Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

Toxicological evaluation of three contaminants of emerging concern by use of the Allium cepa test.

Di(2-ethylhexyl)phthalate, triclosan and propylparaben are contaminants of emerging concern that have been subjected to extensive toxicological studies, but for which limited information is currently available concerning adverse effects on terrestrial plant systems. The Allium cepa test, which is considered one of the most efficient approaches to assess toxic effects of environmental chemicals, was selected to evaluate the potential risks of these ubiquitous pollutants. Our data demonstrate that all three compounds studied may in some way be considered toxic, but different effects were noted depending on the chemical and the end point analysed. Results derived from the analysis of macroscopic parameters used in testing for general toxicity, revealed that while di(2-ethylhexyl)phthalate had no apparent effects, the other two chemicals inhibited A. cepa root growth in a dose-dependent manner. On the other hand, although all three compounds caused alterations in the mitotic index of root-tip cells, propylparaben was the only one that did not show evidence of genotoxicity in assays for chromosome aberrations and micronuclei. The results of the present study clearly indicate that sensitive plant bioassays are useful and complementary tools to determine environmental impact of contaminants of emerging concern.

An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines.

The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the existing legislation and the approval of the new European law regarding the production and importation of chemicals, known as REACH. This new legal framework supports the development of alternative methods to animal experimentation encouraging the improvement and/or design of new methodological strategies for the toxicological evaluation of chemical compounds. Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best agreement between obtaining relevant information about the mechanism of toxic action and the use of alternative methods. Following this trend, this work presents an integrated cellular strategy in order to know the toxicity and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The experimental procedures are performed in two steps. The first one involves the systematic analysis of the main cellular targets using proliferation, viability and morphological probes. The second step relies upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic action and the cellular response. The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.

In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.

Carbamazepine induces mitotic arrest in mammalian Vero cells.

We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells.

Cytotoxicity of butylated hydroxyanisole in Vero cells.

Butylated hydroxyanisole (BHA) is perhaps the most extensively used synthetic antioxidant in the food and cosmetic industry, although considerable controversy exists in the literature regarding the safety of this compound. Most in vitro studies describing the effects of BHA have been performed in cancer cells, but it is unclear whether normal cells are equally susceptible to BHA exposure. The present study investigate the toxic potential of BHA in mammalian cells, using biochemical and morphological parameters, which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth inhibition was assessed by using colorimetric assays, whereas cellular alterations after BHA exposure, were evaluated using conventional light and fluorescence microscopy. Low doses of BHA exerted a significant cytotoxic effect, associated with loss of mitochondrial function. As the concentration of BHA was increased, morphological alterations in critical subcellular targets such as lysosomes, mitochondria and actin cytoskeleton, were observed. In parallel, BHA induced an irreversible loss of cell proliferative capacity, preceding apoptosis induction. Thus, the dose-dependent activity of BHA on Vero cells appears to be cytotoxic as well as cytostatic. Our observations, although simplified with respect to the in vivo situations, allowed the assessment of the specific damage at the cellular level, and provide some clue about the effects of BHA in non-tumoral mammalian cells.

Cytotoxic effects in mammalian Vero cells exposed to pentachlorophenol.

The effects of pentachlorophenol have been studied on diverse biological systems both in vivo and in vitro, however the cellular basis of the pronounced cytotoxicity of this organochlorine compound is poorly understood. In this work, morphological and biochemical analyses were carried out to identify the primary targets of pentachlorophenol toxicity in mammalian cells. Our results show that pentachlorophenol is a very potent cytotoxic drug that displays an unusual and interesting mode of action in Vero cells. Although this compound is a powerful uncoupler of oxidative phosphorylation, we present the novel finding that lysosome destabilization is an early cytotoxic response that precedes the mitochondrial dysfunction. In addition, soon after exposure to moderate doses of pentachlorophenol, a significant number of cells initiate an apoptotic death process identified by the condensed and fragmented state of their nuclei. These results demonstrate that there are multiple potential targets of PCP-induced toxicity in mammalian cells, and the need to develop further experimental studies for the risk assessment of this environmental pollutant.

