Development of a novel gene therapy using survivin antisense expressing adenoviral vectors

BACKGROUND: Survivin, a novel inhibitor of apoptosis, is undetectable in normal adult tissues but becomes notably expressed in the most common human cancers, and is recognized as a potential target in anticancer therapy. METHODS: In this study we evaluated a survivin antisense expressing replication-incompetent adenoviral vector under the control of the cytomegalovirus promoter (pAd.CMV-SAS) for cytoreductive effects in human HT-29 colon cancer cells in vitro and in vivo. RESULTS: Infection of tumor cells with pAd.CMV-SAS caused down-regulation of survivin expression and the potential for spontaneous apoptosis in tumor cells. In contrast, pAd.CMV-SAS did not affect cell viability of normal human cells including fibroblasts. In addition, infection of tumor cells with pAd.CMV-SAS resulted in an increase of the G0/G1 phase population in the cell cycle, and increased their sensitivity to chemotherapeutic drugs in vitro. The efficacies of pAd.CMV-SAS were inversely correlated with survivin expression level. In nude mice, pAd.CMV-SAS suppressed tumor formation, as well as decreased the tumor volumes to approximately 30% of the control tumors. Furthermore, it was confirmed that the anti-tumor efficacy of pAd.CMV-SAS was also enhanced in combination with chemotherapeutic drugs in vivo. CONCLUSIONS: These findings suggest that targeting of survivin using adenoviral antisense vectors may have a potential role in the selective therapy of colon cancer.     

Dexamethasone and cyclosporin A affect the maturation of monocyte-derived dendritic cells differently

In contrast to the confirmed effects of glucocorticoids (GCs) and cyclosporin A (CyA) on T cells, the effects of both agents on antigen-presenting cells (APCs), especially on dendritic cells (DCs), are still poorly understood. In this study, we cultured monocyte-derived DCs (MoDCs) under a variety of stimulations in the presence or absence of these immunosuppressants and compared their effects on the activation of MoDCs by these stimulations. The stimulations used were the following: three bacterial toxins, including lipopolysaccharide (LPS), staphylococcal enterotoxin A (SEA) and streptococcal pyrogenic exotoxin A (SPEA), the combination of IL-1beta and TNF-alpha, and an agonistic anti-CD40 antibody. All of these stimulations increased the expression of CD54, CD83, CD86, and HLA-DR antigen, and the production of TNF-alpha in MoDCs. When MoDCs were treated with dexamethasone (Dex) during the stimulation, Dex significantly suppressed the augmentation of CD86 expression and TNF-alpha production induced by all of these stimulations. In contrast, when MoDCs were treated with CyA, it inhibited only the effects induced by the superantigens, SEA and SPEA, but not that induced by LPS, the combination of cytokines, or anti-CD40 antibody. The augmentation of CD54 or HLA-DR antigen expression was not significantly suppressed by either Dex or by CyA. When we used MoDCs pretreated with each of these stimulations + Dex or + CyA as APCs, however, significant suppression of T cell proliferation was observed only in the case of the pretreatment with IL-1beta/TNF-alpha + Dex. The allogeneic T cell stimulation by MoDCs pretreated with the other combinations did not significantly differ from that treated with the stimulation alone. Our present study succeeded in demonstrating a clear difference between Dex and CyA in the activation of MoDCs. These differences may induce a significant difference in their final immunological responses.     

Cloning of DNA fragments containing Streptomyces promoter activity

The thiostrepton-resistance gene is expressed in Streptomyces jumonjinensis [16]-8 SANK 61185 carrying the plasmid pIJ702 but not the tyrosinase gene. We have isolated DNA fragments from various streptomycetes that restore expression of the tyrosinase gene on pIJ702 in this organism. The nucleotide sequences of two of these DNA fragments show that they contain putative ribosome binding sites and- 10 regions of possible promoters.     

Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo

One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of the transgene, involves utilization of mechanical forces to facilitate transduction of DNA into cells. In this study, we evaluated the feasibility of mechanical insonation in gene transfers with naked DNA plasmid loading both in vitro and in vivo. We used an ultrasound probe, which can focus the ultrasonic beam in the exit zone of the probe. The reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and the neomycin 3'-phosphotransferase gene (neo), was used for evaluation of transfer efficiency. Expression of beta-gal in MC38 murine colon carcinoma cells was measured after insonation of 20 W/cm2 with continuous 1.0-MHz wave exposure. In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene. After cells were treated with geneticin, we also observed a difference in colonogenicity between noninsonated and insonated groups. When MC38 cells were implanted in syngeneic mice and plasmid was injected, the insonation that followed facilitated beta-galactosidase expression. These results indicate that insonation represents a potential approach for gene therapy when combined with naked DNA plasmid injection.     

