The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts.

Schizophyllan (SPG), a ß-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1ß/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Solvent stabilizing effects on the order-disorder transition of schizophyllan in aqueous mixtures of carboxylic acids.

Schizophyllan is a triple helical ß-1,3-D-glucan, and shows the cooperative order-disorder transition in the aqueous solution at the triple helix state. In this paper, the solvent stabilizing effects of two carboxylic acids, acetic acid and citric acid, on the cooperative order-disorder transition of aqueous schizophyllan solution were investigated from DSC and SEC-MALS measurements. The transition temperature (Tr) was shifted to higher temperature with increasing the molar fraction of carboxylic acid in the mixture (x). The transition enthalpy (ΔHr) was increased with increasing x. These solvent stabilizing effects indicate that these carboxylic acid molecules were selectively associated with the branched side chains of schizophyllan to stabilize the ordered state. The composition dependencies of Tr and ΔHr were analyzed by the linear cooperative transition theory to estimate the association parameters between the side chains and carboxylic acid. The theoretical parameters obtained were compared with those for the other active substances for the transition to discuss the molecular interactions between the triple helix and carboxylic acid.

Structural analysis of polysaccharide/antisense DNA complexes during cytoplasmic target mRNA hybridization.

We previously demonstrated antisense oligonucleotides (AS-ODNs) delivery system based on the complex formed with poly (dA) and schizophyllan, a type of ß-1,3-glucan. This complex enables efficient intracellular delivery of AS-ODNs. In this communication, we investigated the cytoplasmic translocation of the complex itself and its mechanism of action on mRNA. As a result, we found that the complex moved into the cytoplasm while keeping its structure, and AS-ODN hybridized with the target mRNA. This result encourages pharmaceutical applications of the complex.

Schizophyllan promotes osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

BACKGROUND: Bioactive polysaccharides are a promising way for bone disease prevention with high efficiency. Schizophyllan (SPG) is a polysaccharide derived from a species of fungus with anticancer, antitumor, and anti-inflammatory effects. In the present study, for the first time, the cell proliferation, osteogenic markers, mineral deposition, and osteogenic gene expression of human adipose tissue-derived mesenchymal stem cells (hADMSCs) grown on SPG were evaluated by in vitro assays. METHODS AND RESULTS: The cytotoxicity of SPG was measured using the MTT assay and acridine orange staining. Differentiation of hADMSCs was assessed using alkaline phosphatase (ALP) activity test, cellular calcium content assay, and mineralized matrix staining. To this end, Alizarin red S, von Kossa staining, and the expression of bone-specific markers, including ALP, Runx2, and osteonectin, were used by real-time RT-PCR over a 2-week period. According to the results, SPG at 10 µg/ml concentration was determined as the optimal dosage for differentiation studies. The results of osteogenic differentiation tests showed that compared to the control groups in vitro, SPG enhanced the osteogenic markers and mineralization as well as upregulation of the expression of bone specific genes in differentiated hADMSCs during differentiation. CONCLUSIONS: The results revealed that SPG could be applied as effective factor for osteogenic differentiation in the future. The current study provides insights into the hADMSC-based treatment and introduces promising therapeutic material for individuals who suffer from bone defects and injuries.

Production and Anti-Inflammatory Performance of PVA Hydrogels Loaded with Curcumin Encapsulated in Octenyl Succinic Anhydride Modified Schizophyllan as Wound Dressings.

Amphiphilic polysaccharides can be used as wall materials and applied to encapsulate hydrophobic active chemicals; moreover, there is significant demand for novel medical high-molecular-weight materials with various functions. In order to prepare amphiphilic schizophyllan (SPG), octenyl succinic anhydride (OSA) was chosen to synthesize OSA-modified schizophyllan (OSSPG) using an esterified reaction. The modification of OSSPG was demonstrated through FT-IR and thermal analysis. Moreover, it was found that OSSPG has a better capacity for loading curcumin, and the loading amount was 20 µg/mg, which was 2.6 times higher than that of SPG. In addition, a hydrogel made up of PVA, borax, and C-OSSPG (OSSPG loaded with curcumin) was prepared by means of the one-pot method, based on the biological effects of curcumin and the immune-activating properties of SPG. The mechanical properties and biological activity of the hydrogel were investigated. The experimental results show that the dynamic cross-linking of PVA and borax provided the C-OSSPG/BP hydrogel dressing with exceptional self-healing properties, and it was discovered that the C-OSSPG content increased the hydrogel's swelling and moisturizing properties. In fibroblast cell tests, the cells treated with hydrogel had survival rates of 80% or above. Furthermore, a hydrogel containing C-OSSPG could effectively promote cell migration. Due to the excellent anti-inflammatory properties of curcumin, the hydrogel also significantly reduces the generation of inflammatory factors, such as TNF-α and IL-6, and thus has a potential application as a wound dressing medicinal material.

