Showing papers by "Gdańsk Medical University published in 2001"

Cooperation of Liver Cells in Health and Disease

Abstract: The liver lobule is formed by parenchymal cells, i.e., hepatocytes and nonparenchymal cells. In contrast to hepatocytes that occupy almost 80% of the total liver volume and perform the majority of numerous liver functions, nonparenchymal liver cells, which contribute only 6.5% to the liver volume, but 40% to the total number of liver cells, are localized in the sinusoidal compartment of the tissue. The walls of hepatic sinusoid are lined by three different cell types: sinusoidal endothelial cells (SEC), Kupffer cells (KC), and hepatic stellate cells (HSC, formerly known as fat-storing cells, Ito cells, lipocytes, perisinusoidal cells, or vitamin A-rich cells). Additionally, intrahepatic lymphocytes (IHL), including pit cells, i.e., liver-specific natural killer cells, are often present in the sinusoidal lumen. It has been increasingly recognized that both under normal and pathological conditions, many hepatocyte functions are regulated by substances released from neighboring nonparenchymal cells. Liver sinusoidal endothelial cells constitute the lining or wall of the hepatic sinusoid. They perform important filtration function due to the presence of small fenestrations that allow free diffusion of many substances, but not of particles of the size of chylomicrons, between the blood and the hepatocyte surface. SEC show huge endocytic capacity for many ligands including glycoproteins, components of the extracellular matrix (ECM; such as hyaluronate, collagen fragments, fibronectin, or chondroitin sulphate proteoglycan), immune complexes, transferrin and ceruloplasmin. SEC may function as antigen-presenting cells (APC) in the context of both MHC-I and MHC-II restriction with the resulting development of antigen-specific T-cell tolerance. They are also active in the secretion of cytokines, eicosanoids (i.e., prostanoids and leukotrienes), endothelin-1, nitric oxide, and some ECM components. Kupffer cells are intrasinusoidally located tissue macrophages with a pronounced endocytic and phagocytic capacity. They are in constant contact with gut-derived particulate materials and soluble bacterial products so that a subthreshold level of their activation in the normal liver may be anticipated. Hepatic macrophages secrete potent mediators of the inflammatory response (reactive oxygen species, eicosanoids, nitric oxide, carbon monoxide, TNF-alpha, and other cytokines), and thus control the early phase of liver inflammation, playing an important part in innate immune defense. High exposure of Kupffer cells to bacterial products, especially endotoxin (lipopolysaccharide, LPS), can lead to the intensive production of inflammatory mediators, and ultimately to liver injury. Besides typical macrophage activities, Kupffer cells play an important role in the clearance of senescent and damaged erythrocytes. Liver macrophages modulate immune responses via antigen presentation, suppression of T-cell activation by antigen-presenting sinusoidal endothelial cells via paracrine actions of IL-10, prostanoids, and TNF-alpha, and participation in the development of oral tolerance to bacterial superantigens. Moreover, during liver injury and inflammation, Kupffer cells secrete enzymes and cytokines that may damage hepatocytes, and are active in the remodeling of extracellular matrix. Hepatic stellate cells are present in the perisinusoidal space. They are characterized by abundance of intracytoplasmic fat droplets and the presence of well-branched cytoplasmic processes, which embrace endothelial cells and provide focally a double lining for sinusoid. In the normal liver HSC store vitamin A, control turnover of extracellular matrix, and regulate the contractility of sinusoids. Acute damage to hepatocytes activates transformation of quiescent stellate cells into myofibroblast-like cells that play a key role in the development of inflammatory fibrotic response. Pit cells represent a liver-associated population of large granular lymphocytes, i.e., natural killer (NK) cells. They spontaneously kill a variety of tumor cells in an MHC-unrestricted way, and this antitumor activity may be enhanced by the secretion of interferon-gamma. Besides pit cells, the adult liver contains other subpopulations of lymphocytes such as gamma delta T cells, and both "conventional" and "unconventional" alpha beta T cells, the latter containing liver-specific NK T cells. The development of methods for the isolation and culture of main liver cell types allowed to demonstrate that both nonparenchymal and parenchymal cells secrete tens of mediators that exert multiple paracrine and autocrine actions. Co-culture experiments and analyses of the effects of conditioned media on cultures of another liver cell type have enabled the identification of many substances released from non-parenchymal liver cells that evidently regulate some important functions of neighboring hepatocytes and non-hepatocytes. To