Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics.

Localization of M2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods.

We investigated parvalbumin (PV) and calretinin (CR) containing interneurons in the rat entorhinal cortex. RNA amplification following single cell dissection of immunohistochemically labeled cells from layers II to VI revealed that PV cells, in contrast to CR cells, express the m2 muscarinic receptor (M2AchR) protein. Double immunostaining to confirm the results of RNA amplification indicated that the majority of PV cells contain M2AchR protein, whereas only a small proportion of CR cells do. In contrast, a large number of layer I CR cells, which are mostly Cajal-Retzius cells, were positive for M2AchR. RNA amplification following dissection of these cells also revealed that they contain the M2AchR protein. These findings emphasize that there are significant differences in the expression of different proteins, even among similar neuronal types in the same brain region. This highlights the importance of accurately collecting single cells, and knowledge of anatomical details in molecular biological studies.

Overexpression of PHGPx inhibits hydroperoxide-induced oxidation, NFkappaB activation and apoptosis and affects oxLDL-mediated proliferation of rabbit aortic smooth muscle cells.

Rabbit abdominal aortic smooth muscle cells (SMC) were stably transfected with the cDNA of porcine phospholipid hydroperoxide glutathione peroxidase (PHGPx) by means of a retroviral gene transfer technique, to create a model for studying cellular processes relevant to atherogenesis. The transfected cells (SMC/PHGPx) had approximately 4-fold higher PHGPx activity when cultured in the presence of selenite whereas the parental cells did not show any significant increase in PHGPx or total GPx activity upon selenium supplementation. In situ functionality of PHGPx was validated by inhibition of linoleic acid hydroperoxide-induced toxicity, dihydrorhodamine oxidation, NFkappaB activation and apoptosis. SMC grown in 1% FCS responded to oxidized LDL (oxLDL) with a marked proliferation, as measured by [3H]thymidine incorporation, irrespective of selenium supplementation. In SMC/PHGPx grown with or without selenite under control conditions or exposed to native LDL, thymidine incorporation was generally depressed. Also, oxLDL-induced proliferation was lower in SMC/PHGPx compared to untransfected SMC up to 24 h of incubation. After 40 h, however, selenite supplementation restored maximum proliferation response to oxLDL in SMC/PHGPx. The results suggest a proliferative effect of endogenous hydroperoxides in SMC. They further reveal that hydroperoxy lipids of oxLDL contribute to the induction of proliferation, but also suggest involvement of hydroxy lipids in the response to oxLDL.

Effect of vitamin E on endothelial vasodilator function in patients with hypercholesterolemia, chronic smoking or both.

OBJECTIVES: The purpose of this study was to test the hypothesis that long-term supplementation with Vitamin E improves endothelium-dependent relaxation in hypercholesterolemia patients and/or chronic smoking, two risk factors that have been shown to be associated with increased radical formation. BACKGROUND: Experimental evidence suggests that oxidized low density lipoprotein (LDL) impairs endothelium-dependent relaxation, and vitamin E, a lipid-soluble antioxidant, reduces the oxidation of LDL. METHODS: Thirteen subjects with hypercholesterolemia, 14 smokers and 15 hypercholesterolemic smokers were enrolled in a double-blind, placebo-controlled study. After baseline measurements of plasma autoantibodies against oxidized LDL and assessment of endothelium-dependent relaxation using intra-arterial forearm infusions of acetylcholine, participants within each group were randomly assigned in a 1:2 fashion to receive either placebo or vitamin E for 4 months, when plasma levels of autoantibodies against oxidized LDL and vascular function were reassessed. RESULTS: Vitamin E significantly augmented endothelium-dependent relaxation in hypercholesterolemic smokers but not in patients with either hypercholesterolemia or chronic smoking. At baseline, hypercholesterolemic smokers had significantly higher autoantibody levels against oxidized LDL (compared with the other two groups), which were significantly reduced after 4 months of vitamin E supplementation. There was a significant relationship between improvement in acetylcholine-induced vasodilation and the change in autoantibody titer against oxidized LDL (r = -0.59; p = 0.002). CONCLUSIONS: Long-term vitamin E supplementation improves endothelium-dependent relaxation in forearm resistance vessels of hypercholesterolemic smokers, which are characterized by increased levels of autoantibodies against oxidized LDL. These findings may suggest that the beneficial effect of vitamin E is confined to subjects with increased exposure to oxidized LDL.

Vitamin E decreases hepatic levels of aldehyde-derived peroxidation products in rats with iron overload.

