Polyphenols of Antibacterial Potential - May They Help in Resolving Some Present Hurdles in Medicine?

The phenomenon of antibiotic resistance has been recognized as one of the greatest threats to humanity. Therefore, there is an enormous need to introduce new antibiotics to the medical practice that will effectively eradicate the resistant bacterial strains threatening human health and life. One solution currently being considered as an alternative to antibiotics involves secondary metabolites of plants that can be used in modern antibacterial therapy. Polyphenols represent a broad and diversified group of plant-derived aromatic compounds. Their antibacterial potential has been recognized via specific mechanisms of action, e.g., by inhibition of bacterial biofilm formation, through synergistic effects with the action of currently used antibiotics, and by inhibition of the activity of bacterial virulence factors.

Porcine endogenous retrovirus (PERV) infection of HEK-293 cell line alters expression of human endogenous retrovirus (HERV-W) sequences.

The risk of infections of human recipients after xenotransplantations is now mainly represented by porcine endogenous retroviruses (PERVs) as these particles are part of the porcine genome. As in all vertebrates, human genome harbours its own numerous genetic sequences of retroviral origin; it is estimated that they comprise about 8 % of the human genome. Because some of them play an important role in human physiology, it is valuable to estimate whether the presence of PERVs in human cells influences homeostasis of the human endogenous retrovirus (HERV) expression pattern. The aim of the study was to evaluate whether the expression profile of HERV-W genes changes after infection of cells by porcine endogenous retroviruses. In the experimental settings, human embryonic kidney cell line (HEK-293) was infected by PERV particles and cultivated up to 22th passage after infection. HERV-W gag, pol and env, as well as env from locus 7q21.2 gene expression was monitored by means of realtime reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot techniques. We found that the expression level of HERV-W genes differs in PERV-infected HEK-293 cell cultures in comparison with that from non-infected cultures. Relative HERV-W gene expression also differed significantly between particular passages (P < 0.05). Moreover, we have noticed a high correlation between the HERV-W Env(7q21.2) mRNA and protein level (Spearman rank r = 0.65; P < 0.05) during the course of the experiment. As previously hypothesized, human genomic sequences of retroviral origin may be changed by the presence of porcine endogenous retroviruses.

Expression and localisation of kinin receptors in colorectal polyps.

Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n=18), villous (n=15) and hyperplasic polyps (n=15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/microg total RNA+/-312 SE) which is significantly higher as compared with controls (683+/-197 SE, p<0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852+/-936 SE) as compared with controls (843+/-263 SE, p<0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.

Sequence analysis of proviral DNA of porcine endogenous retroviruses.

Among all species analyzed, the domestic pig seems to be the most appropriate organ donor for xenotransplantation. Porcine endogenous retroviruses (PERVs) are present in genomes of all pigs and are capable of infecting human cells in vitro thus posing a serious threat for xenotransplantation procedures. Despite the abundant distribution of PERVs integrated with porcine genome, the majority of PERV proviral DNA is not capable of expressing viral proteins unless seriously mutated. The aim of the study was to analyze PERV genome for mutations. The study was performed on blood samples from 146 pigs. Long-range polymerase chain reaction (Long-PCR) was performed with primer sets designed within long terminal repeats (LTRs). Long-PCR products of different molecular weights were obtained: 530 bp (33.1% of individuals), 580 bp (76.7%), 933 bp (100%), and 2900 bp (59.8%). Amplimers of 7200 bp were absent in 12.8% of individuals, indicating the lack of intact proviral DNA. Sequence analysis showed that most PERV proviral DNA was significantly mutated, thus suggesting the inability to express functional viral RNA; however, it cannot be ruled out that compensatory recombination processes could occur enabling replication of defective proviruses.

Experimental studies on the stem cell concept of liver regeneration. II.

There is increasing evidence in support of the existence of a hepatic stem or progenitor cell system as well as its participation in the physiological as well as reparative regeneration of the liver and in carcinogenesis. In the present paper it will be demonstrated that under the condition of a simultaneous intoxication with allyl alcohol and tetrachlorcarbon a regenerative process occurs, which is composed of two distinct partial processes: 1. a "proliferative wave" of the persisting, non-necrotic differentiated hepatocytes of the acinus, starting at 24 h after intoxication and ending after >48 h, and 2. a proliferation of cells at the rim of the portal fields, which afterwards enter the acinus and differentiate into hepatocytes, starting at 48 h after intoxication. These results support the participation of a stem or progenitor cell system in the reparative regeneration of the liver.

