Biomedical subjects
Publications and source records attributed to J Brtko.
The induction of mammary gland and other organ tumours by selected chemical carcinogens, 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene, diethylnitrosoamine and azoxymethane is described and their application in experimental carcinogenesis research is discussed.
Genetic immunization or DNA vaccination represents a rapidly developing technology with new perspectives for the prevention and therapy of infectious diseases and it offers new approaches for the treatment of autoimmunity, tumors and even allergy. DNA vaccines are comprised of plasmid DNA which encodes antigen molecules directly in the transfected cells of a target organism. In contrast to protein-induced immune responses, DNA vaccines stimulate both humoral and cell-mediated immune reactions. In the present review we present a palette of unique features of genetic immunization like the effect of CpG motifs, the influence of mode and site of gene delivery and the modulation of immune responses by co-delivery of cytokines, colony stimulating factors, adhesion molecules and other stimulatory molecules. In addition, modulation of the immune response via translation, processing and presentation will be discussed, which in sum demonstrate the elegant possibilities of genetic immunization to induce tailor-made immune responses.
Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.
Among the nutritional factors contributing to maintain health during ageing, fat-soluble vitamins (FSV) are crucial to protect against free radical-generated degenerative processes or impaired efficiency of the immune system. However, no sound scientific evidence is able to confirm specific dietary needs in vitamin A, vitamin E and carotenoids for the healthy elderly. VITAGE project aims at providing such evidence by undertaking studies on male volunteers from 3 European countries, aged between 20-75 years. Biomarkers and variables related to status, metabolism and functions will be measured either in steady-state conditions, or during dietary depletion and repletion in FSV. Original, yet already developed, methodologies will provide clear information about the physiological characteristics of vitamin A, vitamin E and carotenoids. Simultaneously, marketing opportunities for FSV-enriched dietetic foods, specifically designed for the elderly will be determined. The scientific and economical evidence obtained in this project will provide the basis to implement a EU nutritional policy towards the elderly and to develop a new sector of dietetic food products.
In the present work the role of 13-cis retinoic acid and CpG oligodeoxynucleotides (CpG-ODN) in a 1-methyl-1-nitrosourea (MNU)-induced mammary gland carcinoma animal model was investigated. Treatment with both components, applied either alone or in combination, induced a significant decrease of the tumour burden and the volume of tumours only in rats that received CpG-ODN (p = 0.046, compared to the MNU control group). The data indicate that the Th-1 biased immunostimulatory capacities of CpG motifs may play a significant role in induction of protective immune responses against mammary gland tumours in Sprague-Dawley rats.
Nuclear retinoid receptors - retinoic acid inducible transcription factors - participate in pathways influencing many components of the immune system. In the present study in vivo effects of DNA-based immunization of mice on binding parameters of all-trans retinoic acid receptors (RARs) in spleen cell nuclei was investigated. A eucaryotic expression vector encoding the gene for the model enzyme beta-galactosidase of Escherichia coli (pCMV-beta) was used for intradermal injection. Furthermore, immunostimulatory CpG motifs, which stimulate the expression of various cytokines and may serve as a 'danger signal' for the mammalian immune system, were coinjected as oligodeoxynucleotides. The results demonstrate that the concentration of RARs was significantly reduced in the late phase of the primary immune response (21 days after injection of plasmid DNA-indicated by high affinity IgG antibodies and IFN-gamma expression). Coinjection of CpG motifs did not change the course of the humoral response but enhanced and accelerated the proliferative response and expression of IFN-gamma, which correlated with the reduced RARs concentration.
