Short-term treatment with nitrate is not sufficient to induce in vivo antithrombotic effects in rats and mice.

In humans, short-term supplementation with nitrate is hypotensive and inhibits platelet aggregation via an nitric oxide (NO)-dependent mechanism. In the present work, we analyzed whether short-term treatment with nitrate induces antithrombotic effects in rats and mice. Arterial thrombosis was evoked electrically in a rat model in which renovascular hypertension was induced by partial ligation of the left renal artery. In mice expressing green fluorescent protein, laser-induced thrombosis was analyzed intravitally by using confocal microscope. Sodium nitrate (NaNO3) or sodium nitrite (NaNO2) was administered orally at a dose of 0.17 mmol/kg, twice per day for 3 days. Short-term nitrate treatment did not modify thrombus formation in either rats or mice, while nitrite administration led to pronounced antithrombotic activity. In hypertensive rats, nitrite treatment resulted in a significant decrease in thrombus weight (0.50 ± 0.08 mg vs. VEH 0.96 ± 0.09 mg; p < 0.01). In addition, nitrite inhibited ex vivo platelet aggregation and thromboxane B2 (TxB2) generation and prolonged prothrombin time. These effects were accompanied by significant increases in blood NOHb concentration and plasma nitrite concentration. In contrast, nitrate did not affect ex vivo platelet aggregation or prothrombin time and led to only slightly elevated nitrite plasma concentration. In mice, nitrate was also ineffective, while nitrite led to decreased platelet accumulation in the area of laser-induced endothelial injury. In conclusion, although nitrite induced profound NO-dependent antithrombotic effects in vivo, conversion of nitrates to nitrite in rats and mice over short-term 3-day treatment was not sufficient to elicit NO-dependent antiplatelet or antithrombotic effects.

Genetic engineering and molecular characterization of yeast strain expressing hybrid human-yeast squalene synthase as a tool for anti-cholesterol drug assessment.

AIMS: The main objective of the study is molecular and biological characterization of the human-yeast hybrid squalene synthase (SQS), as a promising target for treatment of hypercholesterolaemia. METHODS AND RESULTS: The human-yeast hybrid SQS, with 67% amino acids, including the catalytic site derived from human enzyme, was expressed in Saccharomyces cerevisiae strain deleted of its own SQS gene. The constructed strain has a decreased level of sterols compared to the control strain. The mevalonate pathway and sterol biosynthesis genes are induced and the level of triacylglycerols is increased. Treatment of the strain with rosuvastatin or zaragozic acid, two mevalonate pathway inhibitors, decreased the amounts of squalene, lanosterol and ergosterol, and up-regulated expression of several genes encoding enzymes responsible for biosynthesis of ergosterol precursors. Conversely, expression of the majority genes implicated in the biosynthesis of other mevalonate pathway end products, ubiquinone and dolichol, was down-regulated. CONCLUSIONS: The S. cerevisiae strain constructed in this study enables to investigate the physiological and molecular effects of inhibitors on cell functioning. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast strain expressing hybrid SQS with the catalytic core of human enzyme is a convenient tool for efficient screening for novel inhibitors of cholesterol-lowering properties.

Role of xanthine oxidoreductase in the anti-thrombotic effects of nitrite in rats in vivo.

The mechanisms underlying nitrite-induced effects on thrombosis and hemostasis in vivo are not clear. The goal of the work described here was to investigate the role of xanthine oxidoreductase (XOR) in the anti-platelet and anti-thrombotic activities of nitrite in rats in vivo. Arterial thrombosis was induced electrically in rats with renovascular hypertension by partial ligation of the left renal artery. Sodium nitrite (NaNO2, 0.17 mmol/kg twice daily for 3 days, p.o) was administered with or without one of the XOR-inhibitors: allopurinol (ALLO) and febuxostat (FEB) (100 and 5 mg/kg, p.o., for 3 days). Nitrite treatment (0.17 mmol/kg), which was associated with a significant increase in NOHb, nitrite/nitrate plasma concentration, resulted in a substantial decrease in thrombus weight (TW) (0.48 ± 0.03 mg vs. vehicle [VEH] 0.88 ± 0.08 mg, p < 0.001) without a significant hypotensive effect. The anti-thrombotic effect of nitrite was partially reversed by FEB (TW = 0.63 ± 0.06 mg, p < 0.05 vs. nitrites), but not by ALLO (TW = 0.43 ± 0.02 mg). In turn, profound anti-platelet effect of nitrite measured ex vivo using collagen-induced whole-blood platelet aggregation (70.5 ± 7.1% vs. VEH 100 ± 4.5%, p < 0.05) and dynamic thromboxaneB2 generation was fully reversed by both XOR-inhibitors. In addition, nitrite decreased plasminogen activator inhibitor-1 concentration (0.47 ± 0.13 ng/ml vs. VEH 0.62 ± 0.04 ng/ml, p < 0.05) and FEB/ALLO reversed this effect. In vitro the anti-platelet effect of nitrite (1 mM) was reversed by FEB (0.1 mM) under hypoxia (0.5%O2) and normoxia (20%O2). Nitrite treatment had no effect on coagulation parameters. In conclusion, the nitrite-induced anti-platelet effect in rats in vivo is mediated by XOR, but XOR does not fully account for the anti-thrombotic effects of nitrite.

