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Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three-dimensional cell culture system provides optimal cell-to-cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT-PCR to measure the mRNA expression of cytokines (TNF-α, IL-1β, IL-6, IL-1ra, TGF-β, IL-15, IFN-γ), chemokines (ccl5, cxcl1, cxcl2), and i-NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases).

Accidental intoxication of the infant-juvenile population in households: profiles of emergency care

OBJECTIVE Analyzing profiles of intoxication and accidental poisoning of infant-juvenile population (0-24 years) in the household, treated at a reference facility for Emergency and Primary Care, during the year 2013. METHOD A descriptive, cross-sectional study. Data were analyzed using Epi-Info, by way of simple and bivariate analyzes. The project was approved by the Research Ethics Committee (protocol 405.578). RESULTS There were 45 intoxications, with a prevalence of males (60.0%), aged 1-4 years (71.1%). Among children under one, there was a higher frequency of pesticide poisoning (66.6%), between the ages of 1-4 by cleaning products (34.4%), and between 5-9 years of age by pharmacological substances (66.6%). The primary assistance was provided only at health institutions, with hospital admissions in 24.4% of the cases. CONCLUSION The importance of prevention through legislation is evident, in order to ensure greater safety in packaging of various products, and community awareness to eliminate risks in the household environment.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

A case of suicide by ingestion of caffeine.

Intentional or unintentional caffeine abuse due to excessive intake of beverages or energy drinks containing caffeine is relatively frequent. However, death due to caffeine intoxication is rare and case reports of fatalities from caffeine toxicity are relatively infrequent. In this report, we describe an autopsy case involving a 31 year-old man who intentionally took a large amount of caffeine tablets in the form of a weight loss supplement as part of a suicide plan. Caffeine femoral blood concentration (170 mg/l) was within the toxic and potentially lethal ranges reported in the literature in similar cases. Postmortem biochemistry results suggested depressed glomerular filtration rate and pre-renal failure at the time of death but failed to reveal myoglobinuria, glycosuria, ketonuria or ketonemia. Based on the absence of pathological findings at autopsy and the high blood caffeine level, death was attributed to acute caffeine toxicity. The case emphasizes the usefulness of performing exhaustive toxicology and searching for all potentially relevant information in order to formulate appropriate hypotheses concerning the cause and manner of death.

Need of reduced and harmonized bureaucracy in multi-centre clinical research - a case-report from a Swiss trial

Introduction: We launched an investigator-initiated study (ISRCTN31181395) to evaluate the potential benefit of pharmacokinetic-guided dosage individualization of imatinib for leukaemiapatients followed in public and private sectors. Following approval by the research ethics committee (REC) of the coordinating centre, recruitment throughout Switzerland necessitatedto submit the protocol to 11 cantonal RECs.Materials and Methods: We analysed requirements and evaluation procedures of the 12 RECs with associated costs.Results: 1-18 copies of the dossier, in total 4300 printed pages, were required (printing/posting costs: ~300 CHF) to meet initial requirements. Meeting frequencies of RECs ranged between 2 weeks and 2 months, time from submission to fi rst feedback took 2-75 days. Study approval was obtained from a chairman, a subor the full committee, the evaluation work being invoiced by0-1000 CHF (median: 750 CHF, total: 9200 CHF). While 5 RECs gave immediate approval, the other 6 rose in total 38 queries before study release, mainly related to wording in the patient information, leading to 7 different fi nal versions approved. Submission tasks employed an investigator half-time over about 6 months.Conclusion: While the necessity of clinical research evaluation by independent RECs is undisputed, there is a need of further harmonization and cooperation in evaluation procedures. Current administrative burden is indeed complex, time-consuming and costly. A harmonized electronic application form, preferably compatible with other regulatory bodies and European countries, could increase transparency, improve communication, and encourage academic multi-centre clinical research in Switzerland.

Mechanisms of proteasome inhibitor-induced cytotoxicity in malignant glioma.

The 26S proteasome constitutes an essential degradation apparatus involved in the consistent recycling of misfolded and damaged proteins inside cells. The aberrant activation of the proteasome has been widely observed in various types of cancers and implicated in the development and progression of carcinogenesis. In the era of targeted therapies, the clinical use of proteasome inhibitors necessitates a better understanding of the molecular mechanisms of cell death responsible for their cytotoxic action, which are reviewed here in the context of sensitization of malignant gliomas, a tumor type particularly refractory to conventional treatments.

Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to investigate the influence of glial cells on the neurotoxicity of two organophosphorus pesticides (OPs), chlorpyrifos and parathion. Mixed-cell aggregate cultures were treated continuously for 10 days between DIV 5 and 15. Parathion induced astrogliosis at concentration at which MAP-2 immunostaining, found here to be more sensitive than neuron-specific enzyme activities, was not affected. In contrast, chlorpyrifos induced a comparatively weak gliotic reaction, and only at concentrations at which neurons were already affected. After similar treatments, increased neurotoxicity of parathion and chlorpyrifos was found in aggregate cultures deprived of glial cells. These results suggest that glial cells provide neuroprotection against OPs toxicity. To address the question of the difference in toxicity between parathion and chlorpyrifos, the toxic effects of their leaving groups, p-nitrophenol and trichloropyridinol, were studied in mixed-cell aggregates. General cytotoxicity was more pronounced for trichloropyridinol and both compounds had similar toxic effects on neuron-specific enzyme activities. In contrast, trichloropyridinol induced a much stronger decrease in glutamine synthetase activity, the enzymatic marker of astrocytes. Trichloropyridinol may exert a toxic effect on astrocytes, compromising their neuroprotective function, thus exacerbating the neurotoxicity of chlorpyrifos. This is in line with the suggestion that glial cells may contribute to OPs neurotoxicity, and with the view that OPs may exert their neurotoxic effects through different mechanisms.

Early repolarization, acute emotional stress and sudden death.

We herein report the case of a 36-year-old man who died suddenly after a fight with another man. Forensic investigations included unenhanced computed tomography, postmortem angiography, autopsy, histology, neuropathology, toxicology, and biochemistry and allowed a traumatic cause of death to be excluded. An electrocardiogram recorded some years prior to death revealed the presence of an early repolarization pattern. Based on the results of all investigations, the cause of death was determined to be cardiac arrhythmia and cardiac arrest during an emotionally stressful event associated with physical assault. Direct third party involvement, however, was excluded, and the manner of death was listed as natural. The case was not pursued any further by the public prosecutor.

Simultaneous determination of antidementia drugs by HPLC-MS: validation data of the method and plasma level determinations in 300 patients

Aims: A rapid and simple HPLC-MS method was developed for the simultaneousdetermination of antidementia drugs, including donepezil, galantamine, rivastigmineand its major metabolite NAP 226 - 90, and memantine, for TherapeuticDrug Monitoring (TDM). In the elderly population treated with antidementiadrugs, the presence of several comorbidities, drug interactions resulting frompolypharmacy, and variations in drug metabolism and elimination, are possiblefactors leading to the observed high interindividual variability in plasma levels.Although evidence for the benefit of TDM for antidementia drugs still remains tobe demonstrated, an individually adapted dosage through TDM might contributeto minimize the risk of adverse reactions and to increase the probability of efficienttherapeutic response. Methods: A solid-phase extraction procedure with amixed-mode cation exchange sorbent was used to isolate the drugs from 0.5 mL ofplasma. The compounds were analyzed on a reverse-phase column with a gradientelution consisting of an ammonium acetate buffer at pH 9.3 and acetonitrile anddetected by mass spectrometry in the single ion monitoring mode. Isotope-labeledinternal standards were used for quantification where possible. The validatedmethod was used to measure the plasma levels of antidementia drugs in 300patients treated with these drugs. Results: The method was validated accordingto international standards of validation, including the assessment of the trueness(-8 - 11 %), the imprecision (repeatability: 1-5%, intermediate imprecision:2 - 9 %), selectivity and matrix effects variability (less than 6 %). Furthermore,short and long-term stability of the analytes in plasma was ascertained. Themethod proved to be robust in the calibrated ranges of 1 - 300 ng/mL for rivastigmineand memantine and 2 - 300 mg/mL for donepezil, galantamine and NAP226 - 90. We recently published a full description of the method (1). We found ahigh interindividual variability in plasma levels of these drugs in a study populationof 300 patients. The plasma level measurements, with some preliminaryclinical and pharmacogenetic results, will be presented. Conclusion: A simpleLC-MS method was developed for plasma level determination of antidementiadrugs which was successfully used in a clinical study with 300 patients.

Evaluation of the interactions between multiwalled carbon nanotubes and caco-2 cells

The aim of this study was to determine whether multiwalled carbon nanotubes (MWNCT) are taken up by and are toxic to human intestinal enterocytes using the Caco-2 cell model. Caco-2 cells were exposed to 50 ?g/ml MWCNT (oxidized or pristine) for 24 h, and experiments were repeated in the presence of 2.5 mg/L natural organic matter. Cells displayed many of the properties that characterize enterocytes, such as apical microvilli, basolateral basement membrane, and glycogen. The cell monolayers also displayed tight junctions and electrical resistance. Exposure to pristine and oxidized MWCNT, with or without natural organic matter, did not markedly affect viability, which was assessed by measuring activity of released lactate dehydrogenase (LDH) and staining with propidium iodide. Ultrastructural analysis revealed some damage to microvilli colocalized with the MWCNT; however, neither type of MWCNT was taken up by Caco-2 cells. In contrast, pristine and oxidized MWCNT were taken up by the macrophage RAW 264.7 line. Our study suggests that intestinal enterocytes cells do not take up MWCNT. [Authors]