Retinoic acids and their cognate nuclear receptors exert a substantial regulatory role in cell growth and development as well as in the neuroendocrine system. These effects are primarily mediated by all-trans retinoic acid receptors (RARs), members of the steroid/thyroid hormone receptor superfamily of ligand inducible transcription factors. The present study was undertaken in order to compare the effects of immobilization stress (IMO), laparotomy, and 2-deoxy-D-glucose (2DG)-induced intracellular glucopenia on both nuclear RAR affinity and concentration in the rat liver. IMO when compared to non-stressed group of rats, significantly reduced the RAR maximal binding capacity (Bmax) in liver, with the equilibrium association constant (Ka) remaining unchanged. No significant changes of the RAR Bmax and the Ka, were observed in liver of rats that underwent laparotomy. In contrast, a single dose of 2DG (500 mg/kg) resulted in a significant increase of the RAR Bmax 10 h after 2DG application, with the Ka remaining unchanged. Shorter intervals, 1 or 5 h after 2DG application were ineffective on both the RAR Bmax and Ka. In the 2DG-adapted rats (6 doses of 2 DG, 500 mg/kg; 1 dose/day), decapitated 24 h after the last 2DG dose, the RAR Bmax was found significantly higher when compared to control group of animals. No further effect of the next dose of 2DG to repeatedly injected rats on the RAR Bmax and Ka was observed in animals killed 5 h after the seventh dose of 2DG. 2DG-induced intracellular glucopenia markedly up-regulates RARs in liver, but does not change the affinity of the receptor. Thus, the effect of 2DG on RAR concentration in liver specifically differs from that of IMO or laparotomy.
OBJECTIVE: To induce, evaluate and classify advanced stages of mammary gland tumours induced by MNU. METHODS: Female Sprague-Dawley rats were injected intraperitoneally with 1-methyl-1-nitrosourea (MNU; 50 mg.kg-1) on the day 33, 40, 47, 54 and 61 of age in the first experiment and on 50th and 113th day in the second experiment. On the 117th day (first experiment) and on the 153rd day of age (second experiment) the rats were sacrificed by decapitation and their mammary glands were evaluated both macroscopically and microscopically for the presence of grossly detectable mammary tumours. Mammary tumours were classified according to Russo et al. (1990). RESULTS AND CONCLUSIONS: The final incidence of palpable carcinomas was ranging from 60 % to 76 %. All microscopically evaluated tumours were malignant. Among the total number of lesions classified the percentage of invasive tumours ranged from 35 % to 44 %. No metastases were observed in other organs in MNU treated animals.
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OBJECTIVE: Evaluation of the dynamics of all-trans retinoic acid receptor binding properties in mouse spleen nuclear extracts during a primary immune response against beta-galactosidase. METHODS: Female BALB/c mice, aged between 5 and 6 weeks were immunized intradermally into the shaved back (4 spots each) with 100 microg beta-galactosidase in 100 microl sterile phosphate buffered saline (pH 7.2) and blood was taken by tail bleeding on days 0 (preimmune serum), 4 and 6. Production of antibody in serum and the detection of cytokines (IL-4, IFN-gamma) from proliferation supernatants were determined by ELISA. Antigen-specific proliferation assay of isolated spleen cells was based on [3H]-thymidine incorporation measured in a liquid scintillation counter. Both, the maximal binding capacity (Bmax) and the affinity (Ka) of all-trans retinoic acid nuclear receptors (RAR) were evaluated according to Brtko (1994). RESULTS AND CONCLUSIONS: Injection of beta-galactosidase induced the first detectable antibody responses on day 4 (IgM) and on day 6 (IgG). These points of time, reflecting the early and the mature immune response served to measure the antigen-specific proliferation and production of IL-4 and IFN-gamma in the supernatants of the proliferation cultures as well as all-trans retinoic acid receptor (RAR) binding characteristics in spleen nuclear proteins. The RAR Bmax was significantly (P<0.05) decreased only at the time of the first specific IgG antibody production. CONCLUSIONS: The data obtained indicate the involvement of RAR in the late phase of an in vivo immune response.
Inflammatory cytokines in vitro are believed to be involved in the regulation of type I iodothyronine 5'-deiodinase (5'-DI) activity. The present study was undertaken to investigate in vivo effects of DNA immunization of mice on the 5'-DI activity in the liver. A mammalian expression vector encoding the beta-galactosidase (pCMV-betagal) was used for intradermal immunization. Furthermore, immunostimulatory CpG motifs, which induce the expression of IL-6, IL-12, IL-18, TNF-alpha/beta and IFN-gamma were coinjected as oligodeoxynucleotides. From our data we conclude that the activity of 5'-DI in mouse liver when compared to non-immunized animals (100%) was found to be significantly enhanced by DNA immunization 2 weeks (175.7%) or 3 weeks (192.6%) after the plasmid injection. In addition, the activity of the 5'-DI in mouse liver was markedly enhanced 2 weeks (252.4%) or 3 weeks (243.3%) after the injection when CpG motifs were applied together with the plasmid DNA.