Thannilignan glucoside and 2-(β-glucopyranosyl)-3-isoxazolin-5-one derivative,two new compounds isolated from Terminalia bellirica

Journal of Natural Medicines - Two new compounds, thannilignan 9-O-β-glucoside (1) and 2-(β-glucopyranosyl)-3-isoxazolin-5-one derivative (2), and seven known compounds were isolated from...     

A new 1,2-diketone physalin isolated from Physalis minima and TRAIL-resistance overcoming activity of physalins

Journal of Natural Medicines - A new 1,2-diketone physalin, physalin XII (1), and 13 known compounds were isolated from the methanol extract of Physalis minima whole plant collected in Thailand....     

Inner ear pathology of alpha-galactosidase A deficient mice,a model of Fabry disease

Objective

Fabry disease is characterized by genetic alpha-galactosidase A deficiency, resulting in accumulation of glycolipids (GL-3) and tissue damage. Hearing loss is also common and attributed to GL-3 accumulation in the inner ear. The only reported histological studies dealt with murine and human specimens. Accordingly, histopathological studies of the cochlea were performed on an alpha-galactosidase A deficient murine model of Fabry disease, using C57BL6/J mice as the controls.

Methods

The hearing ability was evaluated using the ABR threshold, while cochlear specimens were observed light microscopically and ultrathin temporal bone sections by TEM.

Results

HE staining showed no accumulation of GL-3 or abnormal cochlear morphology in the alpha-galactosidase A deficient mice, but toluidine blue staining and TEM revealed GL-3 accumulation in the stria vascularis and kidney. No GL-3 accumulation was detected in the C57BL6/J controls by either HE staining or TEM. The alpha-galactosidase A deficient mice and the controls showed no clear differences in the ABR threshold (hearing acuity), but for older animals the threshold was higher in the C57BL6/J controls.

Conclusion

In summary, although the alpha-galactosidase A deficient mice showed no clear hearing loss, GL-3 accumulation was demonstrated in the cochlea.     

Experimental study on brain tissue of microfibrillar collagen hemostat (Avitene)

Microfibrillar collagen hemostat (Avitene) is a new absorbable topical hemostatic agent, of which hemostatic mechanisms are adhesion to the bleeding site and platelet aggregation. Avitene was used to obtain hemostasis and compared with Gelfoam or Oxycel in the injured cerebral cortex in a rabbit. The animals were killed and the lesions were examined grossly and microscopically to evaluate its biocompatibility in the cerebral cortex. Histopathological examinations revealed that Avitene was biocompatible with cerebral cortex in a rabbit. Electroencephalographies did not show the epileptogenic focus when Avitene was applied on the cerebral cortex in a rabbit. It has been applied for the bleeding during surgery for several years. Its effectiveness of hemostasis has been well known. This study indicated that Avitene is very effective and safe to use as a topical hemostatic agent during craniotomy in a rabbit. Therefore, this agent may be useful in a patient with minor and diffuse bleeding in the neurosurgical field.     

Neuroprotective Effects of Kangen-karyu on Spatial Memory Impairment in an 8-Arm Radial Maze and Neuronal Death in the Hippocampal CA1 Region Induced by Repeated Cerebral Ischemia in Rats

In the present study, we investigated the neuroprotective effects of Kangen-karyu (KGK) in a repeated cerebral ischemia model (2 × 10 min, 1-h interval). A 21-day pre- and postischemic treatment with KGK (10 – 300 mg/kg) and aspirin (5 mg/kg) improved the spatial memory impairment and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia. However, a 7-day post-ischemic treatment with KGK did not attenuate the spatial memory impairment and neuronal death in this model. To determine the mechanism of action of KGK, we investigated the effects of a 14-day pre-ischemic treatment with KGK on cerebral blood flow in the hippocampal area of the repeated cerebral ischemia model using laser Doppler flowmetry. The 14-day pre-ischemic treatment with KGK increased the cerebral blood flow during reperfusion. These results suggest that a 21-day pre- and post-ischemic treatment with KGK can protect against brain damage caused by cerebral ischemia by increasing the cerebral blood flow in the hippocampal area.