the key mediators involved in the intercellular communication in the liver belong prostanoids, nitric oxide, endothelin-1, TNF-alpha, interleukins, and chemokines, many growth factors (TGF-beta, PDGF, IGF-I, HGF), and reactive oxygen species (ROS). Paradoxically, the cooperation of liver cells is better understood under some pathological conditions (i.e., in experimental models of liver injury) than in normal liver due to the possibility of comparing cellular phenotype under in vivo and in vitro conditions with the functions of the injured organ. The regulation of vitamin A metabolism provides an example of the physiological role for cellular cross-talk in the normal liver. The majority (up to 80%) of the total body vitamin A is stored in the liver as long-chain fatty acid esters of retinal, serving as the main source of retinoids that are utilized by all tissues throughout the body. Hepatocytes are directly involved in the uptake from blood of chylomicron remnants, and the synthesis of retinol-binding protein that transfers retinol to other tissues. However, more than 80% of the liver retinoids are stored in lipid droplets of hepatic stellate cells. HSC are capable of both uptake and release of retinol depending on the body's retinol status. The activity of some major enzymes of vitamin A metabolism have been found to be many times higher per protein basis in stellate cells than in hepatocytes. Despite progress in the understanding of the roles played by these two cell types in hepatic retinoid metabolism, the way in which retinoids move between the parenchymal cells, stellate cells, and blood plasma has not been fully elucidated. Sinusoidal blood flow is, to a great extent, regulated by hepatic stellate cells that can contract due to the presence of smooth muscle alpha-actin. The main vasoactive substances that affect constriction or relaxation of HSC derive both from distant sources and from neighboring hepatocytes (carbon monoxide, leukotrienes), endothelial cells (endothelin, nitric oxide, prostaglandins), Kupffer cells (prostaglandins, NO), and stellate cells themselves (endothelin, NO). The cellular cross-talk reflected by the fine-tuned modulation of sinusoidal contraction becomes disturbed under pathological conditions, such as endotoxemia or liver fibrosis, through the excess synthesis of vasoregulatory compounds and the involvement of additional mediators acting in a paracrine way. The liver is an important source of some growth factors and growth factor-binding proteins. Although hepatocytes synthesize the bulk of insulin-like growth factor I (IGF-I), also other types of nonparenchymal liver cells may produce this peptide. Cell-specific expression of distinct IGF-binding proteins observed in the rat and human liver provides the potential for specific regulation of hepatic IGF-I synthesis not only by growth hormone, insulin, and IGF-I, but also by cytokines released from activated Kupffer (IL-1, TNF-alpha, TGF-beta) or stellate cells (TGF-alpha, TGF-beta). Hepatic stellate cells may affect turnover of hepatocytes through the synthesis of potent positive as well as negative signals such as, respectively, hepatocyte-growth-factor or TGF-beta. Although hepatocytes seem not to produce TGF-beta, a pleiotropic cytokine synthesized and secreted in the latent form by Kupffer and stellate cells, they may contribute to its actions in the liver by the intracellular activation of latent TGF-beta, and secretion of the biologically active isoform. Many mediators that reach the liver during inflammatory processes, such as endotoxins, immune-complexes, anaphylatoxins, and PAF, increase glucose output in the perfused liver, but fail to do so in isolated hepatocytes, acting indirectly via prostaglandins released from Kupffer cells. In the liver, prostaglandins synthesized from arachidonic acid mainly in Kupffer cells in a response to various inflammatory stimuli, modulate hepatic glucose metabolism by increasing glycogenolysis in adjacent hepatocytes. The release of glucose from glycogen supports the increased demand for energetic fuel by the inflammatory cells such as leukocytes, and additionally enables enhanced glucose turnover in sinusoidal endothelial cells and Kupffer cells which is necessary for effective defense of these cells against invading microorganisms and oxidative stress in the liver. Leukotrienes, another oxidation product of arachidonic acid, have vasoconstrictive, cholestatic, and metabolic effects in the liver. A transcellular synthesis of cysteinyl leukotrienes (LTC4, LTD4, and LTE4) functions in the liver: LTA4, an important intermediate, is synthesized in Kupffer cells, taken up by hepatocytes, converted into the potent LTC4, and then released into extracellular space, acting in a paracrine way on Kupffer and sinusoidal endothelial cells. Thus, hepatocytes are target cells for the action of eicosanoids and the site of their transformation and degradation, but can not directly oxidate arachidonic acid to eicosanoids. (ABSTRACT TRUNCATED)

