Two interactive genes responsible for a new inherited cataract (RCT) in the mouse.

We discovered a mutant mouse, RCT (Rinshoken cataract), with a new congenital cataract in strain SJL/J. The opacity of the lens associated with microphthalmia could be observed visually at 3 to 3.5 months of age. Marked degeneration of the lens, including loss of the fine structure of the lens fibers and swelling of epithelial cells with vacuoles of various sizes in the cortex, but no other defects except photoreceptor degeneration in the retina, was detected. Histological change in the lens was first observed at 2 days after birth. No sex-related differences were detected, and normal phenotypes in the F1 progeny of RCT and normal mice indicated that the cataract was recessive. The chromosomal location of the causative gene was determined by interval mapping by using intersubspecific backcross progeny of RCT and MSM/Ms, an inbred strain from the Japanese wild mouse Mus musculus molossinus. Backcross progeny were divided into three groups according to phenotype: mice (1) with an early-onset cataract, which can be detected visually as in RCT mice, (2) with a late-onset cataract, which can be detected histologically but not visually, and (3) with a normal lens. Three phenotypes were found to be expressed by allele combinations of two recessive genes, rct and mrct (a modifier of rct). The rct locus essential for the onset of the cataract was tightly linked to D4Mit278 on Chromosome (Chr) 4 with no recombination. The mrct locus was closely linked to D5Mit239 (chi2 = 66.3, P << 0.00001) on Chr 5.

Four dominant loci for the vascular responses by the antitumor polysaccharide, lentinan.

Lentinan, beta-1,6;1,3-glucan, showing an antitumor effect against mouse solid type tumors, can induce marked vascular dilation and hemorrhage (VDH) in very localized areas such as the ears, feet, and tails of mice in the early stages after its administration (Maeda et al. 1984). VDH has been found to be one of the T-cell-mediated responses triggered by lentinan. We reported previously that the responsiveness of mice to lentinan with respect to VDH induction is controlled by a dominant gene(s), Ltn2 (formerly), and that no sex difference was observed (Maeda et al. 1991). To determine the chromosomal location of the Ltn2 gene(s), we typed genomic DNAs of 193 N2 segregants of crosses between a high responder MA/MyJ and a low responder AKR/J by the polymerase chain reaction-simple sequence length polymorphism technique using 83 chromosome-specific microsatellite markers. We identified one major gene (Ltnr3) and three minor genes (Ltnr4, Ltnr5, and Ltnr6) responsible for the VDH induction. Ltnr3 was closely linked to D6Mit135 on chromosome 6 (P <0.00000) and Ltnr4, Ltnr5, and Ltnr6 to D9Mit161 on chromosome 9 (P <0.00032), D15Mit147 on chromosome 15 (P <0.00014) and D16Mit4 on chromosome 16 (P <0.00014), respectively.

Polygenic control of the expression of biological activities of an antitumor polysaccharide, lentinan.

Genetic studies were carried out on two in vivo responses of lentinan, delayed type-acute phase responses (DT-APR) and vascular dilation and hemorrhage (VDH). Linkage analyses showed that DT-APR was controlled by two recessive genes, ltnr1 and ltnr2, which were mapped on chromosome 3 and 11, respectively. VDH was also found to be controlled by polygenes. One dominant major gene, Ltnr3, and three dominant minor genes, Ltnr4, Ltnr5, and Ltnr6, were mapped on chromosomes 6, 9, 15 and 16, respectively.

Two genes controlling acute phase responses by the antitumor polysacch aride, lentinan.

Lentinan, a beta-1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) and Mus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence length polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 and ltn1.2) responsible for DT-APR. ltn1.1 is closely linked to D3Mit11 on chromosome 3 and ltn1.2 to D11Nds9 on chromosome 11 (P <0.001). The linkage analysis also suggested that ltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that the ltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.

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.

Genetic control of the expression of two biological activities of an antitumor polysaccharide, lentinan.

