[Can Analysis of Cellular Lipidome Contribute to Discrimination of Tumour and Non-tumour Colon Cells?] / Muze analýza bunecného lipidomu prispet k rozlisení nádorových a nenádorových bunek tlustého streva?

BACKGROUNDS: Colon cancer development is often characterized by abnormalities in lipid synthesis and metabolism, which may influence energetic balance, structure and function of biological membranes, or production of specific mediators and cell signalling. The changes in lipid profile and metabolism (lipidome) may significantly affect cell behaviour and response to therapy. Permanent epithelial cell lines at various stages of cancer development are used for better understanding of this topic on cellular and molecular levels. In our study, we hypothesized that detailed analyses of colon cancer cell line lipidomes may help to identify major alterations in the amount and profile of specific lipid classes/species, which can contribute to their different response to various stimuli. MATERIAL AND METHODS: Cellular lipids were isolated from six human epithelial cell lines derived from tissues at various stages of tumour development. Liquid chromatography coupled with tandem mass spectometry analyses were performed in order to determine amount and mass profiles of all phospholipid (PL), lysophospholipid (lysoPL) and sphingolipid classes. The data was statistically evaluated (cluster and discrimination analyses) with respect to mutual comparison of cell lines and to significantly discriminating lipid types. RESULTS: The results of cluster analysis arranged cell lines in order corresponding to their level of transformation (normal cells, adenoma, carcinoma and lymph node metastasis). The results of discrimination analyses revealed the most discriminating lipid types and distinction in PL: lysoPL ratios. Particularly, significant correlation of the amount and profiles of both specific lysoPL and sphingolipid classes with cell transformation level were observed. Similar approaches are now applied to compare lipidomes of colon epithelial cells isolated from tumour vs. non-tumour samples of colon cancer patients. CONCLUSION: Our results indicate that a) selected cancer cell lines are suitable model for lipidomic studies that can serve as a basis for subsequent clinical research, b) cellular lipidome analyses may help to discriminate tumour and non-tumour cells in clinical samples, where specific types of lipids could serve as biomarkers.Key words: colon cancer - cell lines - liquid chromatography - mass spektrometry - phospholipids - sphingolipids - bioinformatics The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by Czech Health Research Council, grant No. AZV 15-30585A.Submitted: 19. 3. 2018Accepted: 18. 4. 2018.

Phthalates deregulate cell proliferation, but not neuroendocrine transdifferentiation, in human LNCaP prostate cancer cell model.

Phthalate esters are ubiquitous environmental pollutants widely used as plasticizers, which have been shown to interfere with both endocrine regulation and development of reproductive organs. In the present study, we examined the impact of diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) on the proliferation of androgen-sensitive human prostate carcinoma LNCaP cells and related events. The results showed that both compounds were able to inhibit cell cycle progression in a dose-dependent manner. However, only DEHP was found to weakly reduce androgen receptor (AR) protein levels after long-term exposure, while only DBP partially inhibited expression of the prostate-specific antigen (KLK3) gene, a model AR transcriptional target. This indicated that inhibition of cell proliferation was likely independent of any AR modulations. Both phthalates induced suppression of cell proliferation, but none of them affected the levels of markers associated with neuroendocrine transdifferentiation (NED) in LNCaP cells. Taken together, the presented data indicate that phthalates may exert long-term negative effects on the proliferation of prostate epithelial cells derived from the carcinoma model, which are, nevertheless, largely independent of the modulation of AR expression/activity, and which do not alter further processes associated with NED.

2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits beta-catenin-dependent transcription in liver epithelial cells.