Interleukin-6 blockade in ocular inflammatory diseases.

Interleukin-6 (IL-6) is a key cytokine featuring redundancy and pleiotropic activity. It plays a central role in host defence against environmental stress such as infection and injury. Dysregulated, persistent interleukin (IL)-6 production has been implicated in the development of various autoimmune, chronic inflammatory diseases and even cancers. Significant elevation of IL-6 has been found in ocular fluids derived from refractory/chronic uveitis patients. In experimental autoimmune uveitis models with IL-6 knock-out mice, IL-6 has shown to be essential for inducing inflammation. IL-6 blockade can suppress acute T helper type 17 (Th17) responses via its differentiation and, importantly, can ameliorate chronic inflammation. Tocilizumab, a recombinant humanized anti-IL-6 receptor antibody, has been shown to be effective in several autoimmune diseases, including uveitis. Herein, we discuss the basic biology of IL-6 and its role in development of autoimmune conditions, focusing particularly on non-infectious uveitis. It also provides an overview of efficacy and safety of tocilizumab therapy for ocular inflammatory diseases.

Angiotensin-(1-9) enhances stasis-induced venous thrombosis in the rat because of the impairment of fibrinolysis.

INTRODUCTION: ACE2 alternatively converts angiotensin (Ang) II into Ang-(1-7) and Ang I into Ang-(1-9). There is little information in the literature with respect to Ang-(1-9) properties. A number of studies show a link between peptides of the renin-angiotensin system and thrombosis. MATERIALS AND METHODS: We have investigated the influence of Ang-(1-9) on stasis-induced venous thrombosis in the rat. The contribution of coagulation and fibrinolytic systems, angiotensin receptor type 1 (AT1) and MAS receptor in the mode of Ang-(1-9) action was also determined. RESULTS: Ang-(1-9) enhanced thrombosis development, decreased plasma concentration of tissue plasminogen activator and increased the level of its inhibitor (PAI-1). The action of Ang-(1-9) was reversed by selective antagonist of AT1 receptor, but not Ang-(1-7) antagonist. Ang-(1-9) did not bind to the AT1 receptor. CONCLUSIONS: Ang-(1-9) enhances venous thrombosis in the rat because of the impairment of fibrinolysis. The prothrombotic effect of Ang-(1-9) is mediated by Ang II acting via the AT1 receptor.

Periodontal pharmacotherapy - an updated review.

Periodontal disease is mainly associated with the activity of bacteria which adhere to the tooth surface and form specific structure of bacterial biofilm. Periodontal bacteria cause inflammation of the gums and aggressive immune response, affecting the periodontium. The first phase of initial therapy - mechanical removal of dental plaque and calculus - is necessary. If this non-surgical therapy has proved to be unsuccessful, an alternative treatment with antimicrobial agents is then considered. Pharmacotherapy is based on systemic or local antibiotics and/or antiseptics, which are applied according to the severity of the disease. A number of recent periodontal studies present some of the pharmacological agents, that are directed against bacteria or a host immune response, are often chosen as an adjunct treatment option, but none of these antimicrobials were established as 'a gold standard' in the periodontal treatment. This review provides some present recommendation of pharmacological strategies, with particular emphasis on systemic and local antimicrobial therapy of periodontal disease.

Chorioretinitis in a 7-year-old African girl, probably related to JSSc resolving to methotrexate therapy.

Juvenile systemic sclerosis (JSSc) is a rare but severe multi-system connective tissue disease of unknown etiology. It is one of the most difficult to treat rheumatic diseases in childhood and characterized by thickening and fibrosis of the skin and associated with fibrosis of internal organs. Eye involvement has rarely been reported. In a 7-year-old African girl, who presented with chorioretinitis and subsequently developed JSSc we discuss the possible association of chorioretinitis with JSSc and the putative implications of scleroderma vascular disease in the development of this complication and review the existing literature.

