Global DNA methylation in rats´ liver is not affected by hypercholesterolemic diet.

Increased plasma cholesterol levels are listed between the major atherosclerosis risk factors. The final plasma cholesterol levels result from the interplay between the genetic and environmental (diet, physical activity) factors. Little is known, how dietary factor influence epigenetics. We have analyzed, if an over-generation feeding of rat with cholesterol influences total liver-DNA methylation, and if total liver-DNA methylation differ between the different rat strains (Prague hereditary hypercholesterolemic rats, Prague hereditary hypertriglyceridemic rats and Wistar Kyoto rats). The animals were feed with high fat (additional 5 % over normal capacity) high cholesterol (2 %) diet for 14 days. DNA methylation in the liver tissue in different generations was analyzed using the liquid chromatography coupled with tandem mass spectrometry. We have not observed any significant changes in total liver-DNA methylation over the 9 generations of animals feed by fat/cholesterol enriched diet. Additionally, there were no differences in DNA methylation between different rat strains. In animal model, the dietary changes (hypercholesterolemic diet) not significantly influence the total DNA methylation status within the liver.

Experimental models of hyperlipoproteinemia and atherosclerosis.

The first experimental model of atherosclerosis (in rabbits) is more than hundred years old. Several animal species have been used to produce hyperlipoproteinemia and possible atherosclerosis. The gene manipulation produced the most used models recently. This review acknowledges the extensive study of atherosclerotic changes in experimental models of hyperlipoproteinemia and atherosclerosis to come to light thus far and the purpose here is not only to summarize the published data but also to try to add some details of our experience in using these models. In addition to rabbit (the old but also improved model by reno-vascular hypertension) dog, birds, pig, hamster, mice, rat and non-human primate's animal models are described. The gene manipulation produced the most used models two decades ago. Germline genetically engineered (without apoE or LDL receptor genes) animals have become the most used models producing atherosclerotic changes in the aorta. Recent new models also producing atherosclerotic changes but without germline genetic manipulation are also described.

Control of occupational hepatitis B among healthcare workers in the Czech Republic, 1982 to 1995.

Occupational hepatitis B remains a threat to healthcare workers (HCWs) worldwide, even with availability of an effective vaccine. Despite limited resources for public health, the Czech Republic instituted a mandatory vaccination program for HCWs in 1983. Annual incidence rates of acute hepatitis B were followed prospectively through 1995. Despite giving vaccine intradermally from 1983 to 1989 and intramuscularly as half dose from 1990 to 1995, rates of occupational hepatitis B decreased dramatically, from 177 cases per 100,000 workers in 1982 (before program initiated) to 17 cases per 100,000 in 1995. Among high-risk workers, the effect was even more dramatic (from 587 to 23 per 100,000). We conclude that strong public-health leadership led to control of occupational hepatitis B among HCWs in the Czech Republic, despite limited resources that precluded administering full-dose intramuscular vaccine for much of the program. Application of a similar program should be considered for other countries in regions that currently do not have a hepatitis B vaccination program.

Predictors and risk-taking consequences of drug use among HIV-infected women.

OBJECTIVE: To determine rates of drug use among women with HIV, and to examine associations between drug use, health, risk behavior, and sexually transmitted diseases (STD). DESIGN: A longitudinal cohort study of 260 women with confirmed HIV-positive serostatus. METHODS: Each participant contributed a self-report interview, a clinical examination, laboratory testing of cultures for Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and urinalysis for the presence of metabolites of cocaine and opiates. Data were examined on 140 women at 1-year follow-up. Women were defined as drug users if they reported crack, cocaine, or heroin use in the 6 months before the interview or if they had a positive toxicologic test result for cocaine or opiates. RESULTS: 34% of those in the sample were classified as positive for drug use. Drug use was associated with the number of sexual partners, age at first intercourse, prevalence of STDs, and lower quality of life. STDs were present at baseline in 33.7% and 15.5% of drug users and nonusers, respectively. Drug use among this population was also associated at both baseline and follow-up with the likelihood of having a Karnofsky score below 80, and with overall perceived general health. CONCLUSIONS: Drug users in this cohort were more likely to engage in behaviors that place them at risk for STDs, to have elevated STD prevalence, and to have lower perceived health across several indices. Identification of drug use and treatment for it need to be a central component of HIV care for women.

