Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

NK4 gene therapy combined with cisplatin inhibits tumour growth and metastasis of squamous cell carcinoma.

BACKGROUND: NK4 inhibits vascularisation in tumour tissues, thereby arresting tumour growth. However, the antitumour efficacy of individual antiangiogenic molecules expressed in vivo is not sufficiently potent to induce regression in animal models. One of the strategies to overcome this disadvantage is to use chemotherapy. MATERIALS AND METHODS: This study evaluated the efficacy of combining NK4 gene therapy with cisplatin to treat experimental squamous cell carcinomas. For gene therapy, biodegradable cationised gelatin microspheres were used for the controlled release of NK4 plasmid DNA. RESULTS: A combined regimen of antiangiogenic gene therapy and low-dose cisplatin led to a marked decrease in tumour volume and vascularity, and caused increased apoptosis compared to NK4 gene therapy alone. Moreover, combination treatment of NK4 gene therapy and low-dose cisplatin dramatically inhibited the formation of lung metastases. CONCLUSION: NK4 gene therapy combined with low-dose cisplatin may be an effective regimen for treating oral squamous cell carcinoma.

Cold shock domain protein A (CSDA) overexpression inhibits tumor growth and lymph node metastasis in a mouse model of squamous cell carcinoma.

Cancer cells metastasize by entering the lymphatic system. Regional lymph-node dissemination is the first detectable step in the metastasis of oral squamous cell carcinoma (SCC) and is highly correlated to the prognosis of the disease. Cold shock domain protein A (CSDA) is a DNA-binding protein that represses angiogenesis and lymphangiogenesis by directly binding to hypoxia response element (HRE) and serum response element (SRE). In our study we used the cell line NR-S1M, a mouse SCC model with a high rate of lymph-node metastasis. Into these cells we transfected the expression-plasmid coding for full-length mouse CSDA. Of importance, we showed that overexpression of CSDA significantly inhibits the production of VEGF-A and VEGF-C in NR-S1M cells. The overexpression of CSDA in NR-S1M cells inhibited tumor growth, inhibited regional lymph-node metastasis, and reduced the density of blood vessels and lymphatic vessels in the primary tumors in vivo. Our results support the hypothesis that VEGF-A and VEGF-C are crucial regulators of angiogenesis and lymphangiogenesis in NR-S1M cells. Therefore, they are promising targets for CSDA overexpression gene therapy to inhibit tumor growth and lymph-node metastasis in SCC.

Enhanced regeneration of critical bone defects using a biodegradable gelatin sponge and beta-tricalcium phosphate with bone morphogenetic protein-2.

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and beta-tricalcium phosphate (betaTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-betaTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-betaTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-betaTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma.

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.

Synthesis of an optically pure synthetic intermediate of aloperine from a yeast-reductive product.

Optically pure (S)- and (R)-vinylpiperidine 2 and (S)- and (R)-(hydroxyethyl)piperidine 3, which were key intermediates for the synthesis of aloperine, were synthesized from yeast-reductive products.

Essential role of LFA-1 in activating Th2-like responses by alpha-galactosylceramide-activated NKT cells.

NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-gamma) and Th2 (IL-4) responses following stimulation of their invariant Valpha14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Valpha14 ligand alpha-galactosylceramide (alpha-GalCer) remains unclear. To address this issue, LFA-1-/- (CD11a-/-) mice were used to investigate IL-4 and IFN-gamma production by NKT cells following alpha-GalCer stimulation. Intriguingly, LFA-1-/- mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to alpha-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in alpha-GalCer-activated NKT cells from LFA-1-/- mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.