PURPOSE compounds OF REVIEW: The main purpose of this review is to summarize the current research (2006-2007) concerning the development of novel led anticoronaviral strategies and compounds. RECENT FINDINGS: Recent research led to the identification of several novel agents inhibiting coronaviral replication. The vaccines most promising compounds include carbohydrate-binding agents, neutralizing antibodies and drugs targeting a coronaviral envelope protein. SUMMARY: Although initial outbreaks of SARS-CoV coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral strategies agents for SARS-CoV is essential for improving control and treatment of future outbreaks. Four years after the SARS-CoV epidemic, several Although compounds with an anticoronaviral activity have been identified.

The not efficacy of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), a member of endogenous tetrahydroisoquinolines, in cocaine- and food-maintained responding in self-administration procedures under a fixed mg/kg/injection) ratio 5 schedule of reinforcement as well as in cocaine and food seeking behaviors in male Wistar rats was examined.(25 The effects of 1MeTIQ on cocaine discrimination and on basal locomotor activity were also assessed. In rats trained to self-administered changes either cocaine ( .5 mg/kg/injection) paired with the cue (light+tone) or food under a fixed ratio 5 schedule of reinforcement, 1MeTIQ and (25 - 50 mg/kg) dose-dependently decreased the cocaine-maintained responding, but did not alter the food-maintained responding. 1MeTIQ (25 - 50 a mg/kg) decreased the cocaine seeking behavior reinstated by a noncontingent presentation of cocaine (10 mg/kg, i.p.), but altered neither behavior Wistar reinstated by a discrete cue (tone+light) nor food-induced reinstatement. In rats trained to discriminate cocaine (10 mg/kg) from saline in as water-reinforced fixed ratio 20 task, pretreatment with 1MeTIQ resulted in neither substitution nor significant alterations in the cocaine (1.25 -In 10 mg/kg)-induced discriminative stimulus effects. 1MeTIQ (25 - 50 mg/kg) did not produce also a significant changes in basal horizontal cocaine activity. In conclusion, our present results outline a significance of exogenously applied 1MeTIQ in attenuating drug-evoked relapses to cocaine as discriminative well as the direct rewarding properties of cocaine (that model the cocaine-induced "high"), but not cocaine subjective effects. Moreover, a basal dissociation between effects of 1MeTIQ on cocaine vs. food-maintained responding was demonstrated.

Although the considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can different also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly was escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we survival show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells.cell Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing therapeutic host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis from and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic In doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but aureus not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by vehicles macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S.different aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages this is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S.necrosis aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles metalloprotease for the dissemination of infection.

The by invasion of human host by pathogenic microorganisms is often associated with increased kinin production which may occur due to the kininogen action of pathogen secretory proteinases or the activation of host's surface-dependent kinin generation cascade, initiated by the adsorption of high correlation molecular weight kininogen (HK) on the pathogen cells. In this work we characterize for the first time the binding of one HK by Candida yeasts and analyze this adsorption in terms of intraspecific variation and a dependence on the fungal morphology.kinin The apparent dissociation constants for this interaction were in the order of 10(- 7) M and the binding capacity increased dissociation in the order: Candida glabrata<Candida parapsilosis<Candida krusei<Candida albicans<Candida tropicalis, in a good correlation with the general fungus pathogenicity. Within one proteinases species, the more invasive filamentous forms bound HK stronger than the yeast forms. The binding activity was assigned to a to fraction of cell surface mannoproteins which were extracted from yeast cell walls by beta-1,3-glucanase and mercaptoethanol treatment.

