Strain differences in the cytotoxic activity and TNF production of murine macrophages stimulated by lentinan.

The effect of lentinan, a glucan type immunomodulatory polysaccharide was studied on the antitumor cytotoxicity and on the TNF secretion of peritoneal macrophages in inbred H-2 congeneic mouse strains under in vivo and in vitro conditions. The cytotoxic activity and TNF secretion of murine macrophages was found to be elevated by lentinan in vitro and in vivo conditions. The effectiveness of lentinan to induce cytotoxicity and TNF secretion was highly influenced by the genotype of the host. The increased cytotoxicity of macrophages was modified by the H-2 and the background genes. The black background and the H-2a and H-2d haplotypes were associated with high responsiveness, while the albino and agouti background and the H-2b and the H-2k haplotypes with low responsiveness to lentinan treatment. The degree of TNF secretion of macrophages stimulated by lentinan was influenced by the H-2 genes only. Similarly, to the macrophage cytotoxicity the TNF secretion in the H-2a and H-2d haplotypes were found to be high, on the other hand, in the H-2b and H-2k were low.

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 (Shwartzman's-like reaction), lentinan-induced tumor necrosis is T-cell dependent.

Stimulation of microbicidal host defence mechanisms against aerosol influenza virus infection by lentinan.

The ability of polysaccharide immunomodulator lentinan to stimulate non-specific resistance against respiratory viral infections was investigated. Significant protection was conferred by lentinan administered intranasally before lethal influenza virus infection and could be corroborated by a reduction of the lung virus titres. Since the lung is the target organ of influenza virus infection, lentinan was also administered by the intravenous route. Lentinan conferred complete protection against a LD75 challenge dose of virulent influenza virus and significantly prolonged the survival time after a LD100 challenge. The effect on respiratory burst of broncho-alveolar macrophages was investigated by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. Enhanced CL activity was present at an early stage in groups receiving lentinan. Significant nitric oxide activity could also be stimulated by culturing broncho-alveolar macrophages in the presence of lentinan. TNF activity could not be detected in lung lavage but measurable IL-6 was produced already after 6 h in animals administered lentinan alone and in lentinan-pretreated influenza virus-infected mice. Influenza virus alone did not induce measurable IL-6 at 6 h but high activity was present at later time periods.

Recent progress in immunopharmacology and therapeutic effects of polysaccharides.

Lentinan, a (1----3)-beta-D-glucan with (1----6)-beta-D-glucopyranoside branches and its related polysaccharides have marked antitumour activity in allogeneic, syngeneic and autochthonous primary hosts, suppress chemical and viral oncogenesis, and prevent cancer recurrence or metastasis after surgery. Results of the clinical application of lentinan have proven prolongation of life-span of the patients with advanced and recurrent stomach, colorectal and breast cancer with only little toxic side effect. These polysaccharides also increase host resistance to various kinds of bacterial, viral and parasitic infections including AIDS. Lentinan appears to represent Host Defence Potentiators (HDPs), which can restore or augment the ability of responsiveness of the host to lympho-cytokines or other intrinsic bioactive factors through maturation, differentiation or proliferation of the important cells for host defence mechanisms. That is, HDPs might make the physiological constitution highly cancer and infection-resistant, which may be a concept in Oriental Medicine, the fundamental principle of which is to regulate homeostasis of the whole body and to bring the diseased person to his normal state. HDPs such as lentinan are the most appropriate drugs to prevent cancer recurrence, or the manifestation of AIDS symptoms in HIV carriers.

The effect of lentinan on proliferative processes in parenchymal organs of rats--I. The effect on pyrimidine and nucleic acid syntheses.

