Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis

Characterization studies of the wistar rat hepatic cytosolic methlytrienolone (R1881) binding protein

Age and sex differences in adult rat hepatic microsomal drug metabolism are well-known. Although the exact mechanism is unclear, the role of testosterone has been clearly implicated. Since androgen sensitive sex end-organ tissues are known to contain specific androgen receptors which mediate the action of testosterone in these tissues, a similar mechanism may be involved in testosterone sensitive hepatic microsomal drug metabolism. To test this hypothesis, testosterone sensitive liver microsomal benzo(a)pyrene hydroxylase activities and cytosolic androgen binding protein levels were measured in vitro, using different animal models. Methyltrienolone (R1881) was used as the ligand for the cytosolic androgen binding assay, since testosterone was significantly metabolized in vitro. Scatchard analysis of adult male rat liver whole cytosol indicated two R1881 binding isotherms: a high affinity-low capacity binding site, and a lower affinity-higher capacity component. Addition of excess triamcinolone or Cortisol to the assay mixture eliminated the lower affinity binding component. The high affinity component was not present in the immature female or the intact or the 10-day ovariectomized adult female rat; it was present in low quantities in the immature male. Inhibition studies of the high affinity component using different competitors indicated a high specificity to androgens, including testosterone, dihydrotestosterone, androstenedione, R1881 and mibolerone, a moderate specificity to cyproterone acetate, estradiol and flutamide hydroxide, and no specificity to progesterone, triamcinolone, diethylstilbestrol and Cortisol. Saturation analysis of the high affinity component indicated that testosterone, dihydrotestosterone, androstenedione, mibolerone, cyproterone acetate and estradiol produced similar binding kinetics, indicating that these steroids were binding to the same or a similar high affinity site. Flutamide hydroxide yielded displaceable but non-saturable binding. Castration of adult male rats for 18 hrs, 4 or 10 days, or hypophysectomy for 10-17 days did not have a significant effect on the high affinity binding protein kinetics compared to controls. Nor did testosterone administration to these animals alter this protein's binding kinetics. Flutamide or flutamide hydroxide treatment to testosterone-treated adult male rats significantly reduced both liver microsomal benzo(a)pyrene hydroxylase activities and high affinity binding protein levels compared to the flutamide untreated controls. Streptozotocin treatment to adult males caused a marked decrease in the high affinity binding protein levels. These results indicate that the presence of the high affinity component in the adult male rat is not dependent upon gonadal or pituitary hormones in vivo, but it is altered following flutamide, flutamide hydroxide or streptozotocin administration. These studies do not negate the hypothesis that the presence as well asf the agonist occupancy of the high affinity-low capacity androgen binding protein are prerequisites for testosterone-sensitive effects in the liver.Pharmaceutical Sciences, Faculty ofGraduat

Environment Analysis of Contaminated Sites

xxiii,465 hal,;ill,;21 c

In vitro cytotoxicity and genotoxicity studies of titanium dioxide (TiO2) nanoparticles in Chinese hamster lung fibroblast cells

There are increasing safety concerns about the development and abundant use of nanoparticles. The unique physical and chemical characteristics of titanium dioxide (TiO2) nanoparticles result in different chemical and biological activities compared to their larger micron-sized counterparts, and can subsequently play an important role in influencing toxicity. Therefore, our objective was to investigate the cytotoxicity and genotoxicity of commercially available TiO2 nanoparticles with respect to their selected physicochemical properties, as well as the role of surface coating of these nanoparticles. While all types of tested TiO2 samples decrease cell viability in a mass-based concentration- and size-dependent manner, the polyacrylate-coated nano-TiO2 product was only cytotoxic at higher concentrations. A similar pattern of response was observed for induction of apoptosis/necrosis, and no DNA damage was detected in the polyacrylate-coated nano-TiO2 model. Given the increasing production of TiO2 nanoparticles, toxicological studies should take into account the physiochemical properties of these nanoparticles that may help researchers to develop new nanoparticles with minimum toxicity.Peer reviewed: YesNRC publication: Ye

