Lentinan confers protection against type 1 diabetes by inducing regulatory T cell in spontaneous non-obese diabetic mice.

BACKGROUND: Lentinan (LNT) is a complex fungal component that possesses effective antitumor and immunostimulating properties. However, there is a paucity of studies regarding the effects and mechanisms of LNT on type 1 diabetes. OBJECTIVE: In the current study, we investigated whether an intraperitoneal injection of LNT can diminish the risk of developing type 1 diabetes (T1D) in non-obese diabetic (NOD) mice and further examined possible mechanisms of LNT's effects. METHODS: Pre-diabetic female NOD mice 8 weeks of age, NOD mice with 140-160 mg/dL, 200-230 mg/dL or 350-450 mg/dL blood glucose levels were randomly divided into two groups and intraperitoneally injected with 5 mg/kg LNT or PBS every other day. Then, blood sugar levels, pancreas slices, spleen, PnLN and pancreas cells from treatment mice were examined. RESULTS: Our results demonstrated that low-dosage injections (5 mg/kg) of LNT significantly suppressed immunopathology in mice with autoimmune diabetes but increased the Foxp3+ regulatory T cells (Treg cells) proportion in mice. LNT treatment induced the production of Tregs in the spleen and PnLN cells of NOD mice in vitro. Furthermore, the adoptive transfer of Treg cells extracted from LNT-treated NOD mice confirmed that LNT induced Treg function in vivo and revealed an enhanced suppressive capacity as compared to the Tregs isolated from the control group. CONCLUSION: LNT was capable of stimulating the production of Treg cells from naive CD4 + T cells, which implies that LNT exhibits therapeutic values as a tolerogenic adjuvant and may be used to reverse hyperglycaemia in the early and late stages of T1D.

Quality control and immunological activity of lentinan samples produced in China.

Lentinan is widely used as a therapeutic agent for treatment of malignant tumors in clinical practice. The chemical structure of lentinan is highly associated with its biological activity. In this study, the correlation between the structure of lentinan and its immune activity was investigated to assess the function of key parameters that can influence quality control of lentinan. The results showed that the batch-to-batch consistency of two lentinan samples was satisfactory, indicating the stability of production process of lentinan. However, although the chemical composition and triple-helical conformation (THC) of the tested samples were relatively similar, their Mw, polydispersity index (PDI), and Rgz remarkably varied due to different production processes. In vitro immunomodulatory assay reflects that lentinan could stimulate the macrophages phagocytic capacity. Meanwhile, lentinan samples could improve the spleen and thymus indices, promote the proliferation of lymphocytes and adjust for the percentages of CD4+ and CD8+ T cells in vivo. Furthermore, the immunomodulatory effect of lentinan sample B (Mw: 650,700 g/mol) was superior than that of the sample A (Mw: 4,818,700 g/mol). It was noted that the Mw should be detected as a necessary index for quality control of lentinan to ensure stability and effectiveness of the production process.

Effects of tumor-specific antigen induced by lentinan on murine H22 hepatocellular carcinoma immunoprophylaxis.

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the most prevalent tumor types and the third most common form of morbidity in cancer-related deaths worldwide. Lentinan isolated from Lentinus edodes, is known to be a biologically active macromolecule with extremely strong activation of the human immune system such as host-mediated anti-cancer activity. The aim of this study is to investigate the immunoprophylaxis effect of the antigens induced by lentinan on murine hepatocellular carcinoma. MATERIALS AND METHODS: The antigens were prepared by a co-culture method (HCL) and purified by ammonium sulfate fractionation precipitation (Z1, Z2, Z3). The effects of antigens on murine hepatocellular carcinoma immunoprophylaxis were determined in vivo. The cellular immunity of the immunized mice was tested by spleen lymphocyte proliferation tests and peritoneal macrophage phagocytosis assays. The tumor-specific antigen was confirmed by Western blot analysis. RESULTS: Results in vivo revealed that the antigens (HCL/Z1) activated immunoprophylaxis against hepatocellular carcinoma with a better survival status. The survival rates (60%, 100%) of the HCL/Z1 group were better than the model group (p < 0.01). The quantity of lymphocytes in the spleen in the HCL or Z1 groups treated with ConA or LPS were higher than that of the model group (p < 0.01). The phagocytosis ability of macrophages in the HCL or Z1 groups was better than that of the control group or model group (p < 0.01). The characterization of Western blot analysis showed that about 59.6 kDa tumor specific antigen combined with antiserum of immunized mice specifically appeared in antigens. CONCLUSIONS: The newly generated tumor-specific antigen played a key role in the anti-tumor immune response and in activating the immune system. Our results suggest that this protein could serve as a tumor vaccine, and it could generate new ideas for tumor immunoprophylaxis.

Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds.

Little is known about the polarizing potential of currently used human macrophage cell lines, while a better understanding phenomena can support the prediction of effects in vivo based on in vitro analysis. To test the polarization capability of PMA differentiated-THP-1 macrophages (M0), cells were stimulated with 20 ng ml(-1) IFNγ + 1 µg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vivo and ex vivo into the M1 and M2 state, respectively. Apart from several well-known M1 and M2 markers, also new possible markers for M1 and M2 polarization were analysed in this study. The expression of M1 marker genes was up-regulated in IFNγ + LPS stimulated-M0 THP-1 macrophages. The IL-4 stimulated-M0 THP-1 macrophages expressed M2 cell membrane receptor genes. However, M2 chemokine and their receptor genes were only slightly up-regulated which might be due to the complexity of the secondary cell-cell interaction of the chemokine system. Lipopolysaccharides from E. coli (LPS) and food compounds [lentinan, vitamin D3 (vD3) and the combination of lentinan + vitamin D3 (Len + vD3)] were investigated for their polarizing ability on M0 THP-1 macrophages towards either the M1 or M2 state. LPS (700 ng ml(-1)) was able to skew M0 THP-1 macrophages towards the M1 direction since all analysed M1 marker genes were strongly expressed. Lentinan, vD3 and Len + vD3 did not induce expression of either M1 or M2 markers, indicating no polarizing ability of these compounds. Based on the expression of M1 and M2 marker genes we concluded that THP-1 macrophages could be successfully polarized into either the M1 or M2 state. Therefore, they can be used as a new macrophage polarizing model to estimate the polarizing/switching ability of test food compounds.

Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having N-Acetyl-d-glucosamine branches.

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear ß-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a ß-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

Immunological effects of yeast- and mushroom-derived beta-glucans.

Glucans have a long history as nonspecific biological modulators. We compared the effects of three different glucans on immune reactions. Using two different administrations (intraperitoneal and oral) and two different animal models, we showed that yeast-derived Betamune (Biorigin, São Paulo, Brazil) caused significant stimulation of phagocytic activity as well as potentiation of synthesis and release of interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-13, and tumor necrosis factor-alpha. In addition, Betamune inhibited growth of tumor cells in vivo and affected expression of several important genes in breast cancer cells. Compared to adult mice, young animals showed different sensitivity to glucan action.

[Clinical trial of non-specific immunotherapy using Lentinan in advanced or recurrent gastric cancer].

Randomized phase III study of S-1 alone versus S-1 plus Lentinan (LNT) in advanced or recurrent gastric cancer started in February 2007 conducted by the Japanese Foundation for Multidisciplinary Treatment of Cancer. The objective of this study is to evaluate the superiority of S-1/LNT to S-1 alone. The primary end point is to compare over all survival between both treatment groups. Secondary end points include time to treatment failure, the grade and rate of the adverse events, the evaluation of quality of life (QOL), response rate evaluated by RECIST and immunological parameters. QOL is evaluated by Japanese version of FACT-BRM questionnaire. Immunological parameters include serum complements (CH50, C3) and beta-1, 3 binding monocytes. The sample size is estimated at 150 patients per arm. Registration period is 2 years with 2-year follow-up. This study has a chance to prove the efficacy of the non-specific biological response modifier. We will have to cooperate in order to make this study a success.

The beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects on the mouse spleen as Lentinan.

The stimulatory effects of the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond on the mouse spleen were studied for elucidation of the mechanism of their antitumor activity, and their stimulatory effects were compared with Lentinan. The mouse spleen's weight was increased after the intraperitoneal (i.p.) injection of the oligosaccharides compared with the saline group. In addition, routinely hematoxylin and eosin (HE)-stained spleen sections showed that the injection also changed the spleen's histopathology. RNA samples were isolated from splenocytes of oligosaccharides, Lentinan or saline-injected mice. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot showed that the administration of the oligosaccharides or Lentinan enhanced mouse spleen mRNA production of TNF-alpha but not IL-2. The injection also enhanced Concanavalin A (Con A)-induced mouse splenocytes proliferation, but the in vitro administration of the oligosaccharides did not have the proliferation-enhancing effect. Taken together, these results suggest that the synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose and its analogues containing an alpha-(1-->3)-linked bond have similar stimulatory effects as Lentinan. Additionally, they may exert their antitumor effects through the induction of splenocytes mediated immune responses.