The present study investigated the effect of Lentinan on the biochemical events associated with the pyrimidine and nucleic acid syntheses in the liver, kidney, thymus and spleen of rats. Lentinan was used at a dose of 4 mg/kg/day (twice) and in a single dose of 20 mg/kg. The following changes were observed. (1) The utilization of (14C)orotic acid for the synthesis of uridine components of liver acid-soluble extract and RNA uracil was activated after the administration of both doses of the drug. The specific activity of cytidine components of the acid-soluble extract and RNA were, on the other hand, not affected. The same holds true for the kidney. The ratio of the specific activity of cytidine:uridine components of the acid soluble extract as well as RNA decreased after the administration of both doses of the drug. The specific activity of DNA cytosine and thymine are slightly suppressed in the liver after the administration of a high dose of Lentinan; no effect was observed in the kidney. (2) The uptake of (14C)cytidine by the liver was not affected; the specific activity of DNA cytosine and thymine were increased after the administration of a high dose of Lentinan. (3) The uptake of (14C)thymidine by the liver was not affected; the specific activity of liver DNA thymine was increased after the administration of both doses of the drug. In the thymus an increase of specific activity of DNA thymine has also been observed. (4) Repeated doses of the drug (4 mg/kg for 6 consecutive days) increased the weight of the spleen. The specific activity of DNA thymine of the liver and spleen were significantly increased.

Effect of lentinan on superoxide dismutase enzyme activity in vitro.

The effects of lentinan on enzyme induced lipid peroxidation, xanthine-xanthine oxidase-induced cytochrome c reduction, and on the superoxide-dismutase (SOD) enzyme activity and expression of human lymphocytes and erythrtocytes were studied. Lentinan in low concentration decreased SOD activity of lymphocytes and erythrocytes from healthy subjects. In higher concentration (10 micrograms/ml) lentinan increased the pathologically low SOD activity of erythrocytes and lymphocytes of patients with cirrhosis of the liver. No significant antioxidant (free radical scavenger) effect has been observed in NADPH-induced and Fe3+-stimulated lipid peroxidation and in xanthine-xanthine oxidase system.

Effect of lentinan and mannan on phagocytosis of fluorescent latex microbeads by mouse peritoneal macrophages: a flow cytometric study.

Lentinan, an immunopotentiating beta-1,3-glucan polysaccharide stimulated the in vitro phagocytosis of BSA-coated, C3b- or monoclonal immunoglobulin (IgG2b)-coated fluorescent microspheres by resident or thioglycollate-elicited mouse macrophages in a dose-dependent manner. Analysis of flow cytometric data has shown that microbead phagocytosis of resident macrophages, which exhibit a lower basic phagocytic activity than the thioglycollate elicited ones, has been augmented by up to 900% due to lentinan. The percent ratio of phagocytes among peritoneal exudate cells, however, remained unchanged after short-term lentinan stimulation. Preincubation of the cells with lentinan resulted in increased ingestion of the microbeads. Activation of phagocytosis by lentinan is therefore due in part to the direct stimulation of the cells, however, lentinan also serves as supplementary opsonin for C3b-coated beads. Mannan inhibited the ingestion of C3b-coated microspheres by 75%, which was abolished in part when lentinan was also added to the cells. Mannan did not influence the phagocytosis of BSA-coated or IgG-coated beads. Our data, based solely on in vitro studies, suggest a beta-glucan receptor mediated activation of phagocytes by lentinan. These receptors are different from the C3b, Fc or mannose receptors. It is very likely that stimulation of phagocytic activity of macrophages by lentinan may contribute to the antitumor action of this immunopotentiating polysaccharide.

[Evaluation and consideration of BRM-BRM and HDP (host defense potentiators)].

It should first be stressed that the term BRM is wrong and unscientific, since this would include potassium cyanide or cancer chemotherapeutics in the strict sense of the term. Therefore, in this article we discuss the evaluation of Host Defence Potentiators (HDP). IL-2 or TNF should not be included as HDP because their action is local and not selective to cancer cells, similar to the case of cancer chemotherapeutics. IL-2 is not useful without the presence of IL-2-responsive cells in the host. The most important facet of the action of HDP is to increase the response of the host to cytokines or other bioactive substances according to the degree of maturation, differentiation or proliferation of responsive cells in the host defence mechanism. Lentinan appears to represent a unique class of HDP, markedly potentiating host resistance to cancer and bacterial, viral and parasitic infections, and shows prominent antitumor activity in syngeneic and autochthonous hosts, suppressing chemical and viral oncogenesis. The most important target of HDP is complete prevention of recurrence after "curative" surgery fundamentally through growth inhibition and regression of a small number of autochthonous tumor cells scattered in the host. Considering the excellent end-point results for phase III advanced and recurrent gastrointestinal and breast cancer, lentinan seems to be the most hopeful drug against cancer recurrence. The development of various new types of MDP mediating host homeostasis in the immune, endocrine and nervous systems and nutritional states is expected.