The toxic modes of action of non-dioxin-like polychlorinated biphenyls (PCBs) in liver cells are still only partially understood. Several recent studies have indicated that PCBs may interfere with cell membrane protein functions. Therefore, we analyzed in the present study the effects of di-ortho-substituted 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) on proteins involved in the formation of adherens junctions in a model of rat liver progenitor cells - WB-F344 cell line. PCB 153, at micromolar concentrations, induced a gradual degradation of E-cadherin, beta-catenin or plakoglobin (gamma-catenin) proteins. This effect was not due to changes in gene expression, as PCB 153 had no effect on mRNA levels of the above mentioned proteins. Moreover, apart from the reduction of total beta-catenin pool, PCB 153 also decreased levels of the active beta-catenin form, dephosphorylated at residues Ser37 and Thr41, which is the key co-activator of Wnt-induced TCF/LEF-dependent gene expression. Therefore, we also evaluated the impact of PCB 153 on expression of Axin2, a known transcriptional target of canonical Wnt signaling. PCB 153 reduced basal Axin2 mRNA levels and it inhibited induction of Axin2 expression by recombinant mouse Wnt3a. Nevertheless, PCB 153 had no effect on phosphorylation of glycogen synthase kinase-3beta (GSK-3beta), which is supposed to target beta-catenin for its proteasomal degradation. This suggested that GSK-3beta activity is not modulated by PCB 153 and, consequently, not involved in the observed PCB 153-induced decrease of both total and active beta-catenin levels. Protein levels of E-cadherin and beta-catenin were partially restored with lysosomal inhibitor leupeptin, thus suggesting a possible role of lysosomes in the observed degradation of adherens junction proteins. Taken together, the present data suggest that PCB 153 may interfere with functions of adherens junction proteins involved in both cell-to-cell communication and intracellular signaling. Such mechanisms might be involved in the effects of non-dioxin-like PCBs contributing to liver tumor promotion.

Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin.

OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND METHODS: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells. RESULTS AND CONCLUSIONS: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.

Effects of methylated chrysenes on AhR-dependent and -independent toxic events in rat liver epithelial cells.

Methylated chrysenes (MeChry) are important cigarette smoke constituents and 5-MeChry has been listed as possibly carcinogenic to humans. Although a major attention has been in past paid especially to mutagenic, tumor-initiating effects of MeChry, little is known about toxic effects of MeChry related to tumor promotion. As the position of methyl group has been repeatedly observed to determine genotoxic effects of MeChry, we examined both genotoxic and nongenotoxic effects of MeChry, using rat liver cell lines as experimental models. All six MeChry were relatively efficient aryl hydrocarbon receptor (AhR) agonists, with 3- and 6-MeChry being the most potent inducers of the AhR-mediated reporter gene activity. All six compounds disrupted contact inhibition in rat liver epithelial WB-F344 cells, a process previously reported to be AhR-dependent, suggesting that MeChry may interfere with cell cycle control in an AhR-dependent manner. In contrast, only 5- and 6-MeChry were found to acutely inhibit gap junctional intercellular communication (GJIC), another parameter correlating with tumor promoting effects of xenobiotics. Both 5- and 6-MeChry were efficient inducers of mRNA expression of enzymes involved in metabolic activation of polycyclic aromatic hydrocarbons, including cytochromes P450 1A1/1B1 and aldo-keto reductase 1C9. However, only 5-MeChry, and not 6-MeChry, induced significant formation of DNA adducts in rat liver epithelial cells, which corresponded with its ability to induce high accumulation of cells in S-phase. On the other hand, 5-MeChry induced neither apoptosis related to DNA damage nor phosphorylation of p53 tumor suppressor. Taken together, our results suggest that methyl group position may affect both genotoxic and nongenotoxic effects of MeChry, such as formation of DNA adducts and inhibition of GJIC. All MeChry showed a potency to disrupt cell proliferation control, while 5-MeChry was a single compound inducing DNA damage, disruption of cell cycle control and inhibition of GJIC in rat liver cells.

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: a partial role of cytosolic phospholipase A(2) and extracellular signal-regulated kinases 1/2 signalling.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2',4,4'-tetrachlorobiphenyl (PCB 47) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non-ortho-substituted, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A(2) (cPLA(2)) inhibitor, AACOCF(3). Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA(2) and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.

TCDD deregulates contact inhibition in rat liver oval cells via Ah receptor, JunD and cyclin A.