Ethyl 4-isothiocyanatobutanoate--antiproliferative activity in vitro and in vivo.

A new isothiocyanate (ITC) derivate ethyl 4-isothiocyanatobutanoate (E-4IB) induces an immediate dose-dependent inhibitory action on the division of HeLa cells in the concentration range 1.0-0.1 mg/l. Concomitant with the decrease in cell proliferation which follows E-4IB treatment the protein:cell number ratio increases and DNA accumulates. Cells which have lost their ability to divide do not stop their glucose metabolism and only partly stop their glutamine metabolism. The increased content of DNA suggests that cells synthesize DNA without entering into mitosis and that dying cells are in late S or G2 phases prior to death. E-4IB produces a significant growth inhibition of transplanted sarcoma cells B77-RF in rats (at 28 mg/kg). A 57% regression in tumor volume was observed for at least 30 days following the completion of the in vivo treatment. These findings support the presumption that E-4IB is a potential anti-cancer drug. However, further studies are needed for the optimization of its in vivo activity.

[Evaluation of the results of ELISA in the quantitative determination of antibodies against infectious bursitis in poultry]. / Vyhodnotenie výsledkov ELISA pri kvantitatívnom stanovení protilátok proti infekcnej burzitíde hydiny.

Specific antibodies were investigated in serums of chicks vaccinated with live vaccine and revaccinated with inactivated vaccine against the infectious bursal disease virus, using three methods. An ELISA technique was used to determine antibody titres at a fourfold serum dilution; the reaction was evaluated visually. The results were compared with the titres of neutralizing antibodies and percentage of samples with precipitin activity (Fig. 1). The average values of neutralizing antibody titres and ELISA titres were found to have an analogical pattern; a decrease in maternal antibodies on the first days of chick life and an increase in the antibodies after vaccination, accompanied by an increase in precipitin activity, were typical in these tests. Using the different ELISA technique, 138 samples of fowl serum were examined (Fig. 2). The high correlation, r = 0.85, was found between the ELISA titres determined by visual reading of the reaction within the fourfold serum dilutions and the absorbance values determined at single serum dilution. Applying a spectrophotometer programme, a scale of antibody quantification was made up pursuant to the intensity of immunoenzymatic reaction. For the purposes of method reproducibility, the evaluation was made within the average values of absorbance of positive serum on the one hand and of negative serum on the other. The span of these values was divided into ten equal parts, designated by degrees 0 to 9. Corresponding degrees of positivity were assigned to the examined samples in dependence on the determined value of absorbance (Fig. 3). The correlation r = 0.61 was found between the ELISA values and the titres of neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Detection of antibodies to infectious bronchitis of fowl using the ELISA, hemagglutination-inhibition test and agar-gel precipitation test]. / Vysetrovanie protilátok proti infekcnej bronchitíde hydiny metódami ELISA, HIT a AGPT.

Chicks of a conventional poultry flock, Shaver Starcross 288 hybrid, were vaccinated with infectious bronchitis (IB) virus H 120 at the age of 21 days. Three weeks later, the chicks were divided into three groups and separate groups were infected with infectious bronchitis viruses M 41 and D 274 or revaccinated with virus H 120. The content of specific antibodies to antigens prepared from homologous and heterologous viruses of infectious bronchitis used for chick vaccination and infection was investigated at regular intervals in the separate groups of chicks by means of an ELISA technique and haemagglutination-inhibition test (HIT). Serotype specificity of haemagglutination-inhibition test was documented by the results; the specificity was obvious mainly after the first vaccination and two weeks after infection, or after chick revaccination (Fig. 1). The dynamics of postinfective or postvaccinal antibodies, recorded by the ELISA technique, had analogical patterns in the separate groups of chicks, and there were no larger differences in the values determined on the basis of different antigens during the investigation (Fig. 2). A total of 52 group samples of fowl serum was examined by the ELISA technique and agar-gel precipitin test (AGPT) in another part of this study. Ten serums of identical origin represented the separate groups. The result of this examination was evaluated from the percentage of samples with precipitin activity in the group, or from the average value of ELISA. Mutual comparison of the mentioned values indicated that the precipitin activity was limited by the positivity degree of ELISA reaction (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Tumoricidal properties of rat peritoneal macrophages activated with various activators depend on nitrogen oxid synthesis.