Protein phosphatase 2A: variety of forms and diversity of functions.

Abstract: Protein phosphatase 2A (PP2A) comprises a diverse family of phosphoserine- and phosphothreonine-specific phosphatases present in all eukaryotic cells. All forms of PP2A contain a catalytic subunit (PP2Ac) which forms a stable complex with the structural subunit PR65/A. The heterodimer PP2Ac-PR65/A associates with regulatory proteins, termed variable subunits, in order to form trimeric holoenzymes attributed with distinct substrate specificity and targeted to different subcellular compartments. PP2Ac activity can be modulated by reversible phosphorylation on Tyr307 and methylation on C-terminal Leu309. Studies on PP2A have shown that this enzyme may be implicated in the regulation of metabolism, transcription, RNA splicing, translation, differentiation, cell cycle, oncogenic transformation and signal transduction.

Introduction — Morphology of the Liver Lobule

Abstract: The liver plays a unique role as a metabolic center of the body, and also performs other important functions (Table 1). The macroscopic and microscopic structure of the mammalian liver has been recognized relatively early. In 1833, Kiernan proposed that lobes of the pig liver organized around main branches of the portal vein were built up of small polyhedron morphological units of parenchyma, called lobules, with boundaries made of connective tissue (Kiernan 1833). This classical liver lobule is characterized by the presence of a central vein (terminal hepatic vein) located approximately in the middle of the unit, and of areas of connective tissue at its corners, called portal tracts, that contain interlobular branches of hepatic artery and portal vein, biliary ductules, lymphatic vessels and nerves. Portal tracts are bridged by narrow stripes of connective tissue which accompany terminal afferent arterial and venous branches running between the lobules to supply the sinusoids that lead the blood into a central vein draining the lobule. However, in man and rodents, classical lobules cannot be easily recognized because only a sparse amount of connective tissue septa lies between portal tracts.

Clinical impact of molecular and cytogenetic findings in synovial sarcoma.

Abstract: Synovial sarcoma is an aggressive soft-tissue tumor that accounts for up to 10% of soft-tissue sarcomas. Cytogenetically, synovial sarcoma is characterized by the t(X;18)(p11;q11), found in more than 95% of the tumors. This translocation results in rearrangements of the SYT gene in 18q11 and one of the SSX1, SSX2, or SSX4 genes in Xp11, creating a SYT/SSX1, SYT/SSX2, or SYT/SSX4 chimeric gene. It has been shown that patients with SYT/SSX1 fusion genes have a shorter metastasis-free survival than do patients with SYT/SSX2. Previous studies have also suggested that clonal evolution may be associated with disease progression. In the present study, RT-PCR analysis showed that all 64 examined synovial sarcomas from 54 patients had SYT-SSX chimeric genes. SYT/SSX1 was found in 40 tumors from 33 patients, SYT/SSX2 in 23 tumors from 20 patients, and SYT/SSX4 in one case. Two patients had variant SYT/SSX2 transcripts, with 57 bp and 141 bp inserts, respectively, between the known SYT and SSX2 sequences. Patients with tumors with SYT/SSX1 fusions had a higher risk of developing metastases compared to those with SYT/SSX2 fusions (P = 0.01). The reciprocal transcripts SSX1/SYT and SSX2/SYT were detected using nested PCR in 11 of the 40 samples with SYT/SSX1 and 5 of the 23 samples with SYT/SSX2, respectively. Among 20 blood samples, SYT/SSX1 and SYT/SSX2 were detected in one sample each. The t(X;18), or variants thereof, was found cytogenetically in all patients but three. Among 32 primary tumors, the t(X;18) or a variant translocation was the sole anomaly in 10. In contrast, of the seven metastatic lesions that were investigated prior to radiotherapy, only one had a t(X;18) as the sole anomaly; all other tumors displayed complex karyotypes. Cytogenetic complexity in primary tumors was, however, not associated with the development of metastases. Tumors with SYT/SSX2 less often (4/12 vs. 7/15) showed complex karyotypes than did tumors with SYT/SSX1, but the difference was not significant. Combining cytogenetic complexity and transcript data, we found that the subgroup of patients with tumors showing simple karyotypes and SYT/SSX2 fusion had the best clinical outcome (2/8 patients developed metastases), and those with tumors showing complex karyotypes together with SYT/SSX1 fusion the worst (6/7 patients developed metastases). This corresponded to 5-year metastasis-free survival rates of 0.58 and 0.0, respectively (P = 0.02).

Influence of L-carnitine and its derivatives on myocardial metabolism and function in ischemic heart disease and during cardiopulmonary bypass.