The aryl hydrocarbon receptor (AhR) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Activation of the AhR by TCDD leads to its dimerization with aryl hydrocarbon nuclear translocator (ARNT) and transcriptional activation of several phase I and II metabolizing enzymes. However, this classical signalling pathway so far failed to explain the pleiotropic hazardous effects of TCDD, such as developmental toxicity and tumour promotion. Thus, there is an urgent need to define genetic programmes orchestrated by AhR to unravel its role in physiology and toxicology. Here we show that TCDD treatment of rat liver oval cells leads to induction of the transcription factor JunD, resulting in transcriptional upregulation of the proto-oncogene cyclin A which finally triggers a release from contact inhibition. Ectopic expression of cyclin A in confluent cultures overcomes G(1) arrest, indicating that increased cyclin A levels are indeed sufficient to bypass contact inhibition. Functional interference with AhR-, but not with ARNT, abolished TCDD-induced increase in JunD and cyclin A and prevented loss of contact inhibition. In summary, we have discovered a novel AhR-dependent and probably ARNT-independent signalling pathway involving JunD and cyclin A, which mediates TCDD-induced deregulation of cell cycle control.

Dibenzanthracenes and benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver 'stem-like' cells.

Polycyclic aromatic hydrocarbons (PAHs) with molecular weight 278 are a group of PAHs that are mostly not covered by the current monitoring programs, despite their relative abundance in environmental samples and possible carcinogenicity. Although benzo[g]chrysene (BgChry) and dibenz[a,h]anthracene (DBahA) have been for a long time studied as genotoxic, tumour-initiating compounds, little is known about the potential tumour-promoting effects of this group of PAHs. In the present study, we investigated their impact on activation of the aryl hydrocarbon receptor (AhR), induction of enzymes involved in metabolic activation of PAHs, disruption of cell cycle control in confluent cell population and inhibition of gap junctional intercellular communication (GJIC), using the rat liver epithelial cell line WB-F344 as a model of liver progenitor cells. We found that BgChry was the weakest inducer of the AhR-mediated activity, while relative potencies of benzo[b]chrysene (BbChry) and benzo[c]chrysene (BcChry) were comparable to the previously reported values for dibenzanthracenes. All compounds increased expression of cytochromes P450 1A1 and 1B1, and aldo-keto reductase 1C9. BgChry was found to induce high amounts of DNA adducts, which corresponded with induction of p53 phosphorylation at Ser15, apoptosis and accumulation of cells in S-phase of cell cycle, leading to a decrease in cell numbers. All other compounds were found to stimulate cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. We found that only BgChry, and to a lesser extent also BcChry, inhibited GJIC at high concentrations. Taken together, dibenzanthracenes and benzochrysenes, with exception of BgChry, seem to act primarily through deregulation of cell proliferation in liver epithelial cells, which is related to their relatively high AhR-mediated activity. The disruption of cell cycle control might contribute to their carcinogenic effects, as well as to carcinogenicity of complex environmental mixtures containing high levels of PAHs with molecular weight 278.

Strategy aimed at reduction of sputum eosinophils decreases exacerbation rate in patients with asthma.

Under Global Initiative for Asthma guidelines, the clinical control of disease activity and the adjustment of treatment in patients with asthma are based on symptoms, use of rescue medication, lung function and peak expiratory flow measurement (standard strategy). We investigated whether a strategy to reduce the number of sputum eosinophils (EOS strategy) gives better clinical control and a lower exacerbation rate compared with the standard strategy. Fifty-five patients with moderate to severe asthma entered this open, randomized, parallel-group study and visited the out-patient department every 3 months for 18 months. The dose of corticosteroids was adjusted according to the standard strategy or the percentage of sputum eosinophils (EOS strategy). During the study period, the EOS strategy led to a significantly lower incidence of asthma exacerbations compared with the standard strategy group (0.22 and 0.78 exacerbations per year per patient, respectively). There were significant differences between the strategies in time to first exacerbation.

