Fumihiko Takatsuki

The skewing to Th1 induced by lentinan is directed through the distinctive cytokine production by macrophages with elevated intracellular glutathione content

In vivo lentinan (LNT)-elicited peritoneal macrophages (Mps) showed the reduced release of prostaglandins (PGs), IL-10 and IL-6, while it endowed Mps with the elevated capability to produce IL-12 and nitric oxide (NO) upon in vitro triggering, due to the elevated intracellular glutathione (GSH) content in Mps. Deprivation of intracellular GSH completely ablated the production of IL-12. Conversely, lipopolysaccharide (LPS) induced peritoneal Mps with the reduced intracellular GSH content and the reciprocal profile of mediator production. Mps with the elevated intracelluar GSH is arbitrarily termed as reductive Mp (RMp) and that with reduced amount as oxidative Mp (OMp). OMp was converted to RMp when GSH was replenished with glutathione monoethylester (GSH-OEt). The IL-2 administration in combination with LNT exerted the synergistic induction of RMp, resulting in synergistic augmentation of IL-12, NO and reduction of IL-6 production. It was also confirmed that CD4+T cells derived of LNT-administered mice showed augmented IFN-gamma and reduced IL-4 production upon in vitro anti-CD3 stimulation. Taken together it is concluded that skewing of Th1/Th2 balance to Th1 by a beta-(1-3)-glucan, LNT, is directed through the distinctive production of IL-12 versus IL-6, IL-10 and prostaglandin E2 (PGE2) by Mps, depending on intracellular GSH redox status. To the efficient tumor immunotherapy, it may be one of the critical elements to induce a reductive form of Mps in tumor stromal tissues to maintain Th1 response.

In vitro expansion of the murine pluripotent hemopoietic stem cell population in response to interleukin 3 and interleukin 6. Application to bone marrow transplantation

The synergistic action of interleukin 6 with interleukin 3 on the proliferation of a murine hemopoietic stem cell population in a short-term liquid culture system was examined by radioprotective assay. The numbers of colony-forming units in spleen (CFU-S), together with granulocyte/macrophage colony-forming units and viable nucleated cells, were found to increase markedly in culture in the presence of both IL-3 and IL-6, compared with the presence of IL-3 or IL-6 alone. The peak CFU-S value in response to the combination of IL-3 and IL-6 was obtained 6 days after culture initiation, exceeding 5-fold of the input value. Consistent with these data, marrow cells cultured with both IL-3 and IL-6 for 6 days were shown to have a much higher capability of rescuing lethally irradiated mice than did controls. The results may portend the potential clinical use of the combination of IL-3 and IL-6, in particular, in bone marrow transplantation.

Continuous perfusion with interleukin 6 (IL-6) enhances production of hematopoietic stem cells (CFU-S)

The in vivo effect of human recombinant IL-6 on hematopoietic stem cells (colony forming units in spleen, CFU-S) was investigated. Normal mice perfused with IL-6 for 7 days showed an increase in the serum level of IL-6 in a dose-dependent manner. This increase was accompanied by a dramatic enhancement (approximately 8-fold) in the number of spleen CFU-S 7 days after starting perfusion, although heat-treated IL-6 did not exhibit any activities. Enhanced CFU-S number returned to normal at 13 days after cessation of perfusion. These results suggest that IL-6 could be valuable for treating various forms of hematopoietic depletion.

Reconstitution of anti-tumor effects of lentinan in nude mice : roles of delayed-type hypersensitivity reaction triggered by CD4-positive T cell clone in the infiltration of effector cells into tumor

Lentinan, an antitumor polysaccharide used clinically in Japan, requires the intact T cell compartment to manifest its antitumor effects. The aim of the current study was to clarify the mechanisms playing crucial roles in the T cell requirement in the expression of antitumor effects of lentinan. Lentinan treatment of BDF1 mice transplanted intradermally with FBL‐3 induced complete tumor regression and a marked increase in survival time. The antitumor action of lentinan was abolished in mice treated simultaneously with antibodies to CD4 and CD8 antigens, whereas antibody to CD4, CD8 or NK1.1 alone was ineffective. The natural killer, cytotoxic T lymphocyte, and helper T cell activities were already augmented in this FBL‐3/BDF1 system and thus further augmentation of these activities by lentinan was not observed. These activities did not correlate with the antitumor activity of lentinan, as was confirmed in lymphocyte subset depletion experiments. On the contrary, the delayed‐type hypersensitivity (DTH) response against tumor‐associated antigens was triggered by lentinan and was abrogated only in mice treated simultaneously with antibodies to CD4 and CD8 antigens. Furthermore, a non‐cytolytic tumor‐associated antigen‐specific CD4+ T cell clone able to induce the DTH response in concert with lentinan reconstituted the antitumor effects in B6 nude mice when administered with lentinan. These results suggest that, in addition to the augmentation of immune effector cell activity against tumors, infiltration of these cells into the tumor burden initiated by the DTH responses at tumor sites may be involved in eradication of tumors by lentinan.

Curative effects of combination therapy with lentinan and interleukin-2 against established murine tumors, and the role of CD8-positive T cells

The antitumor activity of a combination of an antitumor polysaccharide, lentinan (a β1–3 glucan with β1–6 branches), and interleukin-2 (IL-2) was evaluated against established MBL-2 lymphoma and S908.D2 sarcoma at i.d. sites. Treatment of the MBL-2-tumor-bearing BDF1 mice with lentinan and IL-2 induced complete regression of tumor in 87.5% of mice treated. In contrast, treatments using either lentinan or IL-2 alone failed to induce complete regression of tumor, although temporal growth inhibition of tumor was observed about in half of the mice treated. Improvements of antitumor effects by the combination of lentinan and IL-2 were also observed in the MBL-2/B6 and S908.D2/B10.D2 systems. Expression of the antitumor effects of lentinan/IL-2 treatments required the intact T cell compartment, because the effects were not observed when nude mice were used. In the MBL-2/B6 system, the antitumor action of lentinan/IL-2 treatment was abolished in mice treated with antibody to CD8 antigen, whereas antibodies to CD4 or NK1.1 were ineffective. Furthermore, augmented tumor-specific cytotoxic T lymphocyte (CTL) activity was observed in regional lymph node cells of the mice after lentinan and IL-2 administration. These data indicate that the antitumor effects of lentinan/IL-2 are mediated by CD8+ CTL but not by CD4+ T cells or NK1.1+ NK/LAK cells, and suggest that this combined therapy may be effective against even established tumors that are resistant to IL-2 therapy.

Antitumor and immunological activity of lentinan in comparison with LPS

Lentinan manifests marked antitumor and antimetastatic activity in numerous tumor/host systems, and prevents chemical and viral carcinogenesis. Modulation of immune or vascular functions by lentinan is involved in its antitumor effects. The impact of lentinan on the functions of macrophages is distinct from that of LPS. One of the effects of lentinan on the vascular system is the vascular dilatation and hemorrhage (VDH) reaction, and the effect can be monitored as augmented skin reactions to vasoactive mediators. Lentinan induces the VDH-like reaction at the tumor site, resulting in the induction of hemorrhagic necrosis and complete regression of the tumor. In contrast to LPS-induced tumor necrosis (Shwartzmans-like reaction), lentinan-induced tumor necrosis is T-cell dependent.

Synergistic Antimetastatic Effects of Lentinan and Interleukin 2 with Pre‐ and Post‐operative Treatments

The antimetastatic activity of a combination of lentinan and interleukin 2 (IL‐2) was evaluated against spontaneously metastatic 3‐methylcholanthrene‐induced DBA/2.MC.CS.T fibrosarcoma. Although pre‐operative treatment with either IL‐2 or lentinan alone exerted little effect on the reduction of lung metastasis colony numbers (7.1% or 28.4% reduction, respectively), the combination exhibited a synergistic effect (85% reduction). Furthermore, 3 of 13 mice given the pre‐operative combination treatment achieved complete cure, while no mice given saline did. Although the post‐operative combination treatment also reduced the colony number (71% reduction), it caused little prolongation of survival and no mouse achieved complete cure. Synergistic effects were observed between pre‐ and post‐operative treatments with lentinan and IL‐2: 8 of 12 mice were completely cured. The anti‐metastatic activity was abolished in mice treated simultaneously with antibodies to CD4 and CDS antigens, whereas either CD4, CDS, or NK1.1 antibody alone was ineffective. Analysis of the cellular mechanism involved in the antimetastatic activity revealed the involvement of a tumor‐associated antigen‐specific delayed‐type hypersensitivity response. These data suggest that the life‐prolonging effect of the combination of lentinan and IL‐2 is mediated by antigen‐specific T cells and that the combination of pre‐ and post‐operative therapy with lentinan and IL‐2 may be effective to prevent cancer recurrence and metastasis after surgical resection.