Hepatic iron overload can cause lipid peroxidation with the formation of aldehydic products, hepatocellular injury, and fibrosis. Vitamin E (alpha-tocopherol) may prevent peroxidation-induced hepatic damage. We used confocal laser scanning microscopy, digital image analysis, and immunohistochemical methods to quantitate aldehyde-derived peroxidation products in the liver of rats with experimental iron overload with or without supplemental vitamin E. A strong autofluorescent reaction colocalizing with iron deposits was present in the livers of iron-loaded rats. Fluorescent granules were unevenly distributed in the cytosol of both hepatocytes and Kupffer cells in the periportal regions. Immunohistochemical studies revealed the presence of malon-dialdehyde adducts in the periportal regions of the ironloaded rats. Vitamin E supplementation markedly reduced the fluorescence intensity and the amount of aldehyde-derived peroxidation products and changed the distribution of stainable iron and iron-associated peroxidation products such that their levels were much decreased in Kupffer cells. These results indicate that aldehyde-derived covalent chemical addition products are formed in the liver in iron overload. Vitamin E supplementation markedly reduces the amount of these compounds and changes their cellular distribution. These findings should be implicated in the role of antioxidant therapy in conditions causing iron overload and lipid peroxidation.

Experimental liver cirrhosis induced by alcohol and iron.

To determine if alcoholic liver fibrogenesis is exacerbated by dietary iron supplementation, carbonyl iron (0.25% wt/vol) was intragastrically infused with or without ethanol to rats for 16 wk. Carbonyl iron had no effect on blood alcohol concentration, hepatic biochemical measurements, or liver histology in control animals. In both ethanol-fed and control rats, the supplementation produced a two- to threefold increase in the mean hepatic non-heme iron concentration but it remained within or near the range found in normal human subjects. As previously shown, the concentrations of liver malondialdehyde (MDA), liver 4-hydroxynonenal (4HNE), and serum aminotransferases (ALT, AST) were significantly elevated by ethanol infusion alone. The addition of iron supplementation to ethanol resulted in a further twofold increment in mean MDA, 4HNE, ALT, and AST. On histological examination, focal fibrosis was found < 30% of the rats fed ethanol alone. In animals given both ethanol and iron, fibrosis was present in all, with a diffuse central-central bridging pattern in 60%, and two animals (17%) developed micronodular cirrhosis. The iron-potentiated alcoholic liver fibrogenesis was closely associated with intense and diffuse immunostaining for MDA and 4HNE adduct epitopes in the livers. Furthermore, in these animals, accentuated increases in procollagen alpha 1(I) and TGF beta 1 mRNA levels were found in both liver tissues and freshly isolated hepatic stellate cells, perisinusoidal cells believed to be a major source of extracellular matrices in liver fibrosis. The dietary iron supplementation to intragastric ethanol infusion exacerbates hepatocyte damage, promotes liver fibrogenesis, and produces evident cirrhosis in some animals. These results provide evidence for a critical role of iron and iron-catalyzed oxidant stress in progression of alcoholic liver disease.

Effect of repeated endotoxin treatment and hypercholesterolemia on preatherosclerotic lesions in weaned pigs. Part II. Lipid and glycosaminoglycan analysis of intima and inner media.

We compared the effects of mild hypercholesterolemia and repeated endotoxin infusions on the biochemical composition of aortic intima and inner media of 24 piglets divided into 4 groups 5 days after weaning: controls on normal diet (group I); normal diet and endotoxin (group II); fat-supplemented diet (group III); and fat-supplemented diet and endotoxin (group IV). It was found that mild hypercholesterolemia increased the concentration of arterial esterified cholesterol and the relative amount of the fraction containing chondroitin sulphates A and C in total glycosaminoglycans. Endotoxin infusions partly prevented the increase of serum cholesterol caused by the fat-supplemented diet but had no independent effect on the arterial biochemical composition; nor did they affect the biochemical changes caused by hypercholesterolemia. When the results of all groups were combined, chondroitin sulphates A and C showed a significant positive correlation with the concentration of arterial esterified cholesterol and the percentage of linoleic acid in arterial cholesteryl esters. Serum total cholesterol did not correlate with arterial cholesterol fractions, but the ratio of high density lipoprotein-cholesterol to total serum cholesterol showed a negative association with arterial esterified cholesterol. The present findings indicate that (1) mild hypercholesterolemia is atherogenic in young piglets, and (2) changes in arterial glycosaminoglycan composition might be one of the earliest biochemical alterations in atherogenesis.