In order to make clear whether the expression of biological activities and antitumor polysaccharides are under genetic control, the responses of mice to lentinan, a beta-1,6;1,3-glucan, in the induction of several acute phase proteins (APPs) and T-cell-mediated vascular dilation and hemorrhage (VDH) were investigated. Twenty inbred strains of mice were divided into two groups according to their phenotypes in the induction of APPs when they were administered lentinan i.p. at a dose of 10 mg/kg; sensitive strains showed a marked increase in levels of APPs and resistant strains showed as low a level of APPs as non-treated control mice. No sex-related differences and no relation with H-2 halotypes were found in the responses. Only low-level responses were observed in F1 hybrid mice obtained by crosses between a sensitive and a resistant strain, indicating that the low APP response to lentinan is dominant. The N2 progeny between the F1 and a high responder segregated into high and low responders at a ratio of almost 1:1. These results suggest that a single major gene on an autosome is responsible for the induction of APPs. The induction of VDH also depended on the strains of mice. However, the strain distribution pattern of the VDH phenotype was distinct from that of the APP phenotype, indicating that the VDH-controlling gene was different from the APP-controlling gene. Further analyses with F1 hybrid and backcross progeny mice suggested that the high VDH response was dominant, and that the phenotype was determined by a single major gene.

Denaturation and renaturation of a beta-1,6;1,3-glucan, lentinan, associated with expression of T-cell-mediated responses.

Correlation between the higher structure and biological functions of lentinan, a beta-1,6;1,3-glucan capable of potentiating T- and non-T-cell-mediated responses, were investigated by measurements of optical rotation and some biological responses. The addition of urea or dimethyl sulfoxide decreased specific rotation at 589 nm, [alpha]D, of lentinan in a concentration-dependent manner and the removal of these denaturants resulted in the recovery of [alpha]D values. Measurements of optical rotatory dispersion in the spectral region between 600 and 200 nm showed the change in the higher structure of lentinan more clearly. Denaturation and renaturation of lentinan using urea and dimethyl sulfoxide were associated with the decrease and the recovery of antitumor activity against P-815 mastocytoma and vascular dilation and hemorrhage-inducing activity, found to be T-cell-mediated responses. Lentinan was also denatured by NaOH and the transition of [alpha]D values and optical rotatory dispersion curves were seen in the manner of two concentration-dependent phases. Removal of NaOH led to the recovery of optical rotation of lentinan and its antitumor and vascular dilation and hemorrhage-inducing activity. However, recovery of these bioactivities was more difficult in the case of the higher concentrations of NaOH above 2% than the lower ones. During the process of renaturation of lentinan, random aggregation may take place. An increase of serum acute phase proteins, a non-T-cell-mediated response caused by lentinan, was not affected by the change of the higher structure of lentinan.

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.

Antitumor activity of lentinan in murine syngeneic and autochthonous hosts and its suppressive effect on 3-methylcholanthrene-induced carcinogenesis.

The antitumor effect of lentinan in syngeneic and autochthonous tumor-host systems and its suppressive effect on 3-methylcholanthrene (MC)-induced carcinogenesis were confirmed using DBA/2 and SWM/Ms hosts. The regressive activity of lentinan against the solid form of Sarcoma 180 was the most effective in DBA/2, SWM/Ms, or A/J mice and less effective in C3H/He or C57BL/6 mice. The growth of a syngeneic MC-induced DBA/2.MC.CS-1 fibrosarcoma (native and trypsinized) was markedly inhibited, and the regression of tumors was detected by the i.p. injection of minute amounts of lentinan into DBA/2 mice, which were the most suitable host in lentinan treatment. When DBA/2 mice were used, lentinan was also effective for even autochthonous primary tumors induced within 15 weeks after MC inoculation, but less effective for tumors induced during the 16 to 36 weeks after MC treatment. Lentinan showed a prominent suppressive effect in MC-induced carcinogenesis using DBA/2 and SWM/Ms mice but not effect when BALB/c, C57BL/6, or C3H/He mice were used. The timing of lentinan administration in the latter result was examined using SWM/Ms mice, and lentinan, when it was given daily for 10 days after the third week of MC inoculation, was strikingly effective (33%), but not so effective (63%) when lentinan was given after the sixth week of MC treatment, compared with tumor-occurrence rate in the control group (88%). The reason why DBA/2, SWM/Ms, or A/J mice were suitable hosts for lentinan treatment is not clear, but the natural killer capability or phagocytic macrophage function in these strains seems to have no relation to lentinan action, because A/J mice are deficient in natural killer function, and in these strains of mice the phagocytic function of macrophages is weak. It may be quite possible that these strains of mice are most sensitive to delayed-type hypersensitivity and/or cytotoxic T-cell response in which T-cells and lentinan play important roles. The tumor-host systems presented here provide a good model in which lentinan retains an inhibitory capacity in syngeneic and autochthonous hosts, and such a model offers the possibility for further study of the host defense mechanism against cancer.