Growth and reproduction of the earthworm Eisenia fetida after exposure to leachate from wood preservatives

Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixed with artificial rainwater, to generate leachates containing As, Cr and Cu. Then, leachates were applied to two soils at rates of 13\ufffd169 mg As kg-1 soil (dry weight basis), 12\ufffd151 mg Cr kg-1 and 10\ufffd216 mg Cu kg-1. Metal bioavailability was evaluated after 28 days using the earthworm Eisenia fetida (Savigny). Metal concentrations in earthworm tissue ranged from negligible to 80 mg As kg-1 (dry weight basis), 89 mg Cr kg-1 and 90 mg Cu kg-1, which appeared to be non-lethal to E. fetida. There was less Cu available to earthworms in the Courval soil (pH 7.8) than the Ch\ufffdteauguay soil (pH 6.8), but earthworm previous termgrowth and reproductionnext term were not affected by exposure to Cu from ACQ-treated wood. In contrast, earthworms exposed to As, Cr and Cu from the CCA-treated wood gained weight more quickly in the Courval soil (1.3\ufffd21 mg g-1 initial biomass days) than in the Ch\ufffdteauguay soil (0.2\ufffd7.8 mg g-1 day-1), but fewer than 20% of the cocoons deposited by the faster-growing earthworms hatched by the end of the 56 days ecotoxicology test. It appeared that E. fetida can allocate more energy to previous termgrowth than reproduction,next term delaying cocoon development and hatching in some situations. Further information is needed on the soil factors that may induce such behavior, as it can affect the interpretation of results from the earthworm ecotoxicology test.NRC publication: Ye

Biotransformation of 2,4,6-trinitrotoluene (TNT) by Enchytraeids (Enchytraeus albidus) in vivo and in vitro

2,4,6-Trinitrotoluene (TNT) is toxic to soil invertebrates, but little is known about its toxicokinetic behavior in soil. Tissue residue analysis was used to evaluate whether the presence of TNT and its reduced metabolites in soil invertebrates was due to uptake of these compounds from the soil into the organism, or due to microbial transformation of TNT associated with the organism followed by uptake. Adult white potworms (Enchytraeus albidus) were exposed to non-lethal concentrations of TNT in amended artificial soil for 21 d, or to TNT in solution for 20 h. Soil exposure studies confirmed earlier reports that TNT was transformed in enchytraeids in vivo to 2- and 4-aminodinitrotoluenes. However, enchytraeid exposure to TNT in solution led to the additional presence of 2,4-diaminonitrotoluene as well as 2- and 4- hydroxyamino-dinitrotoluenes and azoxy-compounds, suggesting that TNT can be metabolized in vivo in the absence of soil. Incubation of unexposed enchytraeid homogenates with TNT led to a protein-dependent appearance of these metabolites in vitro after gtoreq 16 h incubation. Cellular fractionation studies indicated that most of this activity resided in the 8000 x g pellet, and was completely inhibited by broad-spectrum antibiotics. These studies demonstrate that enchytraeids can transform TNT in vivo and in vitro, at least in part, by bacteria associated with the host organism. Crown Copyright Copyright 2004 Published by Elsevier Ltd. All rights reserved.NRC publication: Ye

Rapid isocratic HPLC quantification of metallothionein-like proteins as biomarkers for cadmium exposure in the earthworm Eisenia andrei