Ultrastructural changes induced in HeLa cells after phototoxic treatment with harmine.

In the present work, we have continued our studies on harmine phototoxicity in human tumour cells. The toxicity of harmine in the dark was analysed by a quantitative neutral red uptake assay, and subcellular sensitive targets following harmine photosensitization were de fi ned by electron microscopic analysis of HeLa cells. The results obtained indicated that this compound shows a clear dose-dependent cytotoxic effect in the dark. The combined treatment with suitable doses of harmine and UV radiation was very effective at an early stage, although maximal cell killing appeared 48 h after photodynamic activation. Ultrastructural examination of HeLa cells immediately after the photodynamic treatment revealed lysosomal destabilization and profound cytoplasmic vacuolization that evolved to cytolysis, which is typical of necrotic cell death. It is concluded that harmine could be a valuable photosensitizer whose biological applications merit further evaluation.

Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints.

The occurrence of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry. However, the ecotoxicological effects of pharmaceuticals have still not been researched adequately. Carbamazepine, an anticonvulsant commonly present in surface and groundwater, was studied, using six ecotoxicological model systems with eighteen endpoints evaluated at different exposure time periods. The battery included the immobilization of Daphnia magna, bioluminescence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction and root growth inhibition in the plant Allium cepa. Cell morphology, neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity and glucose-6-phosphate dehydrogenase activity were studied in the salmonid fish cell line RTG-2. The total protein content, LDH activity, neutral red uptake and MTT metabolization in Vero monkey kidney cells were also investigated. The most sensitive system to carbamazepine was the Vero cell line, followed by Chlorella vulgaris, Vibrio fischeri, Daphnia magna, Allium cepa, and RTG-2 cells. EC50 values from 19 microM in Vero cells at 72 h to more than 1200 microM in other systems, were obtained. Comparing the concentrations in water and the toxicity quantified in our assay systems, carbamazepine is not expected to produce acute toxic effects in the aquatic biota under these circumstances, but chronic and synergistic effects with other chemicals cannot be excluded.

An in vitro study on the kinetics, subcellular distribution and phototoxicity of harmine in human tumor cells.

The beta-carboline alkaloid harmine, which was found to possess interesting phototoxic properties in different biological systems, was investigated for photokilling of human tumor cells in vitro. Harmine was readily accumulated by HeLa cells, localized preferably in cytoplasm, being a non toxic compound when used at low concentrations. The photoactivation of harmine-loaded cells with UV radiation showed lysosomal damage, reflected by altered localization of the fluorescent probe acridine orange, as well as an evident cell killing which increase with time up to a maximal value 48 h after the photodynamic treatment.

Effects of flavophospholipol on resistance in fecal Escherichia coli and enterococci of fattening pigs.