Association with Imidazole in the Cooperative Order-Disorder Transition in Aqueous Solution of Schizophyllan.

Schizophyllan, a triple helical polysaccharide, exhibits cooperative order-disorder transition (CODT) in aqueous solutions. The transition transforms the ordered structure (triple helix I) formed between the branched side chains and solvent molecules into the disordered structure (triple helix II) without dissociation of the triple helix. The CODT behaviors in H2O-imidazole mixtures containing HCl with different molar ratios of imidazole/HCl were investigated by adiabatic calorimetry and differential scanning calorimetry on two schizophyllan solutions with different molar masses. The transition temperature (Tr) and the transition enthalpy (ΔHr) significantly depended on both of the mole fractions of imidazole and imidazole/HCl. The composition dependences of Tr and ΔHr in H2O-imidazole mixtures were analyzed with linear cooperative transition theory for the solvent-stabilizing effect in the mixture with active compounds. Theoretical analyses confirmed that both imidazole and imidazolium ions in the solutions competitively interact with the side chain of the triple helix.

Co-production of schizophyllan and cellulolytic enzymes from bagasse by Schizophyllum commune.

Schizophyllum commune is a mushroom-forming fungus well-known for its ability to degrade lignocellulosic materials and production of schizophyllan, a high added-value product for cosmeceutical, pharmaceutical, and biomaterial industries. Conventionally, schizophyllan is produced by submerged fermentation using glucose as a carbon source. In this work, we demonstrate that alkaline pretreated bagasse can be used by Schizophyllum commune as an alternative carbon source for the production of schizophyllan. The influence of different factors was investigated including cultivation time, biomass loading, and culturing media component and a co-product correlation model was proposed. In this lab-scale study, a yield of 4.4 g/L of schizophyllan containing 89% glucose was achieved. In addition to schizophyllan, the cellulolytic enzymes co-produced during this process were isolated and characterized and could find applications in a range of industrial processes. This demonstrates the potential of using agricultural waste as a cheaper alternative feedstock for this biorefinery process.

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.

Enhancement of schizophyllan production in Schizophyllum commune using microparticles in medium.

Schizophyllum commune is a wood-rotting filamentous fungus that secrets a homopolysaccharide called as schizophyllan. Schizophyllan has several applications such as enhanced oil recovery, pharmaceutical materials and an anti-cancer drug carrier. Biomass growth and schizophyllan production increase the viscosity of the cultivation medium, thus resulting in mass transfer limitation for the substrate. In this study, adding talc and aluminium oxide microparticles into the cultivation medium was studied to improve the fungal growth and morphology. The response surface methodology and one factor at a time were applied to find the effects of microparticles with different sizes and concentrations on the schizophyllan production. The optimum concentration and size of aluminium oxide microparticles were obtained as 20 g L-1 and < 30 µm, respectively. Aluminium oxide microparticles in shake flask culture caused to increase the schizophyllan production from 10 to 15 g L-1 and decrease the cultivation time from 10 to 7 days. The production yield also increased from 0.11 to 0.30 g of schizophyllan/g glucose. Bioreactor cultivation showed a twofold increase in schizophyllan production from 1.5 to 3 g L-1. The results of this study suggested a significant increase in the production of schizophyllan using a low-cost "microparticle-enhanced cultivation" without any further optimization of the culture medium.

Efficacy of SPG-ODN 1826 Nanovehicles in Inducing M1 Phenotype through TLR-9 Activation in Murine Alveolar J774A.1 Cells: Plausible Nano-Immunotherapy for Lung Carcinoma.