[Psychological condition of children from pregnancies after assisted reproduction in the third year of life]. / Psychický stav detí z tehotenství po asistované reprodukci ve tretím roce zivota.

OBJECTIVE: To analyse psychological development of children born after assisted reproductive technology. DESIGN: Psychological analysis of the child's development related to the technology of assisted reproduction, length of pregnancy and multiple birth was performed and compared with the control group. SETTING: Institute for the Care of Mother and Child, Prague. METHODS: Out of the total number of 123 children born after assisted reproductive technology during the 1st half of the year 1998 in the Center ISCARE IVF, psychological development was evaluated in 109 children (88.6%). Ninety four children from this sample were assessed using mental, motor and behavior scales of the Bayley Scales of Infant Development II. RESULTS: Mental and motor development of infants born after intracytoplasmatic injection (ICSI) and after in-vitro-fertilisation (IVF) was not significantly different. Fullterm singletons born after assisted reproductive technology did not differ from control fullterm children. There was a developmental delay in both fullterm and preterm children from multiple pregnancies in comparison to control children. No child had serious impairment of psychic functions (developmental index <50). There were no significant differences in behavior records between children born after assisted reproductive technology and control children, but some parents had problems in educational care of their children born after assisted reproductive technology. CONCLUSION: From children born after assisted reproductive technology, those from multiple pregnancies may be at risk for later psychological development. The occurrence of educational problems in assisted reproductive families indicates a need of accessible professional care in this field.

[Effect of early nutrition on growth parameters and psychomotor development of children of very low birth weight]. / Vliv casné výzivy na rustové parametry a psychomotorický vývoj u detí s velmi nízkou porodní hmotností.

OBJECTIVE: The aim of the study was to evaluate influence of early nutrition on growth parameters and psychomotor development of children with very low birth weight (VLBW). DESIGN: A prospective clinical study. SETTING: Institute for Care of Mother and Child, Prague. METHODS: Thirty nine children of birth weight 1,000-1,499 were followed up to one year of their corrected age in a prospective study. The group was divided in two groups according to type of nutrition: 17 children (group A) were fed with milk of own mother - "preterm milk", 22 children (group B) were orally fed with mature milk from the Bank of mother milk - "term milk", which was fortified with BMF preparation (Nutricia, Netherlands). Both groups were comparable in basic anthropometric parameters (weight, lenght, circumference of head and thotax) and in psychosocial characteristics of their mothers. Growths parameters were monitored in weekly intervals for approximatelly eight weeks. In the period between 11th and 15th month of corrected age, the children were evaluated by a clinical psychologist on a blind basis in mental a motor development by using Bayley Scales of Infant Development (BSID-II). Statistical analysis was performed by chi-square test and t-test. RESULTS: No statistically significant differences between the two groups in evaluating the growth parameters were observed. The psychological examination demonstrated statistically significant differences in the motor development. The psychomotor developmental index (PVI) proved to be 84.4 +/- 14.6 in the group A and 94.3 +/- 12.5 in the group B (t-test = 2.28, p<0.05). There was not any statistically significent difference in metal development between the two groups. The mean mentel developmental index (MVI) was 98.2 +/- 10.2 in the A group and 101.0 +/- 13.3 in the group B. CONCLUSION: Result of the study indicate favorable effect of fortification of breast milk in VLBW newborns, especially in view of the observed favorable influence of fortfication on motor development of the children.

Peri- and post-operative course of cytokines and the metabolic activity of neutrophils in human liver transplantation.