Thyroid carcinomas no longer accessible to radio-iodide or TSH-suppressive T4 therapy, due to loss of thyroid-specific functions, might be sufficiently re-differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re-differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid-carcinoma cell lines as model systems. [3H]-RA binding assays with nuclear extracts from follicular thyroid-carcinoma cell lines FTC-133 and -238 revealed high-affinity binding sites for RA. Electrophoretic mobility shift and super-shift assays using a DR2 ("direct repeat" 2) RA response element demonstrated DNA-binding of RARalpha, RARgamma, RXRalpha and RXRbeta in nuclear extracts of FTC-133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA-binding by TRalpha1, TRalpha2, and TRbeta. Northern-blot analysis showed low expression of RXRbeta mRNA in FTC-133 and of TRalpha1 mRNA in FTC-133 and FTC-238 cells. Using RT-PCR, we detected mRNA for RARalpha, RARbeta, RARgamma, RXRalpha, and RXRbeta in the 4 cell lines and in human thyroid-carcinoma samples. RARbeta mRNA was reduced in FTC-238 cells and RXRbeta mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA-receptor-mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid-carcinoma cell lines or tissues, albeit with cell-line and tumor-dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding.
Because chronic ethanol ingestion may perturb thyroid function, we evaluated the effect of 4-wk of oral 10% ethanol ingestion on the hypothalamic-pituitary-thyroid (HPT) axis and septal thyrotropin-releasing hormone (TRH) in 200-g male Wistar rats. Animals were divided into three groups: absolute control receiving tap water and food ad libitum; ethanol group receiving food ad libitum and 10% ethanol as the sole source of drinking fluid; pair-fed group receiving tap water and an amount of food corresponding to the consumption of ethanol group. After 4-wk of treatment, the body weight of the ethanol group was 7% and of the pair-fed rats 19% lower than that of the absolute controls. Both chronic ethanol treatment and food deprivation produced a decrease in plasma thyroid-stimulating hormone (TSH). Pair-fed rats also had a lower plasma T3. Type I iodothyronine 5'-deiodinase activity in the liver was increased in the pair-fed and even more in the ethanol-treated group. The content and secretion in vitro of TRH from the hypothalamic paraventricular nucleus and median eminence were unchanged. TRH content in the septum was increased in both the ethanol and pair-fed groups. TRH secretion from the septum in vitro was lower in the pair-fed, but unchanged in the ethanol group. These data suggest that 4-wk of peroral ethanol intake affects thyroid function mostly at the extrahypothalamic level and that there is a contribution of concomitant food deprivation. Both ethanol treatment and food deprivation increased TRH content in the septum.
In vertebrates, thyroid hormone and its cognate nuclear receptors are involved in a complex arrangement of physiological and developmental function. Since thyroid hormone has also been shown to affect immune responses, we investigated the DNA binding status of T3 receptors of spleen nuclear extracts in a) rats with adjuvant arthritis (AA); b) adrenalectomized rats (ADX), and c) animals with adjuvant arthritis followed by adrenalectomy (AA + ADX). A marked diminution in the functional binding of nuclear thyroid hormone receptors to DR4 thyroid hormone responsive DNA element was found in the spleens of AA and AA + ADX rats when compared to a control group or ADX rats. The data based on in vivo experiments suggest that the nuclear receptor--thyroid hormone responsive element complex status within the cell nucleus may be altered in adjuvant arthritis.