Angiotensin-(1-9), the product of angiotensin I conversion in platelets, enhances arterial thrombosis in rats.

Angiotensin (Ang) (1-9) is the renin-angiotensin-system peptide found in the plasma of healthy volunteers and after angiotensin-converting-enzyme inhibitors therapy. In vitro experiments proved that Ang-(1-9) may be produced from Ang I. In our study, we tried to expand the poor data about the in vivo properties of Ang-(1-9). We revealed that Ang-(1-9) enhanced electrically stimulated arterial thrombosis in the carotid artery of Wistar rats. Losartan, a selective blocker of AT1 receptor for Ang II, abolished the prothrombotic activity of Ang-(1-9). This peptide increased plasma level of fibrinogen, augments fibrin generation, and similarly to Ang II, potentiated collagen induced platelet aggregation. Using HPLC, we found that after incubation of Ang-(1-9) with platelet homogenates or after intravenous administration this peptide is converted to Ang II. We concluded that Ang-(1-9) exerts an Ang II-like prothrombotic effect due to the conversion to Ang II in the circulatory system of rats and that platelets are involved in this process.

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment.

AIMS: Statins - inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase - are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs. METHODS AND RESULTS: We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins. CONCLUSION: The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.

[Clinically oligosymptomatic and diagnostically doubtful brain tumors in patients of the Neurology Department CSK MSW]. / Skapoobjawowe klinicznie i niepewne diagnostycznie guzy mózgu w materiale wlasnym oddzialu neurologii CSK MSW.

Three cases of brain tumours are reported in patients with clinical symptoms much too scant in relation to the extent of the lesion. MRI examinations were decisive for correct diagnosis and for tumour localization. Early brain tumour diagnosis by MR imaging may influence favourably prognosis in such cases.

Activation by reduced glutathione of methotrexate transport into isolated rat liver cells.

The uptake of methotrexate (MTX) by isolated rat hepatocytes and its changes under the influence of exogenous GSH have been studied under various conditions: GSH concentration, pH of incubation medium, preincubation of cells prior to MTX and GSH addition, ionic composition of the incubation medium (standard saline, Na+-free, Na+ and K+-free, or ion-deficient), after prior treatment of cells by membrane -SH blockers (p-CMBS, 4-CMB and DIP2+) and ATP. It was found that GSH strongly accelerated MTX uptake. This effect depended on GSH concentration and on preincubation of cells. The GSH effect was not dependent on medium pH in spite of an observed close relationship between pH of incubate and MTX transport itself. Activation by GSH of MTX transport was connected to an increase in intracellular K+. It was also noted that while blockers of membrane -SH groups like p-CMBS and 4-CMB inhibited MTX uptake and increased the intracellular Na+/K+ ratio, both effects were partially overcome by GSH. After treatment by DIP2+, Na+/K+ ratio was unaffected, but MTX uptake inhibited. Still GSH abolished inhibition. Added ATP also inhibited MTX uptake and caused loss of cellular K+ and accumulation of Na+. Here neither effect could be reversed by GSH; consequently, high cellular amounts of K+ and MTX accumulated by previous action of GSH were depleted on subsequent ATP addition. MTX uptake was low in sucrose medium. But in this ion-deficient medium, GSH had the greatest stimulatory effect on MTX uptake. It is concluded that binding GSH can affect the redox state of the -S-S-/-SH groups of the cellular plasma membrane and that this effect of GSH might demonstrate involvement of the redox state in the control of MTX permeability.

The effect of pyrogen induced fever on pharmacokinetics of rifamycin SV.

The fever was evoked in rabbits by single iv injection of pyrogen-standard from E. coli, and rifamycin SV was administered at the peak of the fever (T = 1.7 degrees C). The rifamycin blood level was determined during the fever stability and changes of kinetics of the antibiotic were investigated. It was found that distribution and elimination of rifamycin were decreased during fever. The consequence of the inhibition of the antibiotic elimination was the increase of its level in blood and tissue compartments.

Effect of some peptides on distribution of rifamycin SV in rabbits.

The effect of insulin, glutathione, trypsin and somatotropin on processes of distribution and elimination of rifamycin SV were investigated using pharmacokinetic analysis. It was found that the peptides, especially glutathione and trypsin, enhance the distribution of rifamycin in rabbits and cause an increase of amount of this antibiotic in the central compartment.