Peri- and post-operative (up to day 7 after surgery) neutrophil chemiluminescence and the plasma concentrations of interleukin 6 (IL-6), IL-8, IL-10 and tumour necrosis factor alpha (TNF-alpha) were evaluated in the blood of patients undergoing liver transplantation. IL-6, IL-8 and IL-10 levels increased during early reperfusion and then returned to normal mostly within the first post-operative day. TNF-alpha was increased during the whole period observed. Spontaneous as well as activated neutrophil chemiluminescence was depressed in early reperfusion and remained low during the whole period followed. Samples collected during early reperfusion provided positive correlation for IL-6 vs IL-8 as well as for IL-6 and IL-8 vs chemiluminescence. The data were also evaluated with respect to the outcome of transplantation. Since IL-8, IL-10 and TNF-alpha levels increased significantly during the first post-operative week, mainly in a group of patients who developed serious complications within the first month after surgery, we proved a connection between peri- and early post-operative induction of cytokine release and the outcome of liver allograft transplantation.

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay.

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.

Monitoring river sediments contaminated predominantly with polyaromatic hydrocarbons by chemical and in vitro bioassay techniques.

Extracts of sediment samples collected from the Morava River and its tributaries (Czech Republic) were examined for mutagenic, dioxin-like, and estrogenic activities. Moreover, the human leukemic HL-60 cell line was tested as a potential model for the detection of effects of environmental contaminants on cell proliferation and differentiation processes. Analytical data indicate that the sediments were contaminated predominantly with polycyclic aromatic hydrocarbons (PAHs) and phthalate esters. The sums of concentrations of 16 U.S. Environmental Protection Agency priority PAHs ranged from 0.8 to 13.2 micrograms/g and those of phthalates reached up to 3,000 ng/g, while only low levels of chlorinated hydrocarbons were found. The main goal of the present study was to determine effects of PAH prevalence on in vitro bioassays, with special emphasis on dioxin-like activity. The dioxin-like activity was tested using a reporter gene assay based on chemical-activated luciferase expression (the CALUX assay). Significant dioxin-like activity (2.6-40.1 micrograms/g benzo[a]pyrene equivalents and 5.9-48.2 ng/g 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents) was detected in all samples, and the results obtained with various exposure times or with both crude and PAH-deprived extracts indicate that the response was probably caused almost exclusively by the presence of high concentrations of PAHs. This corresponds with results of chemical analyses and indicates that various exposure times would allow a discrimination between dioxin-like activities of persistent compounds and easily metabolized aryl hydrocarbon (Ah) receptor inducers. Only sediment extracts containing the highest concentrations of PAHs were mutagenic, as determined by the umu assay. Estrogenic activity was found in several samples (4.75-22.61 pg/g estradiol equivalents) using cells stably transfected with an estrogen-responsive element linked to a luciferase promoter. Noncytotoxic doses of extracts had no effects on HL-60 cell proliferation, while two of the tested crude extracts significantly enhanced their all-trans retinoic acid-induced differentiation. These activities were not associated with phthalate esters and/or PAHs. Our results indicate that cellular and biochemical in vitro assays based on various specific modes of action may yield data complementary to results of mutagenicity tests and that they could be useful in environmental risk assessment. High levels of PAHs are apparently associated with dioxin-like and mutagenic activities rather than with estrogenic activity.

Inhibitors of arachidonic acid metabolism potentiate tumour necrosis factor-alpha-induced apoptosis in HL-60 cells.

We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-alpha (TNF-alpha) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF3; cytosolic phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-alpha-induced apoptosis, while arachidonic acid, AACOCF3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-alpha. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-alpha-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-alpha in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.

Modulation of death receptor-mediated apoptosis in differentiating human myeloid leukemia HL-60 cells.

Differentiating myeloid cells may become resistant to various apoptotic stimuli. In the present study, dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA) were found to modulate the sensitivity of HL-60 cells to death receptor-mediated apoptosis in a time-dependent manner. During the early stages of differentiation, DMSO treatment increased the response of HL-60 cells to tumor necrosis factor alpha; (TNF-alpha), but enhanced responsiveness was lost during later differentiation stages. In contrast, ATRA treatment induced resistance to TNF-alpha-induced apoptosis. HL-60 cells were resistant to Fas-mediated apoptosis but were sensitized by culturing in serum-free conditions. Similar to its effect on TNF-alpha sensitivity, DMSO pretreatment augmented the response to Fas-mediated signaling, which coincided with increased expression of Fas on DMSO-pretreated cells. However, during the later stages of DMSO-induced differentiation, sensitivity to anti-Fas antibody-induced apoptosis declined significantly, although Fas expression was still elevated. The reduced sensitivity to anti-Fas treatment partially correlated with increased Fas-associated phosphatase-1 mRNA expression. Thus, regardless of either Fas up-regulation or potentiation of TNF-alpha-mediated apoptosis during early DMSO-induced differentiation, a slow increase in resistance to apoptosis mediated through these death receptors occurs during DMSO-induced differentiation, which contrasts with the rapid induction of resistance following treatment with ATRA.