Abstract: Carnitine and its derivatives have recently been shown to protect cardiac metabolism and function in ischemic heart disease and other clinical conditions of myocardial ischemia. Potential mechanisms of this effect include an increase in glucose metabolism, a reduction of toxic effects of long-chain acyl-CoA and acyl-carnitine in myocytes, an increase in coronary blood flow and anti-arrhythmic effect. It has also been shown that propionyl-l-carnitine which penetrates faster than carnitine into myocytes is effective in inhibiting production of free radicals. Beneficial effects of carnitine supplementation have been demonstrated under a variety of clinical conditions such as acute cardiac ischemia, during extracorporeal circulation, in carnitine-dependent cardiomyopathy as well as in patients with chronic circulatory failure and in cardiogenic shock. However, further studies are required before carnitine administration could be recommended as a routine procedure in ischemic heart disease or before cardiopulmonary bypass.

Statin-stimulated nitric oxide release from endothelium.

Abstract: BACKGROUND There is increasing evidence that loss of endothelium-derived NO is a major factor in cardiovascular complication events, and that NO might exert antiatherosclerotic actions. The beneficial effects of HMG CoA reductase inhibitors (statins) therapy in atherosclerosis outweigh those expected from simply lowering low-density lipoprotein (LDL) cholesterol, and may be related to the direct action in the endothelium. Based on these concepts, in the studies described here, the effect of new statin derivatives on nitric oxide (NO) and superoxide (O2-) release in bovine endothelial cells was tested. MATERIAL AND METHODS Highly sensitive electrochemical NO and O2--microsensors were placed near the surface of endothelial cells, and the concurrent kinetics of NO and O2-- release were measured in situ. RESULTS All tested statins stimulated NO release. The peak concentration of NO after stimulation with 1 Kmol/l Lovastatin, 1 Kmol/l Atorvastatin, 1 Kmol/l Pravastatin, or 1 Kmol/l Simvastatin was about 77%, 73%, 72%, and 44% lower, respectively, as compared with the NO peak concentration after stimulation with 1 Kmol/l calcium ionophore A23187 (receptor-independent agonist). The tested statins stimulated NO release in a modest way, which resulted in diminishing O2- generation during activation of nitric oxide synthase. Moreover, the kinetics of O2- release after administration of the statins suggested that these compounds may also scavenge O2-. The NO/O2- peak concentration ratio after the NOS agonists administration was as follows: 7.51 for CaI, 6.56 for Lovastatin, 6.00 for Atorvastatin, 4.17 for Pravastatin and 6.25 for Simvastatin. CONCLUSIONS The tested statins, i.e. Lovastatin, Atorvastatin, Pravastatin and Simvastatin demonstrate variable potency to enhance the NO/O2- concentration ratio after stimulation of NOS, resulting in an increase of NO bioavailability in endothelial cells.

One week of treatment with esomeprazole-based triple therapy eradicates Helicobacter pylori and heals patients with duodenal ulcer disease.

Abstract: Background Proton pump inhibitor (PPI) monotherapy is commonly continued for 3 weeks after Helicobacter pylori eradication with PPI-based triple therapy regimens to ensure duodenal ulcer (DU) healing. This randomized, double-blind, multicentre study evaluated whether only 1 week of triple therapy wi

Leptin interacts with heart rate but not sympathetic nerve traffic in healthy male subjects.

Abstract: Objective Administration of leptin to animals increases sympathetic nerve activity and heart rate. We therefore tested the hypothesis that plasma leptin is linked independently to muscle sympathetic nerve activity (MSNA) and heart rate in healthy humans. Methods We measured plasma leptin, plasma insulin, body mass index (BMI), percent body fat, waist : hip ratio, MSNA, heart rate and blood pressure in 88 healthy individuals (50 men and 38 women). Results In men, plasma leptin concentration correlated significantly with BMI (r 0.75, P < 0.001), percent body fat (r 0.70, P < 0.001), waist : hip ratio (r 0.69, P < 0.001), insulin (r 0.37, P 0.009), and age (r 0.38, P 0.006). Only BMI and waist : hip ratio were linked independently to plasma leptin concentration (r 0.78, P < 0.001). Plasma leptin concentrations also correlated with heart rate (r 0.39, P 0.006) and mean arterial pressure (MAP; r 0.38, P 0.007), but not with MSNA (r 0.17, P 0.24). After adjustment for BMI and waist : hip ratio, plasma leptin concentration correlated significantly only with heart rate (r 0.29, P 0.04), and not with MAP (r 0.21, P 0.14). Individuals were divided into high-leptin and low-leptin subgroups on the basis of plasma leptin concentrations adjusted for BMI and waist : hip ratio. Those with high leptin concentrations had significantly faster heart rates than those with low leptin. MAP and MSNA were similar in both subgroups. No relationship between leptin and either heart rate or MSNA was evident in women. Conclusions In normal men, heart rate, but not MSNA, is linked to plasma leptin concentration. This sex-specific relationship between heart rate and plasma leptin is independent of plasma insulin, BMI, waist : hip ratio and percentage body fat. J Hypertens 19:1089‐1094 & 2001 Lippincott Williams & Wilkins.