Regression induced by lentinan, of peritoneal carcinomatoses in a model of colon cancer in rat.

Lentinan has been tested in a model of colon cancer in rats. Peritoneal carcinomatoses were induced in BDIX rats by i.p. injections of syngeneic cells isolated from a colon carcinoma, and established in a permanent cell line. The treatment consisted of five i.p. injections, 2 days apart, of 2 mg lentinan/kg at a concentration of 200 micrograms/ml. This was started on day 14 after tumor cell injection, when the rats bore numerous nodules of 1-5 mm. Lentinan significantly inhibited the growth of carcinomatoses. Eleven out of the 20 rats treated with the best lentinan therapy were tumor free at autopsy on day 42. Lentinan significantly increased the life span of carcinomatous rats. The half life following tumor cell injection was 42 days in the control and 70 days in the treated group. Four out of 10 treated rats were still alive on day 210. They were tumor free at autopsy, whereas all the controls died between the 40th and the 70th day. The effectiveness of lentinan was dependent on the number and frequency of the injections. A dose effect was obtained and a strong influence of the concentration was shown.

The effect of lentinan on the resistance of mice to Mesocestoides corti.

CBA/H mice were given the immunomodulator lentinan in multiple, ascending doses before (prophylactic) or after (therapeutic) inoculation with tetrathyridia of Mesocestoides corti or as a single prophylactic dose. The latter was without effect, but increasing multiple prophylactic and therapeutic doses of lentinan resulted in a marked reduction in the numbers of parasites in the peritoneal cavity, particularly in those mice that received lentinan therapeutically. In mice that received multiple doses of lentinan, liver granulomas were larger than in controls and there was more collagen deposition and fibrosis. Encapsulated parasites were dead or dying, and such damage appeared to be mediated by increased numbers of macrophages and giant cells.

Antitumor and metastasis-inhibitory activities of lentinan as an immunomodulator: an overview.

The antitumor and metastasis-inhibitory activities, mode of action, and clinical application of lentinan, a strictly purified beta-1,6:beta-1,3-glucan, are reviewed. Lentinan exerts a prominent antitumor effect and prevents chemical and viral oncogenesis. The antitumor action of lentinan is host-mediated. Compared to other well-known immunostimulants, such as bacille Calmette Guérin (BCG), Corynebacterium parvum, and lipopolysaccharide (LPS), lentinan appears to represent a unique class of immunopotentiator, a T cell-oriented adjuvant. Lentinan triggers the increased production of various kinds of bioactive serum factors associated with immunity and inflammation, such as IL-1, CSF, IL-3, vascular dilation inducer, and acute-phase protein inducer, by the direct impact of macrophages or indirectly via lentinan-stimulated T cells, which results in the induction of many immunobiological changes in the host. Augmented IL-1 production amplifies the maturation of immature effector cells to mature cells capable of responding to lymphokines such as IL-2 and T cell-replacing factors. Because of this mode of action, intact T cell compartments for antitumor activity of lentinan are required. Lentinan has little toxic side effects. Excellent results were obtained in a 4 year follow-up of the randomized control study of lentinan in phase III on patients with advanced and recurrent stomach and colorectal cancer.

Macrophage-mediated acute-phase transport protein production induced by lentinan.

A new bioactive factor capable of stimulating the production of acute-phase transport proteins, haptoglobin, hemopexin and ceruloplasmin, was found in mouse serum soon after the administration of lentinan, an immunomodulatory polysaccharide. This factor (APPIF) was produced by macrophages, and may regulate the production of acute-phase transport proteins in hepatocytes. The mice given the serum obtained from donor mice 2-6 h after an injection of 10 mg/kg of lentinan showed a marked increase of the acute-phase transport proteins in their serum 4 days after the serum injection. Pretreatment with the antimacrophage agents, carrageenan and mouse Ia antiserum, before the lentinan treatment to donor mice inhibited the production of acute-phase transport proteins in the recipient mice. Thymus or T-cells had no role in the production of APPIF. As the activity of APPIF disappeared after pronase treatment of the serum, APPIF seems to be a peptide compound. Appearance of APPIF is considered to be one of the earliest manifestations of the mode of action of lentinan in addition to its augmented production of vascular dilatation and hemorrhage inducing factor and interleukin-1. The correlation between these inflammatory and immune responses in earlier stages of the host defence mechanisms is also discussed.

