Repolarization of glioblastoma macrophage cells using non-agonistic Dectin-1 ligand encapsulating TLR-9 agonist: plausible role in regenerative medicine against brain tumor.

AIM OF THE STUDY: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic ß-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type. MATERIALS AND METHODS: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6). RESULTS: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1ß post treatment. CONCLUSION: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Induction of humoral and cellular immune response to hepatitis B virus (HBV) vaccine can be upregulated by CpG oligonucleotides complexed with Dectin-1 ligand.

A persistent hepatitis B virus (HBV) infection is characterized by a lack of or a weak immune response to HBV, which may be reflective of tolerance to HBV. Efficient induction of HBV-specific immune response leads to the clearance of HBV in patients with a chronic HBV infection. CpG oligodeoxynucleotides (ODN) has a powerful adjuvant effect in HBV vaccination. A recent report demonstrated that the immunization by B/K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand, schizophyllan (SPG), namely K3-SPG, was more effective in the induction of antigen-specific immune response than that by K3. In this study, we examined the efficacy of K3-SPG as a HBV vaccine adjuvant. Wild-type (WT) mice and HBV transgenic (HBV-Tg) mice were subcutaneously immunized with hepatitis B surface antigen (HBsAg) alone, HBsAg and K3, or HBsAg and K3-SPG. The vaccination with HBsAg and K3-SPG significantly enhanced humoral and cellular immune response to HBV antigen compared to the other vaccinations in WT and HBV-Tg mice. K3-SPG induced the accumulation of dendritic cells (DCs) into draining lymph node and the activation of DCs. The expression of cytokines and chemokines related to Th1 and Th2 responses was upregulated after the vaccination including with K3-SPG. In conclusion, these results indicated that the vaccination using K3-SPG may overcome tolerance even in patients with chronic HBV infection.

A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease.

Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the ß-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD.

Leishmanial CpG DNA nanovesicles: A propitious prophylactic approach against visceral leishmaniasis.

Unmethylated CpG motifs with phosphothioate backbone trigger TLR9 to elicit innate immune response characterized by the production of Th1 cytokines. The use of CpG DNA as an adjuvant has established its role in potentiating the humoral and cell mediated vaccine specific immune response. However, none of the synthetic oligodeoxynucleotides (ODNs) know and used till date are associated with the parasite itself. Our group identified a novel CG rich sequence of 14 base pairs from Leishmania donovani genome (Ld CpG ODN) and established it as a TLR9 agonist. The present study was designed to ascertain the adjuvanticity of Ld CpG ODN with soluble leishmanial antigen in experimental model of L. donovani. During the study Schizophyllan (SPG), a fungal polymer was used for encapsulating Ld CpG ODN for efficient endosomal delivery. The synthesized nanovehicles were of nearly 100 nm and localized within endosomes as confirmed by confocal microscopy. Immunization studies displayed the superior ability of synthesized nanovehicles co-administered with parasite antigen in augmenting innate immune response in comparison to ODN, nanoparticles or soluble antigen alone. The response included generation of ROS, NO and iNOS expression followed by proinflammatory cytokine milieu with reduced parasitic load within liver, spleen and bone marrow. These immune-tailored particles in combination with parasitic antigens elicited significant generation of cell mediated response owing to the presence of high levels of CD8+ T-cells and lymphocyte proliferation. Moreover, vaccination regime with synthesized adjuvant also activated humoral immunity by escalating the levels of IgG2 followed by reduced levels of anti-leishmanial IgG and IgG1 antibodies. The findings support the efficacy of Ld CpG ODN as a potential adjuvant against visceral leishmaniasis.

Dietary schizophyllan reduces mitochondrial damage by activating SIRT3 in mice.

