Manipulation of Nitric Oxide Levels via a Modified Hydroxyethyl Starch Molecule.

INTRODUCTION: Although the improving effect of nitric oxide (NO) donors has experimentally been demonstrated in shock, there are still no NO donor medications clinically available. Thiol-nitrosothiol-hydroxyethyl starch (S-NO-HES) is a novel molecule consisting of NO coupled to a thiolated derivative of hydroxyethyl starch (HES). It was aimed to assess the ability of S-NO-HES to serve as an NO donor under a variety of in vitro simulated physiologic conditions, which might be the first step to qualify this molecule as a novel type of NO donor-fluid. METHODS: We studied the effect of temperature on NO-releasing properties of S-NO-HES in blood, at 34°C, 37°C, and 41°C. Ascorbic acid (Asc) and amylase were also tested in a medium environment. In addition, we evaluated the activity of S-NO-HES in the isolated aortic ring and Langendorff-perfused heart setup. RESULTS: The NO release property of S-NO-HES was found at any temperature. Asc led to a significant increase in the production of NO compared to S-NO-HES incubation (P < 0.05). The addition of amylase together with Asc to the medium further increased the release of NO (P < 0.05). S-NO-HES exerted significant vasodilatory effects on phenylephrine precontracted aortic rings that were dose-dependent (P < 0.01). Furthermore, S-NO-HES significantly increased the heart rate and additionally reduced the duration of the cardiac action potential, as indicated by a reduction of QTc-B values (P < 0.01). CONCLUSIONS: We demonstrated for the first time that the S-NO-HES molecule exhibited its NO-releasing effects. The effectiveness of this new NO donor to substitute NO deficiency under septic conditions or in other indications needs to be studied.

Effects of N-acetylcysteine (NAC) supplementation in resuscitation fluids on renal microcirculatory oxygenation, inflammation, and function in a rat model of endotoxemia.

BACKGROUND: Modulation of inflammation and oxidative stress appears to limit sepsis-induced damage in experimental models. The kidney is one of the most sensitive organs to injury during septic shock. In this study, we evaluated the effect of N-acetylcysteine (NAC) administration in conjunction with fluid resuscitation on renal oxygenation and function. We hypothesized that reducing inflammation would improve the microcirculatory oxygenation in the kidney and limit the onset of acute kidney injury (AKI). METHODS: Rats were randomized into five groups (n = 8 per group): (1) control group, (2) control + NAC, (3) endotoxemic shock with lipopolysaccharide (LPS) without fluids, (4) LPS + fluid resuscitation, and (5) LPS + fluid resuscitation + NAC (150 mg/kg/h). Fluid resuscitation was initiated at 120 min and maintained at fixed volume for 2 h with hydroxyethyl starch (HES 130/0.4) dissolved in acetate-balanced Ringer's solution (Volulyte) with or without supplementation with NAC (150 mg/kg/h). Oxygen tension in the renal cortex (CµPO2), outer medulla (MµPO2), and renal vein was measured using phosphorimetry. Biomarkers of renal injury, inflammation, and oxidative stress were assessed in kidney tissues. RESULTS: Fluid resuscitation significantly improved the systemic and renal macrohemodynamic parameters after LPS. However, the addition of NAC further improved cortical renal oxygenation, oxygen delivery, and oxygen consumption (p < 0.05). NAC supplementation dampened the accumulation of NGAL or L-FABP, hyaluronic acid, and nitric oxide in kidney tissue (p < 0.01). CONCLUSION: The addition of NAC to fluid resuscitation may improve renal oxygenation and attenuate microvascular dysfunction and AKI. Decreases in renal NO and hyaluronic acid levels may be involved in this beneficial effect. A therapeutic strategy combining initial fluid resuscitation with antioxidant therapies may prevent sepsis-induced AKI.

Differentiated control of deranged nitric oxide metabolism: a therapeutic option in sepsis?

Derangement of nitric oxide (NO) metabolism represents one of the key mechanisms contributing to macro- and microcirculatory failure in sepsis. Sepsis-related therapy combining fluid resuscitation with administration of vasopressor and inotropic agents, however, does not guarantee correction of maldistributed nutritive perfusion between and within organs. Therefore, the differentiated and selective pharmacologic modulation of NO-mediated vascular function could play a useful role in hemodynamic management of patients with sepsis. This viewpoint carefully evaluates the potential role of intentionally using partially opposing effects of NO donors and NO synthase inhibitors to complement current therapy of hemodynamic stabilization in patients with sepsis.

Present status and perspectives of cell-based therapies for liver diseases.

In recent years the interest in liver cell therapy has been increasing continuously, since the demand for whole liver transplantations in human beings far outweighs the supply. From the clinical point of view, transplantation of hepatocytes or hepatocyte-like cells may represent an alternative to orthotopic liver transplants in acute liver failure, for the correction of genetic disorders resulting in metabolically deficient states, and for late stage liver disease such as cirrhosis. Although the concept of cell therapy for various diseases of the liver is widely accepted, the practical approach in humans often remains difficult. An international expert panel critically discussed the recent published data on clinical and experimental hepatocyte transplantation and the possible role of stem cells in liver tissue repair. This paper aims to summarise the present status of cell based therapies for liver diseases and to identify areas of future preclinical and clinical research.

In vitro MR imaging of regulated gene expression.

PURPOSE: To design and evaluate a construct that allows regulated expression of the magnetic resonance (MR) imaging reporter gene human tyrosinase under control of the tetracycline response element. MATERIALS AND METHODS: A breast cancer cell line (MCF-7) was transfected with a plasmid that codes for the tetracycline-controlled transactivator and a new construct. In this construct, the reporter gene human tyrosinase is under control of the tetracycline response element, thus allowing suppression of gene expression by adding doxycycline (tetracycline switched off). A reverse transcription polymerase chain reaction was conducted to evaluate gene expression. Additionally, immunohistochemical investigation of tyrosinase and melanin staining was undertaken to analyze the presence of these molecules. After culture in an iron- and holotransferrin-enriched medium, cells were imaged in a 1.0-T clinical MR imager by using a surface coil and T1-weighted spin-echo and gradient-echo sequences. RESULTS: Two stable transfected cell clones were established. Cells cultured with doxycycline showed no background expression of the human tyrosinase gene, whereas withdrawal of doxycycline resulted in detectable tyrosinase messenger RNA expression. Gene expression results in a detectable tyrosinase protein level and melanin content. Increased signal intensity on T1-weighted MR images in cells that expressed the reporter gene was observed in comparison to genetically identical cells with the reporter gene switched off. CONCLUSION: Our construct enables MR imaging of regulated tyrosinase gene expression in vitro.