splenomegaly and lymphadenopathy was observed. Splenomegaly was markedly enhanced in MAIDS mice treated intraperitoneally with IL-4 or IL-10. When MAIDS mice were treated with Z-100, their survival rates were significantly increased compared to those of controls. Splenic T cells from control mice produced type-2 cytokines (IL-4 and IL-10). However, a decreased production of type-2 cytokines by splenic T cells from MAIDS mice treated with Z-100 was demonstrated. Conclusion: Z-100 could decrease the severity of the LP-BM5 MuLV infection through the regulation of MAIDS-associated type-2 T-cell responses.

The effect of combination therapy of radiation and Z-100, an arabinomannan on tumor growth in mice

The effect of radiation combined with intraperitoneal administration of Z-100, an immunomodulatory arabinomannan extracted fromMycobacterium tuberculosis, was studied using Meth A fibrosarcoma in BALB/c mice and metastasis of Lewis lung carcinoma, 3LL, in C57BL/6 mice.In mice bearing Meth A fibrosarcoma, a moderate degree of growth inhibition was observed in the group of single therapy with Z-100 or radiation (10 Gy). When radiation was combined with Z-100, the tumor growth was significantly inhibited. In mice bearing 3LL, slight inhibition of pulmonary metastasis was observed in the group of single therapy, while significant degrees of inhibition of primary tumor growth and pulmonary metastasis were observed in the combination group. This suggests the usefulness of combined use of Z-100 in radiation therapy.