Cytolytic activity of mineral oil elicited rat peritoneal macrophages activated by lipopolysaccharide (LPS) and/or rIFN-gamma, rIL-2, Zymosan and PMA (4-beta-phorbol-12-beta-myristate 13-alpha-acetate) was detected in the presence of various concentrations of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. Significant amount of NO2- was detected in the peritoneal macrophages cultured with 0.4 mmol of L-arginine 8 days and the last 24 h with LPS at a concentration of 1 microgram/ml. No significant differences were found between activated peritoneal macrophages obtained from normal (healthy) and/or from tumor bearing rats to induce tumoricidal activity and NO2- production under the same experimental conditions. The results showed that the major cytolytic mechanism against BP6-Tu2 and U 937 tumor cell lines is L-arginine-dependent nitrogen oxide synthesis of activated rat peritoneal macrophages.

Tumor necrosis factor as a potent effector molecule for tumor cell killing by activated rat peritoneal macrophages in vitro.

Tumor necrosis factor (TNF) sensitive (BP6-Tu2) and less or insensitive (B77, MC-1) target cell lines were used to examine the role of TNF in lytic phases of activated peritoneal macrophages and/or direct (murine)-TNF alpha cytolytic activity against these targets. Using antibody to murine TNF, it was shown that lysis of BP6-Tu2 cells was TNF-mediated. This macrophage lytic activity was markedly diminished or even abolished by anti-TNF alpha antiserum. Results indicated that TNF alpha is the critical effector of macrophage mediated killing of these targets. The tumoricidal activity and its level was dependent upon the length of cultivation of macrophages, induction period and concentration of activators. The findings not only support the thesis that macrophages possess various means of coping with tumor cells but also suggest that the mechanism becoming operative is determined predominantly by the pathway of macrophages activation and the properties of the tumor-cell type.

In vitro activation of tumoricidal properties of rat peritoneal macrophages using lipopolysaccharide.

The peritoneal macrophages from normal Lewis rats were characterized by their capacity to phagocytose, by the presence of nonspecific esterase and Fc receptors. In vitro, these macrophages were maintained in culture 7 and/or 21 days, respectively, and for the last 24 h (activation period) were cultured with 0.1-4.0 micrograms/ml of lipopolysaccharide (LPS) and were found tumoricidal against different rat fibrosarcomas, at a ratio of 50:1 (BP6-Tu2, MC-1 and B77). Macrophage-mediated tumor cytolysis was determined using 51Cr-release assay. The sensitivity of used tumors to macrophage-mediated cyto-toxicity was different. In vivo the transfer of the activated macrophages together with the mentioned tumor cells to rats inhibited the growth of tumors. The adoptive transfer of macrophages activated with "activators" might lead to a new kind of immunotherapy of neoplastic diseases.

[Antitumor immunity in patients with melanoma of the uveal tract]. / Protinádorová imunita u pacientov s melanómom uveálneho traktu.