Metallothioneins (MTs) are regarded as sensitive biomarkers of cadmium (Cd) exposure in a number of organisms. An isocratic high-performance liquid chromatography (HPLC) method using the fluorophore ammonium-7-fluorobenz-2-oxa-1,3-diazole-4-sulfonate (SBD-F) and UV detection was developed for the quantification of MT-like proteins (MTLPs) in earthworm. This method was developed using a rabbit MT (MT-1) standard, and optimized concentrations of reagents including EDTA, SBD-F, and tris(2-carboxyethyl)phosphine (TCEP). The fluoro-tagged MT-1 (SBD-MT) and MTLP (SBD-MTLP) co-eluted at 7.2\ufffd7.3 min retention time on HPLC chromatograms. The optimized method was applied to quantify MTLPs in earthworms exposed to increasing concentrations of Cd (0, 2.7, 26.7, and 267.5 ?g g-1 of wet artificial soil) for 1\ufffd4 weeks. The amounts of MTLPs increased significantly in a dose- and time-dependent manner except in controls and in earthworms exposed to the lowest concentration of Cd. Since earthworm MTLPs are temporal- and dose-responsive to Cd exposure, they can be used as biomarkers to assess the impact of Cd contamination in soils. The method appeared to be rapid, sensitive, and suitable for direct quantification of total earthworm MTs.NRC publication: Ye

Developmental toxicity of glyceryl trinitrate in quail embryos

BACKGROUND: Although glyceryl trinitrate (GTN) is used extensively to treat angina and heart failure, little is known about its effects on the conceptus during organogenesis. The goal of these studies was to investigate the effects of GTN in a model organism, the quail (Coturnix coturnix japonica) embryo. METHODS: To identify the effects of GTN on quail embryo development, fertilized quail eggs (n = 10-12 eggs/group) were injected with GTN (0, 4.4, 44, or 440 ?M) at Hamburger-Hamilton (HH) stage 0, 9, or 19 and examined 7 days later. Next, HH 9 embryos were injected with GTN (0, 0.88, 4.4, 8.8, 44, 88, and 440 ?M, in 20 ?L per egg) and examined 24-hours, 48-hours, or 72-hours postinjection. Finally, the developing eye on one side was exposed to GTN (44 ?M) ex ovo and the tissue was probed for the presence of nitrated proteins. RESULTS: In ovo GTN exposure induced a dose-dependent increase in the number of malformed viable quail embryos with a maximal effect in HH 9 embryos. Microphthalmia, craniofacial, heart, and neural tube defects were elevated in GTN-exposed embryos. An increase in nitrated proteins was observed in the developing eye region of embryos exposed ex ovo to GTN. CONCLUSIONS: GTN treatment induced a variety of malformations in quail embryos. The presence of nitrated proteins suggests that organic nitrates, such as GTN, generate reactive nitrogen species. We hypothesize that GTN perturbations in the redox status of the embryo may underlie its developmental toxicity.Peer reviewed: YesNRC publication: Ye

Genotoxicity of 2,4- and 2,6-dinitrotoluene as measured by the Tradescantia micronucleus (Trad-MCB) bioassay

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6 h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200 mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180 mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20 mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135 mg/l, respectively. Longer exposure (30 h) without recovery time at 150 mg/l of 2,4-DNT and 180 mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.NRC publication: Ye

Rhizotoxicity of cadmium and copper in soil extracts (effects--plants, soil, invertebrates, microflora)

Dissolved organic matter (DOM) influences metal speciation in soil solutions and, hence, metal toxicity. Root-elongation experiments were conducted to examine the effect of soil solution components, such as Ca, H, and DOM, on metal rhizotoxicity. A biotic ligand model (BLM) was tested for its ability to predict the rhizotoxicity of Cd and Cu in soil extracts. It was hypothesized that the concentration of Cd and Cu bound to functional groups at the root surface estimated using a BLM would be a better predictor of rhizotoxicity than the free-metal ion activity in solution. Both metals became less toxic at higher DOM, Ca, and H concentrations. Solution speciation and the effect on root growth explained most of the variability observed in the DOM experiments, but not in the cation experiments. It was concluded that Ca and H inhibited the rhizotoxicity of both metals tested. Rhizotoxicity data correlated better with estimates of metal-root complexes that have been estimated with a BLM than with free-metal ion activity or with total metal concentrations. The BLM seems to be a promising approach for predicting metal availability in soils and for assessing the associated risk.NRC publication: Ye