Growth hormone gene polymorphism and reproductive performance of AI bulls.

Relationships between the growth hormone gene RFLP polymorphism and bull sperm characteristics were the objects of the present study. DNA was extracted from blood or sperm samples collected from 113 AI bulls and submitted for polymerase chain reaction (PCR) followed by digestion with Alu I restriction enzyme. The bGH genotypes were visualized on 10% polyacrylamide gel. The analyzed population of AI bulls consisted of dairy (Holstein Fresian [HF] crossbred [HF x Polish Black and White]) and beef breeds (Limousine, Charolaise, Piemontese, Angus and Hereford). The frequency of the Leu allele was 0.86 among dairy bulls and 0.38 in beef bulls (0.14 and 0.62 for the Val allele, respectively). Eight sperm characteristics and Day 60 non-return rates (NRR) were analyzed. The 3 genotype groups (LL, VV and LV) and the effect of production type (dairy or beef) on sperm characteristics were considered. None of the traits showed significant variability in relation to the bGH genotype, although a tendency was observed for LL bulls to have a lower ejaculate volume and VV bulls higher NRR. Moreover some statistically significant associations with production type were noticed: beef bulls were superior in sperm concentration and non-return rate, whereas dairy bulls excelled in individual fresh sperm motility.

Experimental studies on the stem cell concept of liver regeneration. I.

There are increasing references for the existence of a hepatic stem or progenitor cell system as well as its participation in the physiological as well as reparative regeneration of the liver and in carcinogenesis. For the physiological regeneration the existence of a dynamic "cell-renewal" system finds increasing consideration and in the "streaming liver concept" (Zajicek et al. 1985) its functional expression. This concept is still under discussion. The present paper tries to check this animal-experimentally (Wistar rats) under use of two different thymidine analogues (3H-thymidine and Bromodeoxyuridine). In different time intervals after labelling (1 h, 14, 30, 60, 90, 120 d) a shift of the labelling bias or a migration of the hepatocytes in the liver acinus (Rapaport) in portovenous direction could be shown. The average migration speed is 0.575 microm or 0.0315 cell positions per day, the cell production rate is one in 31.5 days. The present paper results support the inclusion of a stem or progenitor cell system into the physiological regeneration of the liver and allow the classification into the "streaming liver concept" (Zajicek et al. 1985).

Histological and biochemical changes induced by total bile duct ligation in the rat.

The aim of the present investigation was to assess in a correlated biochemical and morphological study the dynamics of fibrogenesis after bile duct ligation and to compare the time course of alterations with those occurring in thioacetamide induced liver fibrosis. The data show that, after bile duct obstruction, the deposition of connective tissue elements and formation of ductular proliferates rapidly set in. The index of fibroplasia correlated well with the changes of the OH-proline concentration of the liver. Comparing the biliary fibrosis with the thioacetamide induced liver fibrosis, the progress of the former occurred more rapidly, even though in both cases only a few necroses were observed. Therefore, we suggest that in biliary fibrosis other mechanisms are responsible for the rapid onset of production of extracellular material and proliferative processes than in thioacetamide-induced liver fibrosis.

The cellular reproduction in physiological and reparative liver regeneration.

The liver regeneration of male rats was examined autoradiographically under physiological and reparative conditions. The reparative regeneration was induced by a single injection of allylalcohol intraperitoneally. In both forms of regeneration a displacement of tritiated thymidine-marked hepatocytes from the periportal field to the perivenous field of the liver acinus could be recognized. The velocity of the cell migration after allylalcohol administration was higher than under physiological conditions. Physiological and reparative liver regeneration do not exclude each other but obviously coexist. A more intensively to be examined hepatocellular stem cell concept gains importance.

[Isolation and characterization of hepatocytes from chronic cholestatic damage to rat livers after bile duct ligation]. / Isolierung und Charakterisierung von Hepatozyten aus chronisch cholestatisch geschädigten Rattenlebern nach Gallengangsligatur.