Over the years 1977-1987 142 patients with malignant melanoma of the uvea were admitted to the Department of Ophthalmology, Teaching Hospital, Comenius University in Bratislava. Of the 119 patients treated by enucleation of the bulb 74.8% were alive after a mean follow up period of 5.9 years, whereas 25.2% of these patients died of consequences of metastases of the melanoma of the eye. The highest death rate was recorded in the first and second year following enucleation (57%) and then in the fourth year (17%). After the eighth year following enucleation of the bulb not a single case of death from consequences of metastases of melanoma was recorded. Eight patients with localization of the tumor in the anterior part of the uveal tract (iris, ciliary body) were treated by microsurgical removal of the tumor from the bulb. Two patients developed relapse of the tumor and the eyeballs had to be enucleated. After a mean follow up period of 6.6 years all these patients are alive and so far no signs of metastases have been observed. Moreover, the visual acuity has remained good in the patients whose eyes were not enucleated. Electrocoagulation was applied in six patients. Within a year the eyes had to be enucleated in three of them due to tumor progression. In two patients the growth of the tumor seems to have been stopped, yet regression of the tumor has not been observed. Complete healing was recorded only in one patient. After a mean follow up period of 4.5 years all the patients are alive without signs of metastases. A series of eight patients refused any kind of treatment of melanoma. Over a follow up period of 8.5 years six of them (75%) died of consequences of metastases within an average of 3.2 years from the establishment of diagnosis. Although the majority of these cases had small and middle sized melanomas, at present only two patients (25%) are still alive. In 127 patients with histologically proved malignant melanoma of the uvea the LAI test for the determination of uveal tumor antigen was also performed. The test was positive in 78.7% of the patients and negative in 23.3%. In a control group of patients with nontumorous eye diseases the LAI test was positive only in 2.9% and negative in 91.1%. Four months after enucleation of the bulb the LAI test was repeated and positivity was found to persist in 57.8% of the patients.(ABSTRACT TRUNCATED AT 400 WORDS)

[Detection of surface antigens in uveal melanoma using monoclonal antibodies]. / Detekcia povrchových antigénov u uveálnych melanómov pomocou monoklonálnych protilátok.

Monoclonal antibodies (MA) were obtained by fusion of a myeloma line from mice with spleen cells of mice previously immunized by live human cells of uveal melanoma of an epithelioid type (UMEL-H). From the cell fusion hybridomas were obtained which produced antibodies against immunized uveal melanoma cells. The binding capacity of MA was assessed by the sandwich radio-immuno (RIA) test using indirect immunofluorescence. Extensive specific tests revealed that MA are strongly bound to uveal melanoma, the cells of which were used to immunize the mice, but also fresh allogenic uveal melanoma cells UMEL-K which were also of the epithelioid type. A smaller binding activity was observed with the cell line of the uveal melanoma VUP-1 which was started 16 years ago and is formed by 92% by epithelioid cells. A smaller binding activity of MA was observed with the line of skin melanoma cells (HMB-2, B-HM8). The bond of MA with carcinomatous cells, fibroblasts, uveal and retinal cells from eyes of healthy humans was negative. The preliminary investigations indicate an antigenic homogeneity of tumour antigens of melanoma cells of the same histological type and the antigenic variability of uveal and skin melanomas.

Restoration of immune response by levamisole in B-77 virus induced tumor-bearing rats.

The immunorestorative effect of levamisole was observed in Lewis rats inoculated with a syngeneic B-77 virus induced tumor and immunized with sheep erythrocytes. In tumor-bearing rats the cell-mediated cytotoxicity, the delayed footpad reaction and the antibody production were suppressed. While the cytotoxicity suppressed in tumor-bearing rats was not restored by levamisole, delayed footpad reaction and antibody production were enhanced as compared with the normal reaction, or were restored to normal levels by levamisole, respectively. Tumor growth was significantly suppressed in rats treated with levamisole. The presence of tumor changed the uptake of 3H-uridine labeled lymphocytes in the spleen and peripheral blood. Restorative effect of levamisole on T lymphocytes was different in the studied organs.

Cell surface antigens of human uveal melanoma identified by monoclonal antibodies.

Hybridoma cells were derived from a mouse immunized with freshly obtained primary human uveal melanoma cells. Supernatants from the resultant hybridoma clones were screened for positive antibody binding to uveal melanoma cell membranes and negative binding to membrane preparations of fibroblasts, retinal and uveal cells of healthy donors using sandwich radioimmunoassay and indirect immunofluorescence. Extensive specificity tests showed that the antibodies produced by ten clones bound strongly to fresh, and/or short-time cultivated primary human uveal melanoma tumour cells (UMEL-H, UMEL-K). Weaker binding occurred in a human uveal melanoma cell line (VUP-1), and/or in human skin melanoma cell lines (HMB-2, B-HM8). Binding assay of carcinoma cells, fibroblasts, uveal and retinal cells was negative. Intensive screening of this type is now under way.

A preliminary report on monoclonal antibodies against human uveal melanoma.

Mouse Sp2/10 myeloma cells were fused with spleen cells from mice that had been immunized with freshly obtained primary human uveal melanoma cells. Hybrids that produced antibodies binding to the uveal melanoma cells, but not to fibroblasts, uveal or retinal cells of healthy donors, were cloned. Extensive specificity tests showed that the antibodies produced by the ten clones bound strongly to fresh or short-time cultures of primary human uveal melanoma tumor cells (UMEL-H, UMEL-K). Weaker binding occurred with a human uveal melanoma cell line (VUP-1), and with human skin melanoma cell lines (HMB-2, B-HM8), respectively. Binding assays with carcinoma cells, fibroblasts, uveal and retinal cells were negative. An intensive screening of this type is now under way.

[Characteristics of paramyxoviruses isolated from pigeons in Czechoslovakia]. / Charakteristika holubích paramyxoviru izolovaných v CSSR.

Nine strains of paramyxovirus isolated from racing pigeons in southern Bohemia, Moravia and western Slovakia in 1983 were identified by the haemagglutination-inhibition test with antisera to seven types of paramyxovirus and three types of influenza A virus as PMV-1, Newcastle disease virus, in all cases. The haemagglutination activity and pathogenicity of the isolates for chicken embryos, chicken fibroblast cultures, and chickens of different age were determined. The mean death time of chicken embryos (MDT/MLD) was 52.8 to 95.4 h, the average being 75.7 h. The intracerebral pathogenicity index (ICPI8) was on an average 1.42 +/- 0.10 (P = 0.95). Experimental infection of chickens at the age of one, two, three and eight weeks did not cause any clinical disease but increased the level of haemagglutination-inhibiting (HI) serum antibodies up to 1 : 256 within three weeks. The course of heat inactivation of pigeon viruses at the temperature of 56 degrees C was practically identical with the inactivation of the velogenic viscerotropic strain California/1082/71. On the basis of the results, the pigeon isolates may be considered the Newcastle disease virus of velogenic viscerotropic type whose pathogenicity for chickens has been reduced to the level of mesogenic strains by long-time passaging in pigeons.

Peripheral blood leukocyte adherence inhibition (LAI) reactivity before and during progressive growth of B77 tumor in rats.

Sequential changes in B77-rat fibrosarcoma (B77-RF) extract-induced leukocyte adherence inhibition (LAI) reactivity of peripheral blood leukocytes (PBL) were investigated in rats from the time of tumor inoculation through progressive growth and/or surgical excision of tumor. The time-course profiles of LAI reactivity of PBL in tumor bearing rats correlated with the stage of tumor growth. LAI reactivity of PBL in tumor bearing rats increased during 5-15 days after cell inoculation. Simultaneously, using in vitro 3H-uridine incorporation method for detection of T-cell population, the significantly increased number of immunocompetent lymphocytes was found. During progressive tumor growth (16 days and more after tumor cell inoculation) the LAI reactivity decreased. In this period moderate depletion of immunocompetent population of lymphocytes was observed. A significant decline in LAI reactivity was noted 10 days after a thorough excision of the tumors as compared to that prior to the surgical intervention. The kinetics of the observed time-course appearance of LAI reactivity and the demonstration of a rapid response following tumor challenge indicate further that LAI is a reliable in vitro measure of in vivo tumor immunity.