Bioactive and degradable scaffolds made from bioactive glass-polycaprolactone with a mineralized surface and a well-defined three-dimensional (3D) pore configuration were produced using a robotic dispensing technique. Human adipose-derived stem cells (hASCs) were cultured on the 3D scaffolds, and the osteogenic development of cells within the scaffolds was addressed under a dynamic flow perfusion system for bone tissue engineering. The bioactive glass component introduced within the composite assisted in the surface mineralization of the 3D scaffolds. The hASCs initially adhered well and grew actively over the mineralized surface, and migrated deep into the channels of the 3D scaffold. In particular, dynamic perfusion culturing helped the cells to proliferate better on the 3D structure compared to that under static culturing condition. After 4 weeks of culturing by dynamic perfusion, the cells not only covered the scaffold surface completely but also filled the pore channels bridging the stems. The osteogenic differentiation of the hASCs with the input of osteogenic factors was stimulated significantly by the dynamic perfusion flow, as determined by alkaline phosphate expression. Overall, the culturing of hASCs upon the currently developed 3D scaffold in conjunction with the dynamic perfusion method may be useful for tissue engineering of bone.

Recent studies have suggested that photo-oxidative lesions produced by photodynamic therapy (PDT)-treated tumors are recognized by the host as altered self, (4) prompting a strong inflammatory and immune response.(5) In addition, the use of PDT vaccines has been studied to generate antitumor immunity and control tumor growth, suggesting that further improvements can be achieved in the optimization of the protocols for the generation of PDTgenerated cancer vaccines.(6,7) Because of the inflammatory/immune response triggered by PDT, this therapy has been shown to be particularly suitable for combination with a variety of immunotherapy based treatments, including angiogenic growth factors, matrix metalloproteinases, cytokines and adoptive transfer of immune cells. (8 -12) Overexpression of these molecules within PDTtargeted tissue can adversely affect tumor response. Therefore, experimental protocols combining PDT with procedures targeting these molecules are being examined in an effort to improve treatment efficacy.Oligodeoxynucleotide (ODN) containing unmethylated cytosine-phosphate-guanosine (CpG) motifs was originally isolated from components of bacterial DNA.(13) CpG-ODN immunotherapy has been studied as a strategy for tumor prevention as well as for treatment of immune disorders. (14,15) A variety of studies have shown that CpG-ODN can activate B cells, monocytes and natural killer cells, and induce a Th1-like pattern of cytokine production.(16-18) The CpG sequences drive macrophages to secrete interleukin-12, a potent inducer of interferon-γ (IFN-γ) production in vivo from natural killer cells. IFN-γ production drives Th1-type immune responses by inducing differentiation of type-1 Th cells, which see antigen in the presence of IFN-γ from the uncommitted T-cell pool. (19,20) Moreover, ODN enhances humoral responses and induces the development of enhanced cytotoxic T lymphocytes (CTL) activity. (17,21) ODN has been studied extensively as a strong immunomodulatory agent. (22)(23)(24) The aim of the present study was to further characterize the immunotherapeutic significance of the combination of CpGoligodeoxynucleotide and TC-1 tumor cell lysates induced by PDT. We examined the in vivo antitumor effect of PDT (or freezing/thawing)-generated cell lysates plus ODN injection and the immune response in a mouse model. Our results showed that PDT-cell lysates plus ODN showed a significant suppression of tumor growth at both prophylactic and therapeutic levels, compared to PDT (or F/T)-cell lysates or ODN alone. Materials and MethodsPreparation of PDT-generated tumor cell lysates. Radachlorin (25) was purchased from RADA-PHARMA group (RADA-PHARMA, Moscow, Russia). The light source was a diode laser with a 662 ± 3 nm wavelength (Won-PDT D662, Won Technology, Daejeon, Korea). The irradiation power in vitro was fixed to be 6.25 J/cm 2 at 20 mW/cm 2 for 5 min irradiation measured at a distance of 3 cm from the exit slit.To generate cell lysates by Radachlorin/PDT, TC-1 cells carrying human papillomavirus (HPV) 16 E7 (ATC...

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

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The aim of this study was to investigate the anti-inflammatory properties of each fraction of Hericium erinaceus (HE). The ethanol extract from HE was partitioned with different solvents in the order of increasing polarity. The treatment with 10-100 μg/mL of each fraction did not reduce RAW 264.7 cell viability except ethyl acetate fraction. Among the various extracts, the chloroform fraction showed the most potent activity against nitric oxide (NO), prostaglandin E(2) (PGE(2)) and reactive oxygen species (ROS). The western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that chloroform fraction from HE (CHE) significantly reduced the protein level of iNOS and cyclooxygenase-2 (COX-2) or mRNA levels of iNOS in lipopolysaccharide-induced macrophages. Furthermore, CHE inhibited the translocation of nuclear factor (NF)-κB p65 subunit, phsophorylation of I-κB, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in a dose-dependent manner. Furthermore, the activation of both activator protein-1 (AP-1) and NF κB in the nucleus were abrogated by CHE with luciferase assay. In conclusion, these results indicate that CHE may provide an anti-inflammatory effect by attenuating the generation of excessive NO, PGE(2), and ROS and by suppressing the expression of pro-inflammatory genes through the inhibition of NF-κB and JNK activity.