Alveolar macrophages are the first line of defense against intruding pathogens and play a critical role in cancer immunology. The Toll-like receptor (TLR) family mediates an important role in recognizing and mounting an immune response against intruding microbes. TLR-9 is a member of the intracellular TLR family, which recognizes unmethylated CG motifs from the prokaryotic genome. Upon its activation, TLR-9 triggers downstream of the MyD-88-dependent transcriptional activation of NF-κB, and subsequently results in abundant inflammatory cytokines expression that induces a profound inflammatory milieu. The present exploratory investigation aimed at elucidating the potency of schizophyllan for entrapping ODN 1826 (SPG-ODN 1826)-mediated stimulation of TLR-9 in provoking an inflammatory-type response in murine alveolar macrophages. Schizophyllan (SPG), a representative of the ß-glucan family, was used in the present study as a nanovehicle for endosomal trafficking of CpG ODN 1826. TEM analysis of SPG-ODN 1826 nanovehicles revealed that the prepared nanovehicles are spherical and have an average size of about 100 nm. Interestingly, SPG-ODN 1826 nanovehicles were competent in delivering their therapeutic payload within endosomes of murine alveolar macrophage (J774A.1) cells. Exposure of these nanovehicles within LPS stimulated J774A.1, resulted in a significant provocation of reactive oxygen species (ROS) (p < 0.01) in comparison to CpG ODN 1826 alone. Moreover, the formulated nanovehicles succeeded in generating a profound Th1-based cytokine profile constituted by enhanced expression of IFN-γ (p < 0.001) and IL-1ß (p < 0.001) inflammatory cytokines. These findings clearly indicated the immunostimulatory potential of SPG-ODN 1826 nanovehicles for inducing the Th1-type phenotype, which would certainly assist in skewing M2 phenotype into the much-desired M1 type during lung cancer.

Oligo-DNA Stoichiometrically Binds ß-1,3-Glucan with the Best Fit Length.

Oligo-deoxyadenylic acid (dAX) forms a novel 1:2 triple-helix with ß-1,3-d-glucan schizophyllan (SPG). We found that dAX meticulously selects the most suitable length of SPG to bind; for example, dA30 only complexes with a short SPG chain having 30, 60, or 90 main-chain glucoses, and they can be easily isolated with each other. This study demonstrated such a novel stoichiometric complex formation by using gel permeation chromatography coupled with multi-angle light scattering and synchrotron small-angle X-ray scattering. These oligo-DNA/polysaccharide complexes can be used as a tool for delivering therapeutic oligonucleotides to immunocytes that express the ß-1,3-d-glucan receptors. The present study provides a robust platform technique to characterize them in terms of modern regulatory science of nanomedicines, which is requisite to transfer drug candidates into clinical trial. Our findings are important for characterizing these complexes as well as for providing a new insight into nucleotide and saccharide chemistry.

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by ß-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8+ T Cells in Nonhuman Primates.

The priming, boosting, and restoration of memory cytotoxic CD8+ T lymphocytes by vaccination or immunotherapy in vivo is an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic acid-based compounds have been shown to act as anticancer monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, a humanized CpG DNA (K3) complexed with schizophyllan (SPG), K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously shown to act as a potent monoimmunotherapeutic agent against established tumors in mice in vivo. In this study we extend the monoimmunotherapeutic potential of K3-SPG to a nonhuman primate model. K3-SPG activated monkey plasmacytoid dendritic cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo. A single injection s.c. or i.v. with K3-SPG significantly increased the frequencies of activated memory CD8+ T cells in circulation, including Ag-specific memory CTLs, in cynomolgus macaques. This increase did not occur in macaques injected with free CpG K3 or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx were lower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, we show that K3-SPG has potent Ag-specific memory CTL response-boosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

Conformation and cooperative order-disorder transition in aqueous solutions of ß-1,3-d-glucan with different degree of branching varied by the Smith degradation.

ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.

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.

Complex Consisting of ß-Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes.

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA257-264) and dA40 and made complexes with SPG. The conjugates with a disulfide bond between OVA257-264 and dA40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA257-264/SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist.

CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing "all-in-one" CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.

Simplified process for preparation of schizophyllan solutions for biomaterial applications.

Schizophyllan is a biopolymer commercially produced for pharmaceutical and cosmetics uses. However, schizophyllan also has potential biomaterial applications. Schizophyllan is conventionally produced from glucose and recovered by diafiltration and ultrafiltration to produce a highly purified product. Here we demonstrate a simplified process for preparation of schizophyllan solutions for biomaterial applications. Schizophyllan was produced in 1.5-L bioreactors from distiller's dried grains with solubles (DDGS), an abundant coproduct of dry grind fuel ethanol production. Downstream processing eliminated filtration and concentration steps, providing solutions containing 4.2 ± 0.3 g schizophyllan/L. Solutions contained high-molecular-weight schizophyllan and exhibited viscosity properties similar to those of commercial schizophyllan. Schizophyllan solutions showed promise as a component of biolubricants in friction and wear tests and by dynamic surface and interfacial tension measurements.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Enzymatic modification of schizophyllan.

An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U ß-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning.