Questionnaire study of prevalence of urinary incontinence in pregnancy and early six weeks.

OBJECTIVE: Detect prevalence of urinary incontinence in pregnant women depending on risk factors. DESIGN: Questionnaire study. SETTING: GONA company s.r.o., Gynaecology and Obstetrics Practise. CASE REPORT: During the annual follow-up, 20 women out of a total reported complaining about the incontinence of power. The trouble was discreet, the women did not limit, they could engage in all activities. They wore inserts as a precaution, but did not shed their fluid intake and were unconcerned by the posible stench of escaping power. Women had the most trouble after 30 weeks of gestation, the condition improved after delivery and none of the interviewees had trouble escaping after six weeks. Three women devoted themselves to rehabilitate after giving birth. CONCLUSION: Pregnancy is a specific condition for a womanś body, so the changes that occur in this area can only mimic the symptoms of incontinence and hyperactive bladder. Prevention before and during pregnancy plays an important role. Collaboration with a physical therapist is appropriate. Preventive strengthening of the pelvic floor reduces the incidence of urinary incontinence.

[Cost of acute heart failure related readmissions]. / Náklady na rehospitalizaci pacientu s akutním srdecním selháním.

AIM OF STUDY: To assess direct in-patient cost and length of stay in the intensive care unit (ICU) and the standard cardiology unit in acute heart failure (AHF) readmissions. RESULTS: Out of 1 759 patients hospitalized with acute heart failure, 223 patients were readmitted to Faculty Hospital Brno-Bohunice (Czech Republic) during study period (61.4% male; mean age 71.2 years) with mean total cost CZK 85 120 (Euro 3 095) per length of stay 9.2 days and interventions. Comparing to the first hospitalization of study cohort (223 pts.) the decrease was recorded in mean room rate, length of stay and need of ICU stay (from 48% to 42% pts.), nevertheless ICU stay increased (from 3.7 days to 4.1 days). The growth of mean cost was recorded in both procedures in angiology (the decrease in number of coronary angiography which is cheaper was more remarkable than PCI decrease in readmitted patients) and arrhythmology (including device: pacemaker, ICD, CRT) which made 57.5% of total readmission costs. CONCLUSION: The difference in mean in-patient cost between the first and second hospitalization was 18%. The antiarrhytmic procedures had the most significant impact on total readmission cost and its variability, butwe assume that these procedures will reduce within next readmissions and their impact will weaken as in angiology procedures.

Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase.

AIMS: To develop a PCR-based method for reliable detection of Escherichia coli that enables its differentiation from biochemically and phylogenetically related bacteria. METHODS AND RESULTS: Using multiplex PCR targeting four genes (cytochrome bd complex, lactose permease, beta-d-glucuronidase, and beta-d-galactosidase) the possibility of specific detection of various control E. coli strains was tested. It was found that four PCR fragments of the predicted size were observed only for E. coli strains, but not for relatives as close as Shigella sp. or other enterobacteria. Not surprisingly, this method enabled us to identify also E. coli strains which did not exhibit the beta-d-glucuronidase activity. Our multiplex PCR was also successfully used for identification of 95 environmental isolates of E. coli. CONCLUSIONS: The developed PCR-based method, in which four genes coding for lactose permease, cytochrome bd complex, beta-d-glucuronidase, and beta-d-galactosidase, serve as target DNA sequences, allows precise and reliable detection of E. coli strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested approach increases the specificity of detection of E. coli since it enables to distinguish E. coli from Shigella sp. and other relative enterobacteria.

Evaluation of methods for isolation of DNA for polymerase chain reaction (PCR)-based identification of pathogenic bacteria from pure cultures and water samples.

Polymerase chain reaction (PCR) provides a reliable detection of pathogenic bacteria in water samples. However, this method can be adversely influenced by the purity of the DNA template. This is a particularly important obstacle when the bacterial DNA is directly extracted from water samples. In this study we compared the suitability of 8 different methods for isolation of bacterial DNA from pure cultures and 10 different methods for isolation of DNA from water samples. The quality of extracted DNA was assessed by PCR amplification of target sequences derived from uid (E. coli and Shigella sp.), tuf (Enterococcus sp.) and hns (Salmonella sp.). Results indicated that there are differences among the methods tested and only a few of them gave satisfactory results. The method based on alkaline lysis of bacterial suspension, which was developed in our laboratory, seemed to be efficient enough for the detection of bacteria from pure cultures. Detection of bacteria directly from water samples was more difficult. The modified method developed by Slusarenko was found as the best of the tested methods.

Direct detection of bacterial faecal indicators in water samples using PCR.

The presence of enteric pathogens in water resources represents a serious risk for public health. Therefore, their precise detection, and especially detection of E. coli, which is obviously regarded as the main indicator of faecal contamination of water, is an essential step in ensuring bacterial safety of water. Numerous PCR protocols for detection of E. coli have been published to date. They are usually based on amplification of regions derived from lacZ (beta-D-galactosidase) and uidA (beta-D-glucuronidase) gene sequences. However, these methods are not universal enough for precise detection of all E. coli strains found in water samples. We developed a novel triplex PCR method for detection of E. coli in which cyd gene coding for cytochrome bd complex was co-amplified along with lacZ and uidA genes. Our triplex PCR approach significantly increases the specificity and reliability of E. coli detection in water samples. This approach allowed us to distinguish Shigella flexneri from E. coli. In addition, we were able to detect even non-coliform Klebsiella and Raoutella spp., some of which can also cause infections to humans.

Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants.

The mode of the cytotoxic activity of three benzo(c)fluorene derivatives was characterized. The observed morphological changes of lysosomes or variations of mitochondrial activity are assumed to be the consequence of cell protection against oxidative damage and/or the part of the damage process. To establish the relationship between the quantity of superoxide (O2*-) generated and the degree of damage resulting from O2*-, a simple system based on measurement of 3-(4-iodophenyl)-2-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) reductase activity in the presence of superoxide dismutase (SOD) was used. The functionality of the chosen battery of in vitro tests was proved using several known superoxide inducers: cyclosporin A (CsA) and benzo(a)pyrene (BP), as well as noninducers: citrinin (CT) and cycloheximide (CH). From the results followed that the cell growth tests are much better indices of toxicity than the other tests. The model system for the evaluation of the protective capacity of antioxidants against superoxide-induced cytotoxicity included simultaneous exposure of HeLa cells to cytotoxic drugs and to quercetin (Qe), an antioxidant of plant origin. The complete abolishment of the inhibition of cell proliferation and clonogenic survival was concluded to be due to the protective effect of the antioxidant. These observations correlated with the decrease of superoxide content as estimated by the INT-reductase assay in the presence of SOD using the same model system, as well as with the increase of intracellular SOD content and its activity.

Immunotoxicity of ethyl-4-isothiocyanatobutanoate in male Wistar rats.

The immunotoxicity of ethyl-4-isothiocyanatobutanoate (E-4IB) using different immuno-pathological parameters and immune function assays in male Wistar rats was evaluated. The rats were administered intraperitoneally 12 times with E-4IB in three varying doses of 21, 28 and 35 mg/kg of body weight, over a period of 36 days. The doses of E-4IB were set according to the results of previous experiments by its anti-proliferative activity in vivo. High and medium doses of E-4IB exceeded the maximum tolerated dose after the 36-day treatment period. Symptoms of toxicity were displayed by a drop in body weight, spleen and thymus weight and in organ and bone marrow cellularity. Haematological changes displayed a dose-dependent decrease in the percentage of lymphocytes and dose-dependent increase in the percentage of polymorphonuclear leukocytes in peripheral blood. The white blood cell count in rats exposed to a high dose of E-4IB was suppressed. The immune system of rats administered 21 mg/kg of E-4IB (low dose) was unaffected. No changes in primary antibody response to sheep erythrocytes, in vitro proliferative response of spleen lymphocytes to mitogens and phagocytic activity of leukocytes were found in those rats. Our findings indicate that this newly developed anti-cancer drug is not immunotoxic.

Stobadine inhibits lysosomal enzyme release in vivo and in vitro.

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.

Expression of disodium cromoglygate 'protective' effects observed during V79 cell proliferation.

Disodium cromoglycate (DSCG) in equimolar concentration of 0.56 mumol/l preincubated with an asynchronous cell population decreases the inhibition of proliferation and results in a 100% elimination of inhibition of colony formation induced by benfluron (BF). DSCG protects the cells from unbalanced growth and unbalanced metabolism (e.g., prevention of increase or decrease in protein cell content, glycolytic activity and in amino acid metabolism) which are both the integrating parts of BF cytotoxic reaction. The protective effect of DSCG is manifested also on synchronous cell populations, particularly in G2 phase (26%). DSCG also stabilizes cell membrane by preventing the alterations of its permeability caused by the cytolytic concentrations of benfluron (at a concentration of 1.05 mumol/l and more).

Cytotoxic activity of macrocyclic metabolites from fungi.

The cytotoxic effect of 7 macrocyclic fungal metabolites on HeLa cells has been determined. The authors noted great differences in the cytotoxic activity of the substances investigated. Least effective had proved the metabolites curvularin and zearalenone which kept exhibiting ED50 at a concentration of 2 X 10(-4)M and at a concentration of 7.5 X 10(-5)M respectively, whereas the most effective cyanein and monorden gave rise to 50% inhibition of multiplication of HeLa cells at a thousandfold lower concentration than zearalenone. Cytochalasins A, B and D brought about 50% inhibition of proliferation of HeLa cells at concentrations of the same order, with ED50 ranging between 1.2-2.1 X 10(-6)M. The different biological activity of the studied macrocyclic fungal metabolites is discussed in connection with their chemical structure.

Sacharose density gradient separation of Ehrlich ascites carcinoma and L-5178Y tumor cells in different cell cycle phases.

Optimal conditions were determined for the distribution of Ehrlich ascites carcinoma (EAC) and L-5178Y mouse tumor cells, proliferating in vivo, by their age within the cell cycle by sedimentation in a buffered linear sacharose density gradient. Measurements of cell size, DNA content and incorporation of tritiated thymidine in successive parts of the gradient confirmed the actual separation of cells of different age: in the upper fractions there were cells in G1 phase, in the middle fractions in S phase and in the lower layers of the gradient there were cells in G2 and/or M phase.

The suitability of different quantitative methods for determination of the cytotoxic activity of agents in cell cultures.

Studies related to the measurement of growth inhibition of HeLa cells by 6-Thioguanine (TG) and Vermiculine (V) have been described. Control and treated cell populations were enumerated as uniform suspensions of whole cells, prepared by standard treatment with trypsin. Determination of cell number, measurements of protein, DNA and RNA as well as of glucose consumption from the culture medium were made throughout the treatment with the studied drugs. It was apparent that the total cell number in control cultures was directly proportional to the total cell protein or nucleic acids. However, in both treated cultures the relationship between these parameters was disturbed as consequences to unbalanced growth which was an integral part of the cytotoxic reaction of the studied agents. From the results presented it followed that only the direct cell enumeration was suitable for the detection of agents' cytotoxicity.

Cytotoxic and cancerostatic effect of 1,4-dithiaanthraquinone-2,3-dicarbonitrile.

1,4-Dithiaanthraquinone-2,3-dicarbonitrile (DTA) has been found to exert a considerable cytostatic effect especially on some of the investigated types of eukaryotic cells, concretely on the HeLa cells, moulds, yeasts, protozoa and algae. In cells of the Ehrlich ascites carcinoma (EAC) DTA after a short exposition causes a parallel inhibition of incorporation of 14C-adenine and 14C-valine, in proportion to its rising concentration. The inhibition of biosynthetic processes thus made manifest, is probably a consequence of the primary DTA intervention into the energy metabolism of EAC cells, particularly in glycolysis. The effect of DTA in concentrations capable of bringing about full inhibition of glucose consumption or lactate formation in EAC cells also results in a loss of their transplantability. On the other hand, DTA also exerts a cancerostatic effect on the Ehrlich ascites carcinoma in mice.

The study of cytotoxic effect of benfluron.

The efficiency of benfluron (B-F) was measured by determining the inhibition of cell proliferation in HeLa and V/79 cells. B-F induced an acute cytotoxic reaction in dependency on the concentration applied. A biphasic unbalanced growth was an integral part of this reaction. In connection with the decrease in cell proliferation which followed B-F treatment at micromolar concentrations, the protein:cell volume ratios increased while at nanomolar concentrations decreased. These variations must be taken into account when expressing and interpreting the results of cell metabolism. The glucose and amino acid metabolism of treated V/79 cells differed markedly when expressed as a function of cell number or as function of protein content per cell.

Study of metabolism and growth limitation of human leukemia cell lines.

The relationship between proliferation and metabolism of four leukemia cell lines (BALL-1, JOK-1, Jurkat, and MOLT-4) in batch culture was studied. The maximum cell density (1.5-2.3 x 10(6) cells/ml) without change of medium was observed on days 6-8 of cultivation. At the same time, the original concentration of glucose in the medium (10 mmol/l) fell to 3.5-4 mmol/l. While BALL-1 and MOLT-4 cells, on day 4 of cultivation, converted 82% of glucose into lactate, on day 7 this value was 50%, or there was no lactate production (MOLT-4), respectively. On the other hand, the values of the coefficient of glycolysis showed that Jurkat and JOK cells converted also other compounds into lactate. Part of the utilized glutamine was employed by all four cell lines: 1. as a precursor of glutamic acid, and 2. as a source of energy. BALL-1 and JOK-1 cells converted part of arginine into ornithine. At the time when the proliferation of the cells ceased, the level of ammonia reached a toxic concentration of 2.0-3.6 mmol/l. Since these cell lines utilized only a part of carbon and nitrogen sources in the medium, it was suggested that the final cell density was limited by a growth inhibitor (i.e. ammonia) and not by a lack of nutrients.

The mechanism of cytolytic and cytostatic activity of benfluron.

Benfluron at concentrations of 0.26 and 0.52 mumol l-1 inhibited the formation of V79 colonies in a concentration-dependent way. Diminution of the size of colonies of the treated cells was accompanied by a decrease in protein content per cell. At the same time an increase in metabolic activity was observed. Benfluron blocked the V79 cells in S and G2 phases of the cell cycle and at higher concentrations induced cell lysis documented by alterations in cell membrane permeability and morphology. The in vitro membrane effect of benfluron involved a biphasic modulation of the cardiac sarcolemmal (Na+ + K+)-ATPase activity.

Ribonucleases in mouse ascitic tumor cells during ageing in vivo and in vitro.

Ribonuclease activities have been determined in cell extracts of mouse L5178Y lymphoma grown in vivo and in vitro. From the obtained results it follows that the level of studied enzymes changes in both systems during ageing of the cells. Moreover, when lymphoma cells are cultured in vitro a marked decrease in specific activity of alkaline ribonuclease II is observed.

The use of cell culture systems for the assessment of general cellular toxicity and to detect the nature and location of free radical damage.

In the process of developing compounds to counteract the damaging effects of free radicals in biological systems it is important to determine the type and the intracellular location of specific toxic events. For that purpose cultured cells are used, because a properly designed cell culture system allows to asses specific damage at the cellular and subcellular level and can contribute to an evaluation of the biochemistry of free radical damage. A wide range of changes in cellular activities resulting from oxidative injury in vitro have been demonstrated. Data from many laboratories indicate that cell culture system coupled with appropriate analytical techniques can be used to explore cellular and biochemical details of damage induced by free radicals. The type of reactive oxygen species used to generate the radicals, the rate of radical production, and the location of action of the toxic species must be taken into account to understand the biochemistry of the system. An effort is made to analyse the considerable progress made in the development of appropriate in vitro models and end-points for use in testing and characterising the nature and location of free radical cytotoxicity.

Comparison of isoproterenol-induced changes in lysosomal enzyme activity in vivo and in vitro.

Isoproterenol was used as a drug which, when administered in high doses, is able to induce lysosomal enzyme activity changes in in vivo conditions. We correlated lysosomal enzyme activity in the absence and presence of isoproterenol, obtained in whole animals and in HeLa and HepG2 cells in tissue culture. In vivo experiments: male Wistar rats (270-300 g) were treated subcutaneously with isoproterenol in various doses. Effect of isoproterenol on lysosomal enzyme activity was assayed in the heart after differential centrifugation. In vitro experiments: Isoproterenol in concentrations 0.1-100 microg/ml was added to HeLa and HepG2 cells and the activity of lysosomal enzyme was measured in the cell homogenate. In the sedimentable and nonsedimentable fractions of the rat myocardium, the isoproterenol-induced changes in the activity of lysosomal enzyme were time-and dose-dependent. In HeLa cells, isoproterenol administration caused a dose-dependent increase of lysosomal enzyme activity, while in HepG2 cells the activity remained unchanged. Thus the isoproterenol-induced changes in lysosomal enzyme activity in the rat myocardium were comparable with the results found in vitro in HeLa cells.

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.