Comparative analysis of phenolic acids in mistletoe plants from various hosts.

Abstract: Phenolic acids present in mistletoe plants collected from various hosts were analysed with the use of HPLC. The following numbers of compounds were found in the mistletoe plant material gathered from respective hosts: Sorbus aucuparia- 12 compounds; Acer plantanoides--14 compounds: Malus domestica, Pyrus communis and Populus nigra--13 compounds each; Quercus robur--15 compounds. Altogether 21 phenolic acids were chromatographically identified in the tested material. The compounds were either free or combined as esters or glycosides. Comparative chromatography revealed qualitative differences in the investigated compounds between the various plant materials. For example o-coumaric acid was only found in mistletoe hosted by Quercus robur. Digallic acid was only found in the plant material hosted by Acer plantanoides. Qualitative and quantitative composition of mistletoes hosted by Malus domestica and Pyrus communis showed considerable similarities as far as phenolic acids were concerned. Moreover. vanillic acid. absent in all other batches of plant material, seemed to be characteristic of the above mistletoes. Quantitative HPLC analysis demonstrated a considerable content of salicylic acid (39.55 mg%) in mistletoe hosted by Sorbus aucuparia. Apart from the above material, this compound was only present in small quantities in plants hosted by Populus nigra (15.63 mg%) and Quercus robur (2.63 mg%).

Atrial fibrillation after coronary artery bypass grafting: does the type of procedure influence the early postoperative incidence?

Abstract: Objective: Atrial fibrillation (AF), the common postoperative complication, has been observed after coronary artery bypass grafting (CABG) in 7‐40% of patients. Cardiopulmonary bypass (CPB), eliminated in off-pump operations (OPCABG) may decrease the incidence of AF, whereas the combination of CABG with heart valve replacement may result in more frequent postoperative atrial fibrillation. The aim of our study was to compare the early postoperative AF incidence rate during ICU stay in three groups of patients: after CABG, OPCABG, and CABG combined with valve replacement. Material and methods: A prospective study of 906 consecutive patients was carried out between January 1999 and January 2000. Clinical profile of 906 patients, including factors having potential influence on postoperative AF did not showed any significant differences between the groups. The presence of arrhythmia history was the reason of excluding 85 patients from the statistical analysis. The observation was performed in each case during ICU-stay, using a HP system for continuos automated arrhythmia analysis. Early postoperative incidence of AF was recorded and compared between three groups of patients: 650 after conventional CABG, 118 after OPCABG, and 53 after CABG combined with valve replacement. Chi-square and a Mann‐Whitney tests, Statistica 5.0 PL were used for the statistical analysis. Results: Atrial fibrillation occurred during the postoperative ICU stay in 9.8% of patients after CABG, in 10.2% after OPCABG, and in 21% after CABG combined with valve replacement. There was no significant difference between CABG and OPCABG groups (Pa 0:965). The confidence interval of the odds ratio ranges from 0.5 to 1.85. Consequently, an increased risk would be possible for both methods. We observed a statistically significant increase of the early postoperative atrial fibrillation incidence rate in patients after CABG combined with valve replacement, when compared with both CABG 1 OPCABG groups (Pa 0:005). Conclusions: (1) Atrial fibrillation is a common postoperative complication after myocardial revascularization procedures which prolongs ICU stay. (2) The study did not show that the incidence of postoperative AF is influenced by the technique of coronary artery bypass grafting: with or without CPB. (3) The prevalence of postoperative AF increase when CABG is combined with valve replacement. q 2001 Elsevier Science B.V. All rights reserved.