A "plasmid-curing effect" of multiresistant Escherichia coli by flavophospholipol, an antibiotic used as an antimicrobial growth promoter (AMGP) in animal feeds, has been reported to occur in vitro and in vivo under experimental conditions. In this study, the effect of flavophospholipol under field conditions was studied. The prevalence and degree (proportion of resistant strains to the total numbers present per gram of feces) of resistance of indicator bacteria, E. coli and enterococci, was determined in fecal samples from three groups of pigs that were fed a commercial finisher feed without any AMGP. Group A was the negative control group without any AMGP, group B received the same feed with 9 mg of flavophospholipol/kg of feed (study group), and group C received the same feed with 15 mg of avoparcin/kg (positive control). Fecal samples from each pig were collected at the start and at the end of the study and assessed for the prevalence and degree of resistance against antibiotics commonly used either for therapy in pig medicine or as an AMGP. Before the start of the study, all pigs were colonized with multiresistant E. coli by mixing three resistant pig isolates through their feed after disturbance of the colonization resistance of the intestinal flora by a 3-day course of lincomycin and spectinomycin. At the end of the study, the overall prevalence and degree of resistance of E. coli in the fecal flora had increased significantly in groups A and C but remained at the same level as at the start of the study in group B. The prevalence of vancomycin resistance was 44 and 41% in groups A and B, respectively, but only very low numbers of vancomycin-resistant enterococci (VRE) per gram of feces were found. In the avoparcin-fed group, the prevalence was 72%, and in 57% of the samples, more than 50% of all enterococci present were vancomycin resistant. The prevalence of resistant Enterococcus faecalis increased only in the flavophospholipol-exposed group, from 23% before the start of the study to 43% at the end of the study. It was concluded that flavophospholipol effectively suppressed the augmentation and dissemination of multiresistant E. coli in the intestinal flora of fattening pigs. Avoparcin use strongly selected for VRE carriage and excretion. Therefore, as neither flavophospholipol nor any related molecule is used therapeutically, no cross-resistance with therapeutic antibiotics exists and no transmissible resistance has been shown; the major decrease in resistance in intestinal E. coli of flavophospholipol-fed animals seemed to outweigh the small increase in the risk of transfer of flavophospholipol-resistant E. faecalis from animals to humans via the food chain.

A test battery for the ecotoxicological evaluation of pentachlorophenol.

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective and quantitative test battery should be developed. The effects of pentachlorophenol were studied using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, bioluminiscence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction in the plant Allium cepa. The inhibition of cell proliferation and MTT reduction were investigated in Vero cells. Neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity were studied in the salmonid fish cell line RTG-2, derived from the gonad of rainbow trout. Pentachlorophenol was very toxic for all biota and cells. The system most sensitive to pentachlorophenol, was micronuclei induction in A. cepa, followed by D. magna immobilization, bioluminescence inhibition in V. fischeri bacteria at 60 min and cell proliferation inhibition of RTG-2 cells at 72 h. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity.

Fast synaptic fatigue in shibire mutants reveals a rapid requirement for dynamin in synaptic vesicle membrane trafficking.

The GTPase dynamin is involved in endocytosis in many cell types, as first revealed by temperature-sensitive paralytic mutations in the Drosophila dynamin gene, shibire (shi), which disrupt synaptic vesicle endocytosis and deplete synaptic terminals of vesicles. Here we report that shi synapses exhibit a fast synaptic fatigue phenotype within 20 ms of repetitive stimulation, which cannot be explained by vesicle depletion, as we confirmed by electron microscopy. These results suggest that, in addition to its well-characterized role in synaptic vesicle recycling, dynamin may be required for short-term maintenance of the readily releasable pool of synaptic vesicles.

Bacterial contamination of fetal fluids at the time of cesarean section in the cow.

Complications after cesarean section delivery in cattle are mainly the result of infections. The bacteria responsible for this infection can be of exogenous or endogenous origin. In this investigation endogenous contamination was studied. Fetal fluid samples of 23 cows were collected from the uterine cavity during cesarean section just after the removal of the calf, by means of a sterile disposable plastic syringe. The uterine flora was cultured, quantitated and presumptively identified by using selective and elective agarplates. Nineteen samples were positive after culture. Eleven samples contained obligate anaerobic bacteria. When the amniotic sac was broken before the obstetrical examination, the total number of bacteria was significantly higher. Vaginal exploration by the farmer had no significant influence on the number of bacteria encountered. Cesarean section is considered a clean contaminated procedure. One must always take into account that the fetal fluids are contaminated with the endogenous vaginal flora. This leads inevitably to contamination of the wound and the peritoneal cavity. Properly antimicrobial prophylaxis is certainly indicated.

Effects of calcisorb on fecal bile acids and fatty acids in human volunteers.