The aim of these investigations was the isolation and characterisation of hepatocytes from chronic injured rat livers after bile duct ligation. In three months old female Uje:WIST rats the distal common bile duct was double ligated. 14 days after the ligation 80% of the rats survived. The content of hepatic OH-prolin was increased in relation to shamoperated control animals. The histological examination showed a marked fibrous and oedematous spreading of the portal tract as well as an extensive bile duct proliferation after 14 days. A correlation between OH-prolin content of the liver and index of fibrosis was found. 14 days after ligation the hepatocytes were isolated according to Seglen (7). The yield of hepatocytes isolated from the liver of cholestatic rats and from control animals was on average 4 x 10(8) cells with a viability of 80%. Ultrastructural the most hepatocytes showed intact organells without injury. We can conclude, that it is possible to isolate hepatocytes from chronic cholestatic rat livers with a good yield and viability as well as with an intact ultrastructure. These cells are suitable for additional metabolic investigations.

[Histological changes in liver tissue in cholestasis]. / Histologische Veränderungen des Lebergewebes bei Cholestase.

Cholestasis is a combination of defined clinical, paraclinical and morphological findings of which icterus is the direct symptom. It is the consequence of the delay or detention of the bile flow. By means of light microscopy different changes can be observed in hepatic tissue: formation of biliary thrombi, intracellular deposition of biliary components, ductal cholestasis with dilatation of the bile-duct lumina and applanation of the bile-duct epithelium, ductular proliferates and furthermore inflammatory mesenchymal reactions with cholangiolitis and cholangitis. These changes strengthen in dependence on the intensity and duration of the existing cholestasis. Bile lakes, bile extravasates, and biliary infarctions may occur in the process. The differentiation between intrahepatic non-mechanically and (extra-)hepatic mechanically conditioned cholestasis is of clinical importance. In the former the production and secretion of bile is disturbed (hepatosis, hepatitis and others), in the latter the extrahepatic bile ducts are above all affected: the bile flow is disturbed by stenosis, compression and obturation. The differentiation between the two kinds of cholestasis is impossible in the first three weeks because both of them develop identical lesion patterns. Distinguishable changes occur only after that. Consequently, findings from liver biopsy can only be a statement of probability. Reliable differentiation between the two kinds of cholestasis is however possible by means of modern technical methods for diagnostic purposes.

Liver function after bilateral nephrectomy.

Consequences of bilateral nephrectomy (NX) for liver functions and for hepatic excretion of various endogenous substances were characterized in rats 24 h after NX. Plasma concentrations of urea, creatinine, fibrinogen, and glutathione increased significantly after NX, whereas the concentrations of total protein, albumin, and lipids decreased. The hepatic excretion of urea, creatinine, phospholipids, cholesterol, and aldosterone significantly increased in uremia, and excretions of protein and glutathione diminished. Active biliary transport can be diminished after NX by the effects of uremic toxins on the liver cells or by the competition phenomena between endogenous substances, which are normally excreted in urine, at the hepatocellular level. Reduced glutathione content and increased lipid peroxidation in hepatocytes have been found. Changes in lipid and protein metabolism after NX can be proved.

Glutathione homeostasis in rats chronically treated with ethanol. Evidence for an increased hepatic GSH export in vivo.

The influence of chronic ethanol feeding to rats on the hepatic glutathione (GSH and GSSG) system (synthesis, catabolism, export) and on the GSH and GSSG concentrations in extrahepatic tissues was investigated. Histological examination of livers from ethanol pretreated rats revealed a minor dilatation of the hepatic sinusoids. After ethanol administration the distribution pattern of gamma-glutamyltranspeptidase (enzymehistochemistry) was nearly unchanged, but the hepatic activity of this enzyme was increased. The ethanol pretreatment led to a decrease in hepatic GSH content. The hepatic activity of the GSSG-reductase were increased after ethanol treatment whereas the activities of the GSH synthesizing enzymes (gamma-glutamyl-cysteinyl-synthetase and GSH-synthetase) were not affected. A strong increase in sinusoidal GSH export was found in the ethanol-pretreated rats. The GSH- and GSSG concentrations of brain, lung, kidney and skeletal muscle were unchanged. It can be concluded that the ethanol-induced alteration of the hepatic GSH metabolism is caused mainly by changes of the sinusoidal membrane of the hepatocytes (direct effect of ethanol on the sinusoidal GSH carrier) leading to an increased GSH export into plasma. This effect should not due to an increased extrahepatic requirement for GSH.