Schizophyllan (SPG), produced by Schizophyllum commune, is an exopolysaccharide with multiple academic and commercial uses, including in the food industry and for various medical functions. We previously demonstrated that SPG conjugated with c-Src peptide exerted a significant therapeutic effect on mouse models of the acute inflammatory diseases polymicrobial sepsis and ulcerative colitis. Here we extended these results by investigating whether SPG exerted a protective effect against mitochondrial damage in the liver via sirtuin 3 (SIRT3) induction, focusing on the deacetylation of succinate dehydrogenase A (SDHA) and superoxide dismutase 2 (SOD2). Liver damage models induced by alcohol or conjugated linoleic acid (CLA, which simulates lipodystrophy) in SIRT3-/-, SOD2-/-, and SDHA-/- mice were used. Results showed that dietary supplementation with SPG induced SIRT3 activation; this was involved in mitochondrial metabolic resuscitation that countered the adverse effects of alcoholic liver disease and CLA-induced damage. The mitochondrial SIRT3 mediated the deacetylation and activation of SOD2 in the liver and SDHA in adipose tissues, suggesting that SPG supplementation reduced ethanol-induced liver damage and CLA-induced adverse dietary effects via SIRT3-SOD2 and SIRT3-SDHA signaling, respectively. Together, these results suggest that dietary SPG has a previously unrecognized role in SIRT3-mediated mitochondrial metabolic resuscitation during mitochondria-related diseases.

Designing an immunocyte-targeting delivery system by use of beta-glucan.

A ß-1,3-d-glucan called Schizophyllan (SPG) can form a novel complex with homo oligodeoxynucleotides (ODNs) via the combination of hydrogen bonding and hydrophobic interactions. Dectin-1 is a major receptor involved in the recognition of ß-1,3-d-glucans and expressed on antigen presenting cells (APCs) including macrophages, dendritic cells, monocytes, neutrophils, and a subset of T cells. Therefore, the SPG/ODN complex can be used as APCs cell-specific delivery of functional ODNs including unmethylated CpG sequences (CpG-ODNs). In fact, CpG-ODN/SPG complex induced high antibody titers when it was administered to cynomolgus monkeys as adjutant of influenza vaccine. These results indicate that SPG can be an excellent immunocyte-targeting drug delivery system.

The targeted delivery of the c-Src peptide complexed with schizophyllan to macrophages inhibits polymicrobial sepsis and ulcerative colitis in mice.

Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a ß-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox.

Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency.

Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.

Immunization with antigenic peptides complexed with ß-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that ß-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases.

Immunotoxicity of soluble beta-glucans induced by indomethacin treatment.

(1 --> 3)-Beta-D-Glucan (beta-glucan) is a biological response modifier that regulates host immune response. However, the side effects of this drug have not been extensively examined. In this study, we found that the combination of a beta-glucan and a nonsteroidal anti-inflammatory drug, indomethacin, induced lethal toxicity in mice. Lethal toxicity of orally administered indomethacin (multiple administration to ICR mice; once a day for 2 weeks) was 0/8 (2.5 mg kg(-1)) and 5/8 (5 mg kg(-1)) (death/total) over 2 weeks. The toxicity was enhanced to 3/8 and 8/8 in mice treated with a clinical beta-glucan preparation, sonifilan (250 microg/mouse, single i.p. administration on day 0). A similar effect was observed for other beta-glucans, including SSG, grifolan, zymosan A and zymocel. Enhanced lethal toxicity resulted from a single p.o. administration of indomethacin on day 5 to day 9 after multiple beta-glucans administration. Interferon-gamma, interleukin-6 and colony stimulating factor concentrations in sera of indomethacin/beta-glucan-treated mice were significantly elevated. These results strongly suggest that indomethacin/beta-glucan treatment induces lethality in mice by maladjusting the cytokine network.

[Studies on metabolism and disposition of sizofiran (SPG), an anti-tumor polysaccharide. II. Effects on hepatic drug-metabolizing enzyme system in rats].

The effect of a single or multiple administration of sizofiran (SPG), an anti-tumor polysaccharide, on a hepatic drug-metabolizing enzyme system was studied in rats. When SPG was given intravenously at a single dose of 0.5 or 10 mg/kg, no alteration was observed in activities of aminopyrine (AP) N-demethylase and aniline hydroxylase, and in cytochrome P-450 (P-450) content in the livers of rats 48 h after dosing. However, only AP demethylase activity decreased by 34% after the administration of 200 mg/kg. Similarly, no change in the hepatic enzyme activities and P-450 content was observed for up to 180 d after a single dose of 10 mg/kg. Subcutaneous treatment of animals with either 10 or 40 mg/kg dose for 3 and 6 months resulted in no alteration in the enzyme activities and P-450 content. These results may indicate that the therapeutically effective dose of SPG has no effect on a hepatic drug-metabolizing enzyme system in rats.

[Effect of schizophyllan on delayed hypersensitivity in the mouse (author's transl)].

The delayed cutaneous hypersensitivity test with picryl chloride was used to demonstrate the effect of sonicated schizophyllan (SPG-S) on cellular immunity in mice. The increase of ear thickness in mice treated with SPG-S (0.5 approximately 10.0 mg/kg) 24 hours before the sensitization with picryl chloride proved to be significantly larger than that in untreated mice on testing with picryl chloride at 7 days following sensitization. Mice were intramuscularly injected with SPG-S (5.0 mg/kg) on -48, -24, 0, +24 or +48 hours of sensitization. Remarkable stimulating effect of SPG-S on the delayed hypersensitivity was recognized in all groups of mice, being somewhat lower in the group treated with SPG-S 48 hours after the sensitization. Stimulating effects of lipopolysaccharide, concanavalin A, pokeweed mitogen and SPG-S on the delayed hypersensitivity were compared. The effect of SPG-S was similar to that of concanavalin A, a nonspecific stimulant for T cells.

[Influence of schizophyllan, streptomycin and rifampicin on histopathological changes in mice infected with tubercle bacilli (author's transl)].

Experimental tuberculosis in mice infected with streptomycin-resistant Mycobacterium tuberculosis SCHACHT strain was treated with streptomycin or rifampicin alone and in combination with schizophyllan. The histopathogical tests of various organs of the treated mice were carried out. 1) In the group of mice treated with streptomycin alone, the moderate focal proliferation of RE cells were seen at the beginning of infection. However, durable activation of RE cells and the prolongation of life-span could not be recognized as compared with control animals. 2) The treatment with streptomycin-schizophyllan combination appeared to be somewhat more effective than schizophyllan alone, the phagocytic capacity being more strongly stimulated. 3) In the group treated with rifampicin alone, the therapeutic effect could be exhibited by the direct antibacterial action of rifampicin, but the activation of RE cells was slight. When rifampicin was discontinued, the growth of tubercle bacilli was rapidly resumed, and the durable therapeutic effect seemed not to be expected. Degeneration of hepatic cells tended to develop. 4) In the group treated with rifampicin-schizophyllan combination, the antibacterial effect of rifampicin appeared to be potentiated by the strong activation of RE cells by schizophyllan, showing the durable therapeutic effects.

[A long-term survivor of primary hepatoma--a case suggesting the superiority of a multi-drug (OK-432, SPG, PSK) over mono-drug immunotherapy].

The studied patient (70 years old, male) had primary hepatoma with rupture of the liver. We resected his left lobe partially and were able to save him. Since a residual tumor was found after surgery, we injected a total of 30 mg of MMC into the SCA 3 times. Under continuous administration of OK-432, the patient survived for 3 years and 6 months in remission. Because the tumor grew gradually, we started a multi-drug immunotherapy (OK-432 5 KE/2 W/intradermal, PSK/3.0 g/day/P.O., SPG/20 mg/2 W/I. M.) which we have advocated. In 6 months the tumor regressed to 1/3 of its original size. The patient is currently in good condition 4 years and 7 months after the operation. This case demonstrated the superiority of multi-drug immunotherapy over single-drug immunotherapy.

[Concomitant intraperitoneal therapy with the antitumor polysaccharide Sizofiran and rG-CSF for ovarian cancer].

The concomitant administration of Sizofiran (a macrophage activator) and rG-CSF (which promotes neutrophil proliferation and activation) caused marked activation of intraperitoneal antitumor immunity. It promoted the induction of IL-2 receptor expression as well as the proliferation and activation of neutrophils, and also caused an increase in LAK and NK activity, which resulted in a clinical antitumor effect. Therefore, this concomitant regimen was found to be useful as maintenance therapy for ovarian cancer.

[Clinical evaluation of SPG (schizophyllan) as a therapeutic adjuvant after surgery of gastric cancer--controlled study by an envelope method].

SPG, a beta-1, 3 glucan extracted from cultured Schizophyllum commune Fries, was clinically evaluated for its efficacy in adjuvant immunochemotherapy for postoperative gastric cancer by the SPG cooperative study group comprising 43 hospitals. On the day of operation and the next day, patients were given intravenously 0.4 mg and 0.2 mg/kg of mitomycin C, respectively. Subsequently, they were randomly allocated to either the SPG group or control group. From the 10th to 20th postoperative days, the SPG group was intramuscularly given SPG at a dose of 20 mg twice a week or 40 mg once a week in combination with tegafur, while the control group was given only tegafur. Significant prolongation of life span of the SPG group was confirmed in Stage III. Minor side effects due to SPG were noted in 2.6% (5/190).

[Immunochemotherapy with tegafur and schizophyllan for stomach cancer--report of 2 cases].

Two cases of gastric cancer were treated with immunochemotherapy using Tegafur and Schizophyllan. In one case, a marked reduction in the size of metastatic liver tumor and disappearance of most subjective symptoms were observed. The response continued even 8 months after the initial treatment. In the other case, no marked symptoms except occasional dysphagis have been noted for 2 years and 2 months since diagnosis, although the primary tumor size has remained unchanged. Thus, it is presumed that the combined immunochemotherapy regimen may be useful in the treatment of advanced gastric cancer.

[Clinical evaluation of schizophyllan (SPG) in advanced gastric cancer (the second report)--a randomized controlled study].

We reported previously that Schizophyllan (SPG) combined with MMC + 5-FU (MF regimen) or Tegafur (F regimen) had a significant life-prolonging effect over chemotherapeutic regimens in patients with inoperable and recurrent gastric cancer in a randomized controlled study. In the present paper, we have further investigated the effect of SPG on life-span followed-up for more than 2 years. A significant life-prolonging effect of SPG was reconfirmed in patients given either the MF regimen (154 patients) or the F regimen (213 patients). Thus, SPG combined with chemotherapeutics has proved to be useful for treating patients with inoperable and recurrent gastric cancer, resulting in a significant prolongation of life-span without serious side effects despite having no influence on tumor size.

[Two cases of cancer with bone metastases treated with local therapy].

Two cases with osteolytic bone metastases from cervical cancer and esophageal cancer treated by local therapy were reported. The first case is a 53-year-old female with bone metastases of left ischium developed 1 year after hysterectomy. She was treated by intratumor injection of sizofiran (SPG) 20 mg/w and radiation therapy. After 4 weeks, ischias pain decreased and bone lesion showed remarkable calcification (PR) 8th months later. The second case is a 58-year-old male with bone metastases of the left tibia and fibula developed 1 year after surgery. He was treated by intratumor injection of SPG 20 mg/w x 4 and OK-432 1.0 KE/w x 8 after radiation therapy. After 4 weeks, pain and swelling of left leg decreased and bone lesions showed remarkable calcification (PR) three months later. We suggest that intratumor injection of SPG and OK-432 with radiation therapy was effective for osteolytic bone metastases.

[A case report of patient with advanced stomach carcinoma of linitis plastica type responding to multi-immunotherapy and chemotherapy].

Multiple-drug (OK-432, PSK and SPG) immunotherapy and chemotherapy provided remission of symptoms for 36 months in a patient aged 21 years suffering scirrhous gastric carcinoma associated with carcinomatous peritonitis in which direct infiltration to the pancreas, retroperitoneum and the left colon was observed. A remarkable improvement with time was observed by endoscopic and roentgenographic observation, and a substantial improvement was also observed in the NK-cell ratios of lymphocyte subsets of the OKT series in relation to immunologic parameters. A tumor necrosis factor [TNF]-like substance was thought to have been induced by multiimmunotherapy in this case.