The Neurofibromatosis Type 2 Gene Is Mutated in Perineurial Cell Tumors : A Molecular Genetic Study of Eight Cases

Abstract: Perineurial cell tumors (PNTs) are rare neoplasms derived from or showing differentiation toward specialized lining cells of the nerve sheath, the perineurial cells. In this study, we have evaluated neurofibromatosis type 2 (NF2) gene alterations in eight PNTs using archival formaldehyde-fixed, paraffin-embedded tissue. Two conventional soft-tissue PNTs from the upper back and chest wall, one retiform soft tissue variant from the scapular region, and five sclerosing PNTs from the fingers and palm were studied. All cases showed histological features of PNTs, and the neoplastic cells were positive for epithelial membrane antigen and negative for S100 protein. The coding sequences (exons 1 to 15) of the NF2 gene were polymerase chain reaction (PCR) amplified and evaluated for mutations by direct sequencing of the PCR products. Five NF2 point mutations, two in the 5′-untranslated region (UTR) and three in exons 3, 6, and 8, were identified in four of eight cases (50%) studied. Exon mutations resulted in changes of predicted amino acids sequences: Asp→Asn at codon 83, Glu→Asp at codon 182, and Leu→Val at codon 241. In two cases (one with a missense mutation in codon 241), the same point mutation in the 5′-UTR at the nucleotide position 8958 was identified. A loss of heterozygosity (LOH) study was performed in three cases. LOH at the NF2 locus was found in one case with a mutation in the 5′-UTR. However, in another case with exon 8 and 5′-UTR mutations, deletion of one allele of the NF2 gene was previously documented by fluorescence in situ hybridization. The coexistence of NF2 gene mutations and LOH at the NF2 locus indicates that the NF2 tumor suppressor gene is altered in PNTs by the two-hit mechanism.

Independent Association Between Plasma Leptin Levels and Heart Rate in Heart Transplant Recipients

Abstract: Background Leptin, the protein product of the ob gene, has been linked to a faster heart rate in animal and human studies. The interaction between leptin and heart rate in the denervated heart is not known. Therefore, we studied the relationship between plasma leptin levels and heart rate in heart transplant recipients. Methods and Results We studied 32 male patients (mean age, 56.5±9.3 years; range, 41 to 74 years) after orthotopic heart transplantation. All subjects underwent a physical examination, anthropometric measurements, blood chemistry analysis, and office blood pressure measurements. A blood sample was collected from each subject while fasting. In univariate analysis, heart rate was related to leptin levels (r=0.47, P=0.007) but heart rate was not related to systolic or diastolic blood pressure, mean arterial pressure, body mass index, or catecholamines. Leptin levels were only strongly associated with heart rate and body mass index (r=0.73, P<0.0001). In multivariate analysis, heart rate was i...

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver

Abstract: Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20–60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

Synergistic Antihypertensive Effects of Nifedipine on Endothelium : Concurrent Release of NO and Scavenging of Superoxide.

Abstract: Recent studies have suggested that part of the vasorelaxation caused by nifedipine, a 1,4-dihydropyridine Ca(2+) antagonist, depends on the endothelium. To study the effect of endothelium-dependent vasorelaxation, the release of NO and superoxide (O(2)(-)) in the presence of nifedipine in isolated cultured rabbit endothelial cells was measured. Highly sensitive electrochemical microsensors were placed onto the cell membrane, and the kinetics of NO and O(2)(-) were measured simultaneously with time resolutions of 0.1 and 0.05 ms, respectively. Nifedipine at its therapeutical concentrations stimulated NO release and scavenged O(2)(-) in endothelial cells. The linear relationship between NO concentration and nifedipine concentration was observed in the range between 0.01 and 1 nmol/L. NO concentration reached a maximum of 200+/-10 nmol/L at 1.2 nmol/L of nifedipine. The NO concentration was approximately 50% and 30% of the concentration measured in the presence of receptor-dependent (acetylcholine) and the receptor-independent (Ca(2+) ionophore A23187) NO synthase (eNOS) agonists, respectively. NO release stimulated by eNOS agonists was followed by the generation of the NO scavenger superoxide. The concentration of O(2)(-) was significantly lower after stimulation with nifedipine (peak 5+/-0.5 nmol/L) than after stimulation with acetylcholine (15+/-1 nmol/L) and Ca(2+) ionophore (25+/-1 nmol/L). The average rate of NO release by nifedipine is relatively slow (17 nmol/L per second). This is in sharp contrast to the fast rate of NO release by acetylcholine and Ca(2+) ionophore (40 and 300 nmol/L per second, respectively). These experiments show that nifedipine, apart from its well-known Ca(2+) antagonistic properties in vascular smooth muscle cells, stimulates the release of significant concentration of NO in endothelium and also preserves NO concentration. Both these effects may be beneficial in the treatment of hypertension.

Quantitative structure-retention relationships with model analytes as a means of an objective evaluation of chromatographic columns.