Assessment of human skin penetration of two irritant herbicides

Two common herbicides; isoproturon and bentazon, are strong skin irritants and cross the skin barrier easily. Assessment of percutaneous absorption of these substances is a very important step in the evaluation of any dermal or transdermal dose, especially among agricultural workers who frequently have dermal exposures during crop treatment. The aims of the study were to determine the permeation rate of human skin for both herbicides in vitro, and histologically evaluate skin damage due to irritation at different concentrations. Skin penetration was assessed using a dynamic flow-through in vitro penetration system and analysis were performed with ion trap LC-MS (acidified water: acetronitile, C18 column). Two concentrations of bentazon (75 and 150 μg/mL) and isoproturon (125 and 250 μg/mL) in saline solution were applied on excised human skin from several donors. Saline water was used as receptor fluid. Collection times were: 4, 8, and 24 hours. After the experiments, the skin was removed and examined by histopathology for apoptosis, acanthosis, acantholysis and epidermolysis. The skin permeation rate, J, was calculated from the slope of the cumulative amount permeated as a function of time. The lag time, tL, was assigned from the time-axis intercept of the extrapolation of this linearity. Our results showed that tL for bentazon and isoproturon for both concentrations tested were similar; 2, 1.5 hours, respectively. Bentazon had a lowerer J compared to isoproturon; 350, 600 ng/cm2/h, respectively. Some acanthosis was observed after 8 hours of exposure to either of the two substances. In conclusion, our in vitro experiments demonstrate that bentazon and isoproturon cross the skin barrier within 2 hours even at very low concentrations, and showed some signs of skin damage. Future tests involve concentrations found in commercial products.

A new alternative method for testing skin irritation using a human skin model: a pilot study.

Studies assessing skin irritation to chemicals have traditionally used laboratory animals; however, such methods are questionable regarding their relevance for humans. New in vitro methods have been validated, such as the reconstructed human epidermis (RHE) model (Episkin®, Epiderm®). The comparison (accuracy) with in vivo results such as the 4-h human patch test (HPT) is 76% at best (Epiderm®). There is a need to develop an in vitro method that better simulates the anatomo-pathological changes encountered in vivo. To develop an in vitro method to determine skin irritation using human viable skin through histopathology, and compare the results of 4 tested substances to the main in vitro methods and in vivo animal method (Draize test). Human skin removed during surgery was dermatomed and mounted on an in vitro flow-through diffusion cell system. Ten chemicals with known non-irritant (heptylbutyrate, hexylsalicylate, butylmethacrylate, isoproturon, bentazon, DEHP and methylisothiazolinone (MI)) and irritant properties (folpet, 1-bromohexane and methylchloroisothiazolinone (MCI/MI)), a negative control (sodiumchloride) and a positive control (sodiumlaurylsulphate) were applied. The skin was exposed at least for 4h. Histopathology was performed to investigate irritation signs (spongiosis, necrosis, vacuolization). We obtained 100% accuracy with the HPT model; 75% with the RHE models and 50% with the Draize test for 4 tested substances. The coefficients of variation (CV) between our three test batches were <0.1, showing good reproducibility. Furthermore, we reported objectively histopathological irritation signs (irritation scale): strong (folpet), significant (1-bromohexane), slight (MCI/MI at 750/250ppm) and none (isoproturon, bentazon, DEHP and MI). This new in vitro test method presented effective results for the tested chemicals. It should be further validated using a greater number of substances; and tested in different laboratories in order to suitably evaluate reproducibility.

First nationwide study on driving under the influence of drugs in Switzerland.

In Switzerland, a two-tier system based on impairment by any psychoactive substances which affect the capacity to drive safely and zero tolerance for certain illicit drugs came into force on 1 January 2005. According to the new legislation, the offender is sanctioned if Delta(9)-tetrahydrocannabinol THC is >or=1.5ng/ml or amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), cocaine, free morphine are >or=15ng/ml in whole blood (confidence interval+/-30%). For all other psychoactive substances, impairment must be proven in applying the so-called "three pillars expertise". At the same time the legal blood alcohol concentration (BAC) limit for driving was lowered from 0.80 to 0.50g/kg. The purpose of this study was to analyze the prevalence of drugs in the first year after the introduction of the revision of the Swiss Traffic Law in the population of drivers suspected of driving under the influence of drugs (DUID). A database was developed to collect the data from all DUID cases submitted by the police or the Justice to the eight Swiss authorized laboratories between January and December 2005. Data collected were anonymous and included the age, gender, date and time of the event, the type of vehicle, the circumstances, the sampling time and the results of all the performed toxicological analyses. The focus was explicitly on DUID; cases of drivers who were suspected to be under the influence of ethanol only were not considered. The final study population included 4794 DUID offenders (4243 males, 543 females). The mean age of all drivers was 31+/-12 years (range 14-92 years). One or more psychoactive drugs were detected in 89% of all analyzed blood samples. In 11% (N=530) of the samples, neither alcohol nor drugs were present. The most frequently encountered drugs in whole blood were cannabinoids (48% of total number of cases), ethanol (35%), cocaine (25%), opiates (10%), amphetamines (7%), benzodiazepines (6%) and methadone (5%). Other medicinal drugs such as antidepressants and benzodiazepine-like were detected less frequently. Poly-drug use was prevalent but it may be underestimated because the laboratories do not always analyze all drugs in a blood sample. This first Swiss study points out that DUID is a serious problem on the roads in Switzerland. Further investigations will show if this situation has changed in the following years.