[Regular respiration during sleep in children in the first months of life]. / Pravidelný dech za spánku u detí v prvních mesících zivota.

Regular respiration is an important parameter of quiet sleep in infants. At the earliest age, the periods of regular respiration are shorter as compared with the periods of EEG, EMG and EOG patterns of quiet sleep. The duration of periods with regular respiration significantly increases with age, and their coincidence with other parameters of quiet sleep becomes higher. The frequency of regular respiration significantly decreases with age. Very short periods of regular respiration, low coincidence with other parameters of quiet sleep, and very high frequency of regular respiration after the 12th week of life may indicate a delay in the central nervous system development.

Correlation between peritoneal fluid and serum antiphospholipid antibodies in women with primary infertility.

OBJECTIVE: To compare the peritoneal and serum levels of antiphospholipid antibodies (aPLs) against the following phospholipids: cardiolipin, L-alpha-phosphatidic acid, L-alpha-phosphatidylethanolamine, 1-alpha-phosphatidyl-DL-glycerol, L-alpha-phosphatidyl-inositol, L-alpha-phosphatidyl-L-serine in immunoglobulin isotypes of G, M, and secretory A. PATIENTS AND METHODS: A group of 107 women with primary infertility were immunologically examined. An ELISA method was used for detection of aPLs. RESULTS: aPL-L-serine and aPL-cardiolipin predominate over peritoneal fluid in all studied Ig isotypes (G, M, secretory A). Levels of aPL-ethanolamine are much higher in peritoneal sIgA and IgM than in sera. There is a predominance of IgG-aPLs-inositol, DL-glycerol, and aPL-acid in serum, and of sIgA in peritoneal fluid. CONCLUSION: Our patients had higher levels of aPLs (IgG, IgM, sIgA) against cardiolipin, serine, and ethanolamine in peritoneal fluid than in sera. The prevalence of aPLs in IgG was against DL-glycerol, phosphatidyl-inositol, and phosphatidic acid. It is quite difficult to estimate whether the aPLs have an influence directly through phospholipid epitopes on uterine mucous membrane, on the surface of oocytes, or on the surface of the early embryo during the fertilization and/or implantation process.

Total antioxidant capacity of serum increased in early but not late period after intestinal ischemia in rats.

The ischemia of small intestine was induced in anesthetized Wistar rats by occluding the superior mesenteric artery for 45 min and then the reperfusion was set. Serum samples were obtained at the end of the ischemic period and also during early (1 to 4 h) and late postischemic period (1 to 4 d). The total antioxidant capacity (TRAP) of serum samples was evaluated using luminol enhanced chemiluminescence. The increased mobilization of phagocytic cells and the release of reactive oxygen species into the circulation was observed from the first and second hour of the postischemic period, respectively. Nevertheless, the activity of natural antioxidant mechanisms of serum was already elicited at the end of the ischemic period. Furthermore, the TRAP of serum increased with the increasing duration of early postischemic period. Among the antioxidants studied, urate and ascorbate concentrations exerted the highest correlation with TRAP, but 31.6% of the total antioxidant capacity remained for the activity of an unidentified antioxidant(s). After being exhausted, the TRAP of serum oscillated around the preoperation level at days 1-4 of the postischemic period. The increase in total antioxidant capacity of serum induced by oxidative stress was sufficient to prevent lipoperoxidation both in serum and intestinal tissue.