The GH4C1 pituitary cell line, an excellent model for a thyroid hormone action study, was used for determination of the relationship between thyroid hormone receptor occupancy and intensity of cell proliferation, prolactin (PRL) production, thyrotropin (TSH) inhibition and 3,5,3,-L-triiodothyronine (T3) receptor down-regulation. Nuclear receptor population was progressively occupied by T3 in concentrations ranging from 0.025 to 10.0 nM T3. Bmax ranged from 0.029 fmol/10(6) cells at the lowest T3 concentration to Bmax = 12.51 fmol/10(6) cells at the highest concentration. Each of the observed biological events is operative within distinct dose-response ranges in cultured GH4C1 cells. The maximal biological response (except the TSH inhibition and T3 receptor down-regulation) does not require the occupation of the whole nuclear receptor population by T3 and the intensity of none of the responses studied was directly proportional to thyroid hormone receptor occupancy.
Retinoic acid receptor alpha (RAR alpha) expression and RAR binding characteristics were investigated in the liver of rats with high-sucrose (HS) diet-induced insulin resistance. Animals were fed a basal (B) or HS (63 cal%) diet with or without fish oil (FO) (30% w/w of total fatty acids) for two weeks. A significant augmentation (p < 0.01) in the RAR alpha mRNA accumulation in rats fed HS diet when compared to rats fed B diet was demonstrated. In comparison with rats fed B + FO diet, a significant increase (p < 0.005) in the RAR alpha expression was found in rats fed HS + FO diet. In [3H]-retinoic acid (RA) binding studies, Scatchard plots confirmed a significant increase (p < 0.05) in the RAR maximal binding capacity (Bmax) only in rats fed HS + FO diet when compared to rats fed B diet. No significant changes in the association constant (Ka) were found among the groups when compared to rats fed B diet. In contrast to RAR alpha, a significant decrease (p < 0.005) in nuclear thyroid hormone receptor alpha 1 (TR alpha 1) expression was found in rats fed HS diet when compared to rats fed B diet. A significant decrease (p < 0.05) in the TR alpha 1 expression was also detected in rats fed HS + FO diet in comparison with rats fed B + FO diet. In addition, an analogous pattern in the expression of the TR isoform (TR alpha 2) was evaluated as well. In conclusion, the high-sucrose diet-induced insulin resistance might be associated with an increased RAR alpha expression and RAR population, and also with a decreased TR alpha 1 and TR alpha 2 mRNA accumulation.
The effect of feeding Wistar rats with high-sucrose (63 wt% of sucrose, HS) or high-fat (30 wt% of fat, HF) diets for two weeks on serum selenium concentration and type I iodothyronine 5'-deiodinase (5'-DI) activity in liver was investigated. No significant differences in serum selenium concentration (as determined by graphite-furnace atomic absorption spectrometry) were found among the groups of rats fed basal, HS, or HF diets. A significant reduction of the 5'-DI activity (p < 0.005-0.05) was found in groups of rats fed either HS or HF diet in comparison with rats fed B diet. In conclusion, it is suggested that decreased 5'-DI activity in HS or HF diet-induced insulin resistance is not due to selenium status, but it may involve other dietary-related factors.
The present study was undertaken to investigate both in vitro effects of methylene blue and its long-term in vivo influence on 3,5,3'-L-triiodothyronine nuclear receptor binding characteristics in rat liver. In vitro, methylene blue at 1 nmol/l was found to inhibit (p < 0.05) the 3,5,3'-L-triiodothyronine (T3) specific binding to its cognate nuclear receptors. Methylene blue at 100 nmol/l significantly (p < 0.05) reduced the thyroid hormone receptor (TR) concentration (expressed as the TR maximal binding capacity). The equilibrium association constant (Ka) of that reduced number of TR resulting from 100 nmol/l methylene blue TR treatment was significantly higher (p < 0.05), when compared to samples without methylene blue. Moreover, the T3 was found to dissociate from the TR-T3 complex in the presence of 1 mumol/l methylene blue. In vivo long-term treatment of rats with 0.125%, 0.25% or 0.5% methylene blue for 28 days did not affect either the TR maximal binding capacity (Bmax) or the Ka values in comparison with rats fed a standard laboratory diet. The data based on both in vitro or in vivo experiments suggest that in spite of a direct and quick in vitro effect of methylene blue on liver TR concentration and/or TR-T3 dissociation, a long-term in vivo treatment of rats by three different methylene blue concentrations does not affect the thyroid hormone receptor status (the concentration and the affinity) in the liver.