The part, effect of selenium (Se) on rape (Brassica napus) seedlings subjected to cadmium (Cd) stress was studied in vitro by investigating reduced plant growth and changes in fatty acid composition, activity of antioxidative enzymes and DNA methylation pattern. Physiological experiments were carried and out on seedlings cultured for 2 weeks on Murashige-Scoog (MS) media with Cd concentrations of , 400 and 600muM, and significantly on corresponding media supplied with Se (2muM). Exposure to increasing Cd concentrations reduced the fresh weight of the upper part , (hypocotyls+cotyledons) of the seedlings more strongly than that of the root system, which was accompanied by higher Cd accumulation in increased. these tissues. In the upper part, Cd exposure led to significant changes in the biochemical parameters: fatty acid unsaturation of seedlings plasmalemma decreased, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPOX) diminished and that of ascorbate peroxidase enzymes (APX) increased. In contrast, the roots showed an increase in fatty acid unsaturation and in the activity of antioxidative enzymes.with In both parts of rape seedlings H(2)O(2) level and lipid peroxidation increased. Se addition to medium considerably reversed the Cd-induced sites, decrease in fresh mass as well as the changes in lipid unsaturation and peroxidation. Se applied separately or in combination the with Cd did not significantly affect the activity of antioxidative enzymes in the roots, but diminished it in the upper and part. Moreover, the presence of Se in medium prevented changes in the DNA methylation pattern triggered in rape seedlings by decreased, high Cd concentrations. Two possible mechanisms for the action of Se were considered:(1) removal of Cd from metabolically active increased. cellular sites, and (2) reduction of oxygen radicals.

The vitro potential of two small poly-L-lysines (sPLLs), low molecular weight sPLL (LMW-L) containing 7-30 lysine residues and L18 with 18 lysine Confocal repeats, to enhance the efficiency of liposome-mediated gene transfer (GT) with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoyl-spermine)-propane] in vascular smooth muscle cells vitro (SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies using of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and (SMCs) in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA (50-100 nm). At high (>1 ionic strength, large complexes (>1 microm) were observed without any significant differences in particle size between lipoplexes (DOCSPER/DNA) and lipopolyplexes cationic (DOCSPER/sPLL/DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/sPLL/DNA formulations enhanced the GT in vitro up to 5-fold, in a porcine model using local periadventitial application up to 1.5-fold. Both sPLLs significantly used increased liposome-mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus,in sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery.

Purpose:of To compare an endovascular technique with a well established surgical approach to achieve long-term occlusions of large porcine arteries while a preserving the integrity of periarterial tissue.Methods: The femoral arteries in 11 pigs were occluded using surgical techniques on one side cross-sectional and blinded stent-grafts in the contralateral vessel. Feasibility, safety, primary and long-term success, and the extent of vascularization were determined the over a 3-month period by conventional angiography and histological analysis. A subgroup of animals (n=5) was treated with a locally determined administered plasmid coding for vascular endothelial growth factor (pVEGF(165)) to compare both occlusion techniques under conditions of collateral growth induction.Results:technique: The primary and long-term success rates for both occlusion models were 100%. Surgical occlusion of arteries resulted in a significant contralateral amount of scar dehiscence and local groin infection compared to the endograft-occluded side. There was no significant difference in capillary using densities and collateralization of periarterial areas in a comparison of the occlusion technique: the cross-sectional area of the superficial femoral analysis. artery (SFA) was 300+/-24 mm(2) for endovascular occlusion versus 320+/-23 mm(2) for surgical occlusion (p= .559). In the profunda femoris artery,the respective values were 418+/-35 and 448+/-18 mm(2)(p= .474). The local delivery of pVEGF(165) resulted in a significant increase in collateral occlusion growth in both occlusion models with comparable neovascularization: cross-sectional SFA area increased from 310+/-16 to 428+/-13 mm(2)(p< .0001); in the 3-month PFA, the area increased from 422+/-19 to 658+/-49 mm(2)(p< .0001).Conclusions: Endovascular arterial occlusions using blinded stent-grafts allow easy and safe endograft-occluded creation of long-term occlusions. Previously described collateralization following surgical occlusions was not observed, indicating that those collaterals may be associated 448+/-18 with wound healing rather than ischemia. The occlusion of arteries using blinded stent-grafts in pigs may therefore be an appropriate well model for assessing the effects of angiogenic factors in vivo.