Abstract: The performance of several previously designed model series of test analytes has been tested to characterize in an objective, quantitative manner modern stationary phases for reversed-phase high-performance liquid chromatography (RP-HPLC) using quantitative structure-retention relationships (QSRRs). Three QSRR approaches and three respective series of test analytes recommended for studies of the molecular mechanism of chromatographic retention are employed: the reduced linear solvation energy relationship (LSER)-based model of Abraham, a model employing structural descriptors from molecular modeling, and a model relating retention to the n-octanol-water partition coefficient log P. All of the models and test analytes proposed provide reliable QSRR equations. Those equations discriminate in quantitative terms individual columns and chromatographic systems and can be interpreted in straightforward rational chemical categories. In view of QSRRs, the differences in the intermolecular interactions between a given stationary phase and a structurally defined analyte rationalize the observed differences in retention. The QSRR models (previously derived retrospectively) are demonstrated to work well on new sets of RP-HPLC data. At the same time, it has been confirmed that the three test series of analytes have properly been designed and can be recommended for comparative studies of analytical columns. QSRRs once derived on a given column for model analytes can be used to predict the retention of other analytes of a defined structure. That in turn can facilitate the procedure of the rational optimization of chromatographic separations.

Cardiovascular variability characteristics in obstructive sleep apnea

Abstract: Patients with obstructive sleep apnea (OSA) are at increased risk for cardiovascular disease. Altered cardiovascular variability is a prognostic indicator for cardiovascular events. This review examines the evidence that OSA is accompanied by alterations in cardiovascular variability. This alteration is evident even in the absence of hypertension, heart failure or other disease states, and may be linked to the severity of OSA. The presence of clear-cut abnormalities in time and frequency-domain measures of blood-pressure and heart-rate variability in normotensive OSA patients provides intriguing evidence for the concept of an etiologic interaction between sleep apnea and cardiovascular disease. Mechanisms that could contribute to altered cardiovascular variability in patients with sleep apnea include abnormalities in chemoreflex, baroreflex and endothelial function.

Combined chemotherapy and radiation in locally advanced nonsmall-cell lung cancer

Abstract: The efficacy of radiotherapy in locally advanced non-small-cell lung cancer is limited. One attempt to improve survival uses a combination of radiation and chemotherapy. These two modalities can be applied in sequence or concurrently, but results from phase III trials of combined therapy versus radiation alone have been inconsistent. Early studies were mostly negative, but more recent trials using platinum-based regimens have shown some survival benefit for combined treatments. The positive impact of chemotherapy has also been shown in a meta-analysis. In recent studies, concurrent chemotherapy and radiation appears better than sequential application. However, the benefit of the combined approach is modest and should be balanced against increased early and late toxicity. The role of new agents such as taxanes, vinorelbine, gemcitabine, and topoisomerase inhibitors in combined modality therapy of non-small-cell lung cancer warrants further clinical investigation.

Gradient HPLC in the determination of drug lipophilicity and acidity

Abstract: The linear-solvent strength (LSS) model of gradient elution in high-performance liquid chromatography (HPLC) has been demonstrated to provide parameters of lipophilici- ty and acidity of analytes. pK a and log k w values are determined in three gradient runs. The first two experiments use an aqueous buffered eluent with a wide-range organic modifier gra- dient at pH of buffer, providing suppression of ionization of the analyte. That experiment allows an estimate of contents of the organic modifier in the mobile phase (%B), producing requested retention coefficient, k, for the nonionized form of the analyte. The next experi- ment is carried out with the latter %B and a pH-gradient of the aqueous component of the eluent that is sufficient to overlap possible pK a value of the analyte. The initial pH of the buffer used to make the mobile phase is selected to insure that the analyte is in nonionized form. The resulting retention time allows an estimate of pK a in a solvent of the given %B. The log k w parameter obtained correlated well with the corresponding value obtained by the standard procedure of extrapolation of retention data determined in a series of isocratic measurements. The correlation between log k w and the reference parameter of lipophilicity, log P, was very good for a series of test analytes. The values of pK a were found to correlate with the literature pK a data determined in water for a set of aniline derivatives studied.

The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis.

Abstract: Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.

