Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic Aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment System varying particle number concentration independent of particle chemistry, and an aerosol Deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully differentiated HBE is most appropriate in future toxicity studies.

Pharmacokinetics and pharmacodynamics of γ-hydroxybutyrate in healthy subjects.

AIMS γ-Hydroxybutyrate (GHB) is used as a treatment for narcolepsy and alcohol withdrawal and as recreational substance. Nevertheless, there are limited data on the pharmacokinetics and pharmacokinetic-pharmacodynamic relationship of GHB in humans. We characterized the pharmacokinetic profile and exposure-psychotropic effect relationship of GHB in humans. METHODS Two oral doses of GHB (25 and 35 mg/kg) were administered to 32 healthy male subjects (16 for each dose) using a randomized, placebo-controlled, cross-over design. RESULTS Maximal concentrations of GHB were (geometric mean and 95%CI): 218 (176-270) nmol/ml and 453 (374-549) nmol/ml for the 25 and 35 mg/kg GHB doses, respectively. The elimination half-lives (mean ± SD) were 36 ± 9 and 39 ± 7 min and the AUC∞ values (geometric mean and 95%CI) were 15,747 (12,854-19,290) and 40,113 (33,093-48,622) nmol∙min/ml for the 20 and 35 mg/kg GHB doses, respectively. Thus, plasma GHB exposure (AUC0-∞ ) rose disproportionally (+40%) with the higher dose. γ-Hydroxybutyrate produced mixed stimulant-sedative effects, with a dose-dependent increase in sedation and dizziness. It did not alter heart rate or blood pressure. A close relationship between plasma GHB exposure and its psychotropic effects was found, with higher GHB concentrations associated with higher subjective stimulation, sedation, and dizziness. No clockwise hysteresis was observed in the GHB concentration effect plot over time (i.e., no acute pharmacological tolerance). CONCLUSION Evidence was found of a non-linear dose-exposure relationship (i.e., no dose proportionality) at moderate doses of GHB. The effects of GHB on consciousness were closely linked to its plasma exposure and exhibited no acute tolerance. This article is protected by copyright. All rights reserved.

Neuropeptide Y restores non-receptor-mediated vasoconstrictive action in superior mesenteric arteries in portal hypertension

BACKGROUND & AIMS Vascular hyporeactivity to vasoconstrictors contributes to splanchnic arterial vasodilatation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY), a sympathetic cotransmitter, has been shown to improve adrenergic vascular contractility in portal hypertensive rats and markedly attenuate hyperdynamic circulation. To further characterize the NPY-effects in portal hypertension, we investigated its role for non-receptor-mediated vasoconstriction in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham-operated rats. METHODS Ex vivo SMA perfusion of PVL and sham rats was used to analyse the effects of NPY on pressure response to non-receptor-mediated vasoconstriction. Dose-response curves to KCl (30-300 mM) were used to bypass G protein-coupled receptor mechanisms. Potential involvement of the cyclooxygenase-pathway was tested by non-selective cyclooxygenase-inhibition using indomethacin. RESULTS KCl-induced vascular contractility but not vascular sensitivity was significantly attenuated in PVL rats as compared with sham rats. Administration of NPY resulted in an augmentation of KCl-evoked vascular sensitivity being not different between study groups. However, KCl-induced vascular contractility was markedly more enhanced in PVL rats, thus, vascular response was no more significantly different between PVL and sham rats after addition of NPY. Administration of indomethacin abolished the NPY-induced enhancement of vasoconstriction. CONCLUSIONS Receptor-independent vascular contractility is impaired in mesenteric arteries in portal hypertension. NPY improves non-receptor mediated mesenteric vasoconstriction more effective in portal hypertension than in healthy conditions correcting splanchnic vascular hyporesponsiveness. This beneficial vasoactive action of NPY adds to its well known more pronounced effects on adrenergic vasoconstriction in portal hypertension making it a promising therapeutic agent in portal hypertension.

Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes.

Artemisinin is an antimalarial sesquiterpene lactone that contains a 1,2,4-trioxane heterocycle. Dihydroartemisinin and artesunate demonstrated activity against Echinococcus multilocularis metacestodes in vitro but were not effective in a mouse model. In this study, the in vitro effects of a small library of synthetic ozonides (1,2,4-trioxolanes) were investigated. Initial compound screening against E. multilocularis metacestodes was performed at 20μM, and selected ozonides were further assessed in dose-response studies in metacestode cultures and mammalian cells. Transmission electron microscopy (TEM) was employed to characterise compound-induced structural alterations. At 20μM, the most potent ozonides (OZ401, OZ455, OZ491 and OZ494) led to death of ca. 60-100% of the parasites. Subsequent dose-response experiments demonstrated that OZ401, OZ455 and OZ491, which contain an aminopropylether substructure, were the most potent, with 50% inhibitory concentrations ranging from 11μM to 14μM. Cytotoxicity for these three ozonides, assessed in human foreskin fibroblasts, rat hepatoma cells and green monkey epithelial kidney (Vero) cells, was evident only at high concentrations. TEM demonstrated that OZ401 and OZ491 treatment induced considerable metabolic impairment in metacestodes at 1 day post exposure. At Day 3 post exposure, the germinal layer was severely distorted, although some intact cells were still visible, demonstrating that not all cell types in the parasite tissue were equally affected. Complete destruction of the germinal layer was noted at 5 days post exposure. Synthetic ozonides could represent interesting leads that will be further investigated in a suitable in vivo model of E. multilocularis infection.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response

BACKGROUND Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Active intravenous drug use during chronic hepatitis C therapy does not reduce sustained virological response rates in adherent patients

Reluctance has been expressed about treating chronic hepatitis C in active intravenous (IV) drug users (IDUs), and this is found in both international guidelines and routine clinical practice. However, the medical literature provides no evidence for an unequivocal treatment deferral of this risk group. We retrospectively analyzed the direct effect of IV drug use on treatment outcome in 500 chronic hepatitis C patients enrolled in the Swiss Hepatitis C Cohort Study. Patients were eligible for the study if they had their serum hepatitis C virus (HCV) RNA tested 6 months after the end of treatment and at least one visit during the antiviral therapy, documenting the drug use status. Five hundred patients fulfilled the inclusion criteria (199 were IDU and 301 controls). A minimum exposure to 80% of the scheduled cumulative dose of antivirals was reached in 66.0% of IDU and 60.5% of controls (P = NS). The overall sustained virological response (SVR) rate was 63.6%. Active IDU reached a SVR of 69.3%, statistically not significantly different from controls (59.8%). A multivariate analysis for treatment success showed no significant negative influence of active IV drug use. In conclusion, our study shows no relevant direct influence of IV drugs on the efficacy of anti-HCV therapy among adherent patients.