Lentinan: Rationale for Development and Therapeutic Potential

........ . 1. Biological Activities of Lentinan 1.1 Antitumour Activities . . . . 1.2 Antimetastatic Activities . . 2. Mode of Action of Lentinan in Tumour Destruction 2.1 Vascular Activities of Lentinan .. . 2.2 Immunological Activities of Lentinan ... 2.3 Effects of Lentinan on Tumour Progression. 3. Clinical Studies with Lentinan .... . .. . 3.1 Effects on Survival . . . . ..... . 3.2 Mechanisms of Clinical Effects of Lentinan 3.3 Quality of Life 4. Conclusions ....... .. ... . 121 122 122 123 124 124 124 127 130 130 130 131 131

Lentinan augments skin reaction induced by bradykinin: its correlation with vascular dilatation and hemorrhage responses and antitumor activities

The effects of lentinan, an antitumor polysaccharide, on vascular reactions against vasoactive mediators were investigated in murine systems. Lentinan augmented intradermal reactions against bradykinin. Induction of acute phase proteins (APP) and the vascular dilatation hemorrhage (VDH) reaction on the ears have been reported to reflect the host responses to lentinan. The strain difference in the intensity of skin reactions coincided with those observed in VDH responses and with lentinan-induced antitumor effects against Sarcoma 180. Augmentation of skin reactions was not observed in T-cell-deficient mice. Inhibitors of lipoxygenase, thrombin and plasmin which reduced skin reactions also decreased the incidence of tumor necrosis positive mice among FBL-3-bearing mice treated with lentinan. Furthermore, B10D2 mice treated with fluorouracl (5-FU) and lentinan 10 days after S908.D2 transplantation showed complete tumor regression and augmented skin reactions, whereas augmentation of skin reactions and tumor regression were not observed in mice treated with 5-FU and lentinan 32 days after tumor inoculation. Taken together, these results suggest that these vascular reactions might play crucial roles in antitumor effects of lentinan and that the skin reaction, the convenient method for investigating vascular reactions, is a promising tool to monitor host sensitivity to lentinan in antitumor responses.

Effects of a leucine-enriched amino acid supplement on muscle mass, muscle strength, and physical function in post-stroke patients with sarcopenia: A randomized controlled trial

OBJECTIVES The aim of this study was to investigate the effects of a leucine-enriched amino acid supplement on muscle mass, muscle strength, and physical function in post-stroke patients with sarcopenia. METHODS We conducted an eight-wk, two-parallel group intervention, randomized controlled, blinded outcome assessment among 44 post-stroke older patients with sarcopenia. Sarcopenia was defined as a loss of skeletal muscle mass and decreased muscle strength according to the Asian Working Group for Sarcopenia criteria. The intervention group (n = 21) received a leucine-enriched amino acid supplement; the control group (n = 23) did not. Both groups performed low-intensity resistance training in addition to a post-stroke rehabilitation program. A primary outcome of physical function by using the motor domain of Functional Independence Measure (FIM), and secondary outcomes of appendicular muscle mass (skeletal muscle mass index [SMI]) measured via bioelectrical impedance analysis and muscle strength as handgrip strength were measured at baseline and at the end of the intervention. RESULTS The FIM score increased significantly in both groups over time (P < 0.01), with significantly greater improvement in the intervention group than in the control group (P < 0.045). Handgrip strength also increased significantly over time (P <0.05), with significantly greater improvement in the intervention group (P < 0.01). The SMI increased significantly in the intervention group but not in the control group over time, with significantly greater improvement in the intervention group (median estimated difference, 0.50 kg/m2; 95% confidence interval, 0.01-2.11). CONCLUSIONS We demonstrated that an eight-wk intervention consisting of a leucine-enriched amino acid supplementation and low-intensity resistance training increased muscle mass, strength, and physical function in post-stroke patients with sarcopenia.