Effect of lentinan on pinocytosis in mouse peritoneal macrophages and the murine macrophage cell line C4M phi in vitro.

Lentinan, an immunopotentiating polysaccharide, stimulated the pinocytosis of horseradish peroxidase (HRP) or FITC-dextran by resident or thioglycollate-elicited mouse macrophages from 10 to 50% in a dose dependent manner. Pinocytosis of HRP and FITC-dextran by C4M phi cells, a murine macrophage cell line, exhibiting a lower basic pinocytic activity than peritoneal cells, was augmented up to 310 and 120%, respectively, by lentinan. Mannan inhibited the HRP uptake by peritoneal macrophages via specific mannose receptors. This inhibitory effect was partly abolished, when lentinan was also added to the cells. Mannan was not able to inhibit pinocytosis of HRP by C4M phi macrophages, indicating little or no mannose receptor activity on these cells. Pinocytosis of FITC-dextran was not affected by mannan. Lentinan, opsonized in mouse sera inhibited the uptake of HRP by peritoneal macrophages by 30-35%. Opsonized lentinan and mannan added together caused 60% inhibition of HRP uptake in peritoneal macrophages indicating a possible functional relationship between the mannose and C3b receptors. The results demonstrate that lentinan activates the pinocytic function of macrophages predominantly via specific beta-glucan receptors. These mechanisms may contribute to the antitumor and immunopotentiating action of lentinan and other glucan-type polysaccharides.

[Experimental studies on growth inhibition and regression of cancer metastases].

The most important target in pharmaceutical therapy against cancer is complete suppression of metastases and recurrence after curative surgical operation. It is fundamentally, a growth inhibition and regression of small number of autochthonous tumors scattering in the host, and coexistence between tumor and host is also important. As immunosuppressive anticancer drugs have detrimental effects for patients in such cases, application of strong immunopotentiators such as lentinan should be expected. Lentinan showed a prominent effect on suppression of metastases in experimental systems of clinical models using MH-134 hepatoma, Madison-109 lung carcinoma and DBA/2.MC.CS-1 fibrosarcoma. Suppression of carcinogenesis may be considered as one of experimental methods to prevent metastases in a viewpoint of regression of small number of autochthonous tumor cells. Lentinan given at suitable timing and schedule showed marked prophylactic effect on chemical and viral carcinogenesis. Mode of action of lentinan as T-oriented adjuvant in its antitumor and metastases-inhibitory effects is also discussed. Considering excellent end-point results of Phase III with advanced and recurrent gastrointestinal cancer, lentinan is the most hopeful substance to prevent micrometastases.

Effect of lentinan on the chemiluminescence produced by human neutrophils and the murine macrophage cell line C4M phi.

Lentinan, an immunopotentiating polysaccharide, stimulates the production of chemiluminescence (CL) by human neutrophils and the murine macrophage cell line C4M phi. The CL enhancing effect of lentinan opsonized in human serum is greater than that of lentinan itself. Lentinan's stimulation of neutrophil CL was increased by 1/2 when opsonized in human serum inactivated at 56 degrees C to remove complement, while the CL was increased two fold by lentinan opsonized in whole serum. This indicates that C3b and immunoglobulin contribute separate signals in the activation process mediated by opsonized lentinan. The distinct roles of the C3b and Fc receptors was further illuminated by the finding that an Fc receptor-negative cell line was unresponsive to lentinan opsonized in heat inactivated serum (56 degrees C), whereas it exhibited a five fold increase in CL in response to lentinan opsonized in serum containing complement. Lentinan in an opsonized form can stimulate the production of CL via C3b and Fc receptors. This mechanism may be considered as one mode of action of lentinan and other similar immunopotentiating and antitumour glucan-type polysaccharides.

Superoxide anion, superoxide dismutase and lipid peroxidation in the murine macrophage cell line C4M phi.

A remarkable increase in the production of superoxide radicals and SOD activity was measured in suspension of the murine macrophage cell line C4M phi treated with Lentinan (4-10 X 10(3) micrograms/5 X 10(6) cells). In activated macrophages the decrease of lipid peroxidation could be interpreted as a consequence of enhanced SOD activity.