Glutathione homeostasis and turnover in the totally hepatectomized rat: evidence for a high glutathione export capacity of extrahepatic tissues.

Glutathione (GSH) homeostasis and turnover were investigated in totally hepatectomized (HX) rats. A technique is described to remove the liver totally, with preservation of the hepatic portal and vena caval vasculature. Euglycemia could be maintained with hourly infusions of 50 mg 100 g-1 b.m. of glucose after bolus i.v. injection of glucose at the same dose. The efficiency of the animal model was demonstrated by examination of paraclinical blood parameters: progressive increases in total plasma bilirubin and alkaline phosphatase activity were noted after HX; the other parameters tested were predominantly in the normal range during the observation period of 6 hours. Histological examination revealed an acute but reversible impairment of intestine and kidneys. These results indicate that the surgical procedure and postoperative care were able to secure sufficient physiological conditions for the experiments over a longer period. 3 to 6 hours after HX we observed a decreased but stable plasma GSH level in anhepatic rats (about 50% of the control value). The GSH levels of brain and kidney were not changed. With increasing time period after HX the heart and lung GSH levels were depressed. A small depression of muscle GSH concentration was observed 4 and 6 hours after HX. A progressive increase in the concentration of oxidized glutathione was seen in brain and kidney. Our observations could be indicative for a high GSH export capacity of extrahepatic tissues contributing about 50% of the total GSH influx into circulation. Probably, the skeletal musculature is an important GSH origin for plasma.

Metabolic and hemodynamic responses of bivascularly perfused rat liver to nerve stimulation, noradrenaline, acetylcholine and glucagon in thioacetamide-induced micronodular cirrhosis.

Thioacetamide-induced rat cirrhosis was characterized by single-cell necroses, fibrosis, nodular parenchyma, decrease in parenchymal volume density and an increase in liver weight per body weight so that the total amount of parenchyma was not altered. The glycogen content was normal, and signs of decompensation were not found. Isolated livers were single-pass perfused by way of both the hepatic artery and the portal vein. In the normal livers stimulation of the nerve plexuses around the hepatic artery or portal vein (7.5 Hz; 2 msec) and infusions of noradrenaline (1 mumol/L) by way of either vessel and of acetylcholine (10 mumol/L) by way of the artery only increased glucose output, reduced both portal and arterial flow and increased the intravascular pressures. Glucagon (0.5 nmol/L) augmented glucose release and had no hemodynamic effects. In chronically thioacetamide-injured livers all stimuli caused smaller metabolic alterations per gram of liver weight and decreased portal flow more and arterial flow less with stronger enhancements of intravascular pressures than in the controls. The lowered metabolic responsiveness per gram of cirrhotic liver was largely compensated by the increase in liver weight. Thus despite massive histological alterations and pronounced increases in stimulation-dependent resistances - predominantly in the portal system - cirrhotic rat livers responded in their glucose metabolism to nervous and hormonal stimuli in almost the same manner as normal livers.

Collagen peptidase and type III procollagen peptide serum levels in chronic liver diseases.

The concentration of the N-terminal peptide of procollagen III and the activity of collagen peptidase (PZ-peptidase) were measured in sera from 92 patients with chronic liver disease. In patients with liver cirrhosis and chronic hepatitis with transformation of liver structure, high values were found for both variables compared with hepatoses and chronic hepatitis without transformation. The concentration of procollagen III peptide and the activity of collagen peptidase in serum increased with increasing degrees of fibrosis and, even more markedly, with increasing degrees of mesenchymal activity in the liver.

[7-ethoxycoumarin o-deethylase and fibrosis in chronic liver diseases]. / Die 7-Ethoxycoumarin O-Deethylase und der fibrosierende Prozess bei chronischen Lebererkrankungen.

In the liver biopsy of 100 patients with chronic liver diseases, the activity of 7-ethoxycoumarin O-deethylase (ECOD) was determined as a parameter of hepatic monooxygenase system and was compared with some markers of fibrosis e.g. collagen peptidase and hydroxyproline. ECOD was significantly different in healthy liver, fatty liver, chronic active hepatitis (CAH) and cirrhosis. The importance of the fibrotic process was shown by the significant correlations between ECOD and the signs of fibrosis in the liver biopsy. A connection between ECOD and the markers of fibrosis was not found. Further research is necessary to clarify this difference.