The intake of calcium (Ca) is negatively associated with colorectal cancer (crc) risk. The aim of this study was to investigate in a double-blind, placebo-controlled trial, the effects of the Ca-binder Calcisorb, which is given to kidney stone patients with hypercalciuria type I, on risk factors for crc risk, bile acids (BA), and long-chain fatty acids (LCFA) in fecal water. Results show that the concentration of BA and LCFA in fecal water did not change, although the urinary excretion of Ca and magnesium (Mg) and the concentration of Ca and magnesium in fecal water decreased. The daily excretion of BA and LCFA acids decreased significantly (p < 0.05) during the Calcisorb period. In conclusion, binding dietary Ca and Mg with Calcisorb from a diet with a relatively low amount of fat does not enhance the solubility of BA and LCFA in fecal water.

The sterilization of honey with cobalt 60 gamma radiation: a study of honey spiked with spores of Clostridium botulinum and Bacillus subtilis.

Unprocessed honey is a recognized wound-healing remedy. However, to make clinical use of honey acceptable, it should be sterile. To find the lowest dose of irradiation needed for sterilization, six batches of honey (a-f) were gamma irradiated with 6, 12, 18, 22 and 25 kGy Cobalt-60. After a dose of 25 kGy the antibacterial activity was not altered. Presumably glucose oxidase (EC 1.1.3.4), which produces hydrogen peroxide, is not easily damaged by irradiation. Amylase activity on the other hand was significantly reduced to 19%, 19%, 21%, 22%, 43% in batches a), b), c), d) and f) respectively, whereas no decrease was observed in batch e). All batches spiked with approximately 10(6) spores from Cl. botulinum or B. subtilis per 50 g honey proved to be sterile after irradiation with a dose of 25 kGy. Honey was also spiked with Cl. botulinum at up to 5000 spores per 50 g honey, which is the upper limit of natural contamination. The sterilizing dose in this case was 18 kGy.

Simple beta-carboline alkaloids as nucleic acids fluorochromes.

Treatment of cell smears (chicken blood, Trypanosoma cruzi epimastigotes, Ehrlich ascites tumor cells and mouse spleen) with simple beta-carboline alkaloids induced a strong bluish white fluorescence emission of condensed chromatin and basophilic cytoplasm under ultraviolet excitation. The compounds used were harmine, harmol, harman, norharman, harmalol and harmaline (25-50 micrograms/ml aqueous solutions for 2-3 min). Spectrofluorometric studies on harmine solutions in vitro (emission peak at 420 nm) showed fluorescence quenching at high concentrations as well as in the presence of DNA. Microscopic fluorescence features of these new fluorochromes and possible binding modes to nucleic acids are discussed.

S-adenosyl-L-homocysteine: a non-cytotoxic hypomethylating agent.

The cytotoxic effect caused by the hypomethylating agent S-adenosyl-L- homocysteine (SAH) was compared with that of two drugs commonly used to induce DNA hypomethylation, 5-azacytidine and 5-aza-2'-deoxycytidine. Two in vitro cytotoxicity tests, the tetrazolium MTT assay and the intracellular lactate dehydrogenase (LDH) activity test, suggest that SAH induces hypomethylation without causing any cytotoxic effect. We propose the use of SAH as a non-cytotoxic agent which may be more suitable for inducing experimental DNA hypomethylation.

Effects of a calcium binder on the solubility of bile acids and fatty acids in the large intestine of the rat.

Kidney stone patients with hypercalciuria type I are treated with an oral calcium binder. Lower intakes of calcium (Ca) in the range of 0-1500 mg/day have been associated with an increased incidence of colorectal cancer. The aim of this study is to analyze the effects of feeding ethylene diamine tetraacetic acid sodium salt (EDTA), a strong, non-absorbable binder of Ca, on the solubility of bile acids (BA) and long chain fatty acids (LCFA) in the large intestine of the rat. We have shown that the concentrations of soluble BA and LCFA in the large intestine contents remained constant while the concentration of total BA and LCFA decreased. Therefore, lowering the amount of Ca available for binding BA or LCFA is unlikely to increase the risk of colorectal cancer by that method.