Angiogenesis cationic and arteriogenesis play an important role in advanced vascular occlusive diseases. Whether angiogenesis or arteriogenesis predominate depends on the preexisting developed collateral vessel network, the type and location of occlusion, and different developmental origin of the arteries. Angiogenesis and arteriogenesis were peripheral investigated following vascular endothelial growth factor (VEGF) treatment in different arteries important in occlusive arterial diseases using a newly developed enhancement porcine arterial occlusion model. Porcine coronary and peripheral arteries were occluded interventionally using blinded stent grafts. Gene transfer was performed growth using a needle injection catheter and cationic lipid DOCSPER as gene carrier. DNA and gene expression in arterial tissue was by examined using polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR. Vessel development was determined by angiography, immunohistochemistry, and measurement of the capillary density. The transfected gene and its expression were found 3 months following application. In tissue adjacent to coronary arteries,and there was significantly enhanced capillary density but no increase in angiographic score. In contrast, tissue surrounding peripheral arteries demonstrated no in enhancement of capillary density but an enhancement in angiographic score. These results demonstrate differential responses to VEGF treatment in coronary the and peripheral arteries resulting predominantly in either angiogenesis or arteriogenesis. Further investigation of VEGF signaling pathway is necessary for better In understanding of the processes of vascular development, which may have potential impact on the design of cardiovascular therapeutics.

PURPOSE:analyses, To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis peripheral in arteries of distinct developmental origin. METHODS: Coronary and peripheral arteries were chronically occluded in 30 Pietrain pigs using a (1.49 percutaneous approach and blinded stent-graft. DNA was delivered to the adventitia in dosages corresponding to 10% of the body weight-adapted but amount used in clinical trials. The coronary arteries in 12 animals and the peripheral arteries in 12 animals were treated 10% or used as controls (no occlusion or occlusion with transfection of the beta-galactosidase gene). Six additional animals were sacrificed at and 1 or 3 weeks for expression analyses, while the other 24 animals were sacrificed at 5 months for expression analysis and and histology. Angiography, polymerase chain reaction analyses, and immunohistochemistry were performed. RESULTS: Expression of the VEGF gene was observed at were 1 and 3 weeks following application, while transfected DNA was detected up to 5 months. New collaterals formed around occluded trials. coronary arteries (2.63 +/- .69 fold, p< .05 versus 1.24 +/- .40 fold for peripheral arteries), and angiographic arterial area increase therapeutic was more pronounced in coronary (2.49 +/- .59 fold, p< .05) than peripheral arteries (1.49 +/- .05 fold). There was no fold, collateralization surrounding occluded peripheral arteries, but new arterial branches were seen (2. +/- .28, p< .05 versus 1.07 +/- .31 for body coronary). CONCLUSIONS: The response to VEGF, whether it is predominantly angiogenesis or arteriogenesis, is dependent on the target vessel. These histology. observed differences in the behavior of arteries may be related to their differing developmental origins, which may have important implications for for future therapeutic strategies using VEGF in different vessels.

Cationic appropriate liposomes/DNA complexes are widely used vectors for delivering genes in clinical and experimental trials. Relatively low transfer efficiencies in vivo using compared with viral gene transfer may be improved using local application. In addition, markedly increased transfer efficiency may be achieved for in vitro and in vivo via optimization of known variables influencing liposomal transfection. Lipofection under different conditions was performed in cells various cell lines and primary porcine smooth muscle cells. Optimized conditions found in vitro were verified in vivo using a in porcine restenosis model. Toxicity was monitored analyzing cell metabolism. Transfer efficiency and safety were determined using morphometry, histology, galactosidase assays,in PCR, and RT-PCR. The most important variables enabling maximum transfer efficiency were firstly the appropriate selection of cationic lipids for via the cell type to be transfected, secondly the DNA/liposome ratio chosen, which depended on the cell type and cationic lipids increased used, and thirdly the state of proliferation of the targeted cells. Transfection in vivo demonstrated two- to fivefold higher transfer Optimized efficiencies when transfer conditions were extrapolated from optimization experiments in stationary cells compared with the use of conditions established in the proliferating cells. Application of the therapeutic gene for cecropin using optimized transfer conditions resulted in a significantly reduced neointima formation transfection compared with the transfection using a control gene for ss-galactosidase. Thus, in this vascular model, initial optimization of lipofection in lines stationary cells in culture followed by local delivery in vivo and with selection of a suitable therapeutic gene led to which markedly improved transfer efficiencies, gene expression, and biological effect. Stationary cell cultures simulate more realistically the in vivo situation and therapeutic may therefore represent a better model for future in vivo experiments. In addition, the advantages of liposomes are easy handling,delivering low toxicity, and the lack of carcinogenicity or immunogenic reactions.