Preparation and specificity of antibodies to an anti-tumor beta-glucan, lentinan.

Antibodies against beta-glucan, lentinan from "Shiitake" (Lentinus edodes), were raised in the rabbit by subcutaneous immunization. Our antibodies did not recognize the other polysaccharides such as amylose, dextran, laminarin and galactan. It was proved that lentinan contents in mushroom could be measured by ELISA with the anti-lentinan antisera. Its contents were 3.5 mg/g fresh weight in Lentinus edodes. However, lentinan was not contained in Agaricus brazei, Agaricus bisporus and Ramaria bitrytis.

Immunological analysis and clinical effects of intraabdominal and intrapleural injection of lentinan for malignant ascites and pleural effusion.

Twenty effusions in sixteen patients with malignant peritoneal and/or pleural effusions were treated with intracavitary injection of lentinan. Lentinan was injected at a dosage of 4 mg/week for 4 weeks. In total, sixteen (80%) of twenty lesions demonstrated clinical responses. Performance status was improved in seven patients. The average survival time in responders was 129 days, while, in non-responders, it was 49 days. Serious toxicities were not observed. NK activity of PBMC significantly decreased after lentinan injection. NK activity of PEC in responders was augmented significantly. Anti-Daudi and lymphokine activated killer activity were also augmented or maintained after lentinan injection.

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.

Induction of cytotoxic peritoneal exudate cells by T-cell immune adjuvants of the beta(1 leads to 3) glucan-type lentinan and its analogues.

Eight distinct polysaccharides (PS) of beta(1 leads to 3) glucan type were tested for their capacity to render murine peritoneal exudate cells (PEC) cytotoxic. After intraperitoneal injection of lentinan, pachymaran and HE-pachyman 3 and 4 highly cytotoxic PEC were induced. Pachyman and HE-pachyman 1 and 2 were of moderate effect, whereas CM-pachymaran and HE-pachyman 3 and 4, highly cytotoxic PEC were induced. Pachyman and HE-pachymacrophages. The induction of PEC-dependent cytotoxicity exhibited a strict dose relationship. Optimal administration of PS resulted in the induction of cytotoxicity, which persisted for more than 25 days. Surprisingly, none of the PS tested was capable of rendering normal or thioglycollate-induced PEC cytoxic under in vitro conditions. It is suggested that the capacity of PS to render in vivo macrophages cytotoxic is related to the potency of these PS to activate the alternative pathway of complement system (APC) in so far as C3b may be the essential component required to render macrophages cytotoxic.

Solid phase activation of alternative pathway of complement by beta-1,3-glucans and its possible role for tumour regressing activity.

The efficiency of some anti-tumour polysaccharides, such as lentinan, pachyman, pachymaran, carboxymethylpachymaran and hydroxyethylpachyman to trigger the alternative pathway of complement activation (APC) was investigated in detail, in order to clarify whether solid phase activation of APC by these polysaccharides will occur or not. From the eight polysaccharides tested, all were found to be potent activators of the alternative pathway except for carboxymethylpachymaran, regardless of their potency of inhibition of sarcoma 180 (S 180) transplanted in mice. This activation was observed both for the insoluble as well as the soluble part of the same polysaccharide. The turnover of C3, C5 and factor B showed no difference among these seven active polysaccharides. The polysaccharide particles (PX), isolated after treatment with C4d GPS showed prominent C3 consuming activity, which disappeared during prolonged incubation at 37 degrees. The activity of the decayed enzyme could be regenerated by treatment with the purified factor B. The stability of the particulate enzyme PX was comparable to the zymosan-complex (ZX) previously described.

In vivo and in vitro macrophage activation by systemic adjuvants.

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and from M.smegmatis (IPM), and lipopolysaccharide from E.coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted from Lentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential. BCG, IPM and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levamisole was unable to stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.