Aluminum, NO, and nerve growth factor neurotoxicity in cholinergic neurons

Abstract: Several neurotoxic compounds, including Al, NO, and β-amyloid may contribute to the impairment or loss of brain cholinergic neurons in the course of various neurodegenerative diseases. Genotype and phenotypic modifications of cholinergic neurons may determine their variable functional competency and susceptibility to reported neurotoxic insults. Hybrid, immortalized SN56 cholinergic cells from mouse septum may serve as a model for in vitro cholinotoxicity studies. Differentiation by various combinations of cAMP, retinoic acid, and nerve growth factor may provide cells of different morphologic maturity as well as activities of acetylcholine and acetyl-CoA metabolism. In general, differentiated cells appear to be more susceptible to neurotoxic signals than the non-differentiated ones, as evidenced by loss of sprouting and connectivity, decreases in choline acetyltransferase and pyruvate dehydrogenase activities, disturbances in acetyl-CoA compartmentation and metabolism, insufficient or excessive acetylcholine release, as well as increased expression of apoptosis markers. Each neurotoxin impaired both acetylcholine and acetyl-CoA metabolism of these cells. Activation of p75 or trkA receptors made either acetyl-CoA or cholinergic metabolism more susceptible to neurotoxic influences, respectively. Neurotoxins aggravated detrimental effects of each other, particularly in differentiated cells. Thus brain cholinergic neurons might display a differential susceptibility to Al and other neurotoxins depending on their genotype or phenotype-dependent variability of the cholinergic and acetyl-CoA metabolism. © 2001 Wiley-Liss, Inc.

Selectivity tuning and molecular modeling of new generation packings for RP HPLC

Abstract: Retention mechanism, selectivity and physicochemical properties together with theoretical surface simulation experiments of laboratory-synthesized packing materials for high performance liquid chromatography (HPLC) containing cholesterol molecules immobilized on the high quality silica gel were studied. The chromatographic behavior of the three cholesterol-based columns (SG-CHOL, SG-MIX CHOL/AP and SG-10-CHOL) was examined with special regard to retention and selectivity and compared to reference materials: a typical octadecylsilica column (SG-C18) and an alkylamide bonded phase (SG-AP). Temperature investigations (DSC) and molecular modeling simulations didn't bring an expected confirmation of liquid crystalline properties of materials studied. The observed phase transition was attributed to conformation change within the amide bonding.

Optimisation of the second phase of a two phase growth system for anthocyanin accumulation in callus cultures of Rudbeckia hirta

Abstract: A two-phase growth system composed of modified Schenk-Hildebrandt medium and enriched Miller's medium was used to grow callus of R. hirta L. The second phase of the system has been optimised and the callus synthesised a 12-compound set of anthocyanins, comprising 4.97% of tissue mass (tubular flowers of the plant in nature only contain one compound comprising 0.28%). The beneficial effect of high content of cysteine on anthocyanin accumulation was noted. The predominant element of the anthocyanin set obtained in vitro is cyanidin-3-O-(6-O-malonyl-β-D-glucopyranoside), a relatively stable compound.

Cooperation of Liver Cells in the Synthesis and Degradation of Eicosanoids

Abstract: Eicosanoids, the oxygenated derivates of 20-carbon unsaturated fatty acids (eicosa, “20” in Greek) comprising prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), lipoxins and hydroxyeicosanoid acids (HETE), are immediately released after synthesis by a variety of cells in response to physiological and pathological stimuli. Since the biological activity of eicosanoids is rapidly destroyed both in tissues and in the circulation, they are regarded as mediators that maintain local homeostasis. In normal rat liver, eicosanoids are produced only by nonparenchymal cells (mostly Kupffer cells), whereas hepatocytes respond to the arachidonic acid metabolites, metabolize and excrete them into bile (Keppler 1994).

Medical applications of model-based dynamic thermography

Abstract: The proposal to use active thermography in medical diagnostics is promising in some applications concerning investigation of directly accessible parts of the human body. The combination of dynamic thermograms with thermal models of investigated structures gives attractive possibility to make internal structure reconstruction basing on different thermal properties of biological tissues. Measurements of temperature distribution synchronized with external light excitation allow registration of dynamic changes of local temperature dependent on heat exchange conditions. Preliminary results of active thermography applications in medicine are discussed. For skin and under- skin tissues an equivalent thermal model may be determined. For the assumed model its effective parameters may be reconstructed basing on the results of transient thermal processes. For known thermal diffusivity and conductivity of specific tissues the local thickness of a two or three layer structure may be calculated. Results of some medical cases as well as reference data of in vivo study on animals are presented. The method was also applied to evaluate the state of the human heart during the open chest cardio-surgical interventions. Reference studies of evoked heart infarct in pigs are referred, too. We see the proposed new in medical applications technique as a promising diagnostic tool. It is a fully non-invasive, clean, handy, fast and affordable method giving not only qualitative view of investigated surfaces but also an objective quantitative measurement result, accurate enough for many applications including fast screening of affected tissues.