Mechanism of the action of the inhibitor of reverse transcriptase.

An inhibitor of reverse transcriptase of Moloney leukemia virus was reported previously in the cytoplasm of the cultured cells (1). In this report, the mechanism of the inhibition was examined. The inhibitory activity was completely abrogated by treatment at 55 degrees C for 20 min. Destruction of the reaction products of reverse transcriptase was not observed. Strong inhibition was observed by preincubation of poly rA oligo dT used as a template-primer, together with the inhibitor, but was not by incubation of poly rA or oligo dT with the inhibitor separately. It is suggested that the inhibitor reacts with poly rA oligo dT complex and interferes with attachment of the reverse transcriptase to the template-primer complex.

T-cell mediated vascular dilatation and hemorrhage induced by antitumour polysaccharides.

Antitumour polysaccharide lentinan, capable of potentiating T-cell dependent reactions and some other antitumour polysaccharides such as pachymaran, carboxymethyl-pachymaran and zymosan were found to induce vascular dilatation and hemorrhage(VDH) in CD-1 normal mice starting the following day after a single injection. Polysaccharides which do not have the tumour-regressing activity, several immunopotentiators such as BCG, lipopolysaccharide, dextran sulfate and concanavalin A, and chemical mediators of inflammation such as histamine, serotonin and prostaglandin E1 did not induce VDH in mice. This response seems to be mediated by T-cells and macrophages, because VDH was not observed in CD-1 nu/nu mice treated with lentinan, and carrageenan, an antimacrophage drug, inhibited the appearance of VDH by lentinan. Furthermore, carrageenan inhibited the production of the VDH-inducing serum which was caused by lentinan, capable of passively transferring VDH to another mouse.

Current status and perspectives of immunomodulators of microbial origin.

The chemical structure and characteristics, the antitumour activity, the antibacterial, antiviral, antifungal, antiparasitic activities, immunological properties and mode of action of immunomodulators of microbial origin, especially antitumour polysaccharide lentinan, have been discussed immunopharmacologically in comparison with pachymaran, schizophyllan, DiLuzio's yeast glucan, Coriolus preparation PS-K, Streptococcal preparation OK-432, Nocardia rubra cell wall skeleton N-CWS together with BCG and Corynebacterium parvum. Lentinan exerts its inhibitory action not only on allogeneic tumours but also on syngeneic and autologous tumours, and prevents chemical and viral carcinogenesis. The phase III randomized control study of lentinan in cancer patients with gastric and colo-rectal cancer showed the clinical efficacy of lentinan in prolonging the life span and improving host immune responses. Experimental and clinical application demonstrated the advantage of N-CWS over BCG-CWS, and a recent study on OK-432 showed similar results to those obtained with C. parvum. In future work on microbial immunomodulators, it will be essential to find a parameter of the host-tumour relationship which will indicate the protocol for application of immunomodulators, and to find a new-type of selective immunostimulant. For this purpose, exhaustive studies on lymphokines, monokines and lymphocyte tropic hormones are indispensable.

Antitumor activity of degraded products of lentinan: its correlation with molecular weight.

Lentinan, an antitumor polysaccharide from Lentinus edodes, was degraded to seven fractions by treatment with formic acid. The low molecular-weight fractions (I and II) showed no antitumor activity against sarcoma-180 solid-type tumor and the absorption maximum of Congo Red did not shift in their presence in 0.1M sodium hydroxide. The medium molecular-weight fraction III, which required the increase of doses (5 or 10 mg/kg) for inhibition of tumor growth, caused a little shift. On the other hand, the absorption maximum of Congo Red shifted largely by the presence of high molecular-weight fractions (IV approximately VII) which showed the inhibition ratio of over 95% in a dose of 1 mg/kg. Participation of molecular weight in the antitumor activity of polysaccharides which contain (1 leads to 3)-beta-D-glucan main chain was discussed.