Ilaria Palmi

Cell‐Mediated Immune Response in MDMA Users After Repeated Dose Administration

Abstract: Acute administration of 3,4‐methylenedioxymethamphetamine (MDMA, “ecstasy”) produces time‐dependent immune dysfunction in humans. Recreational use of MDMA generally includes repeated drug consumption, often in association with other drugs, such as alcohol and cannabis. In the laboratory setting, repeated MDMA administration to healthy MDMA consumers produced a time‐dependent immune dysfunction similar to that observed with the ingestion of a single dose, and the first of the two administrations paralleled the time‐course of MDMA‐induced cortisol stimulation kinetics and MDMA plasma concentrations. A significant decrease in CD4 T‐helper cells with simultaneous increase in natural killer (NK) cell and a decrease in functional responsiveness of lymphocytes to mitogenic stimulation was observed. Response to the second dose was either long‐lasting compared with the first dose or disproportionate and did not show any parallelism with cortisol and MDMA plasma concentrations. This circumstance extended the critical period during which immunocompetence is highly impaired as a result of MDMA use. Accumulation of MDMA in the body of a poor metabolizer induced higher immunomodulatory effects with statistically significant differences in NK cell function compared with extensive metabolizers. When basal values of lymphocyte subsets were examined in a population of recreational MDMA users participating in different clinical trials, alterations in several immunological parameters were observed. The absolute number of lymphocytes, in particular T lymphocytes and CD4 T‐helper cell subsets, showed a trend toward reduced values, although cell counts were within normal limits. By contrast, NK cells in MDMA consumers were reduced to one‐third of those from healthy persons. A statistically significant decrease in affected immune parameters was recorded during a 2‐year observation period in a subgroup of recreational MDMA users. These permanent alterations in immunologic homeostasis may result in impairment of general health and subsequent increased susceptibility to infection and immune‐related disorders.

Quantification of Delta9-tetrahydrocannabinol and its major metabolites in meconium by gas chromatographic-mass spectrometric assay: assay validation and preliminary results of the "meconium project".

A rapid and simple procedure based on gas chromatography-mass spectrometry (GC-MS) is described for determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in meconium using Δ8-tetrahydrocannabinol (Δ8-THC) and deuterated THC-COOH as internal standards. The biological matrix was subjected to liquid-liquid extraction after enzyme hydrolysis for conjugated analytes. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The method was validated in the range 20 to 500 μg/g using 1g of meconium per assay. The method was applied to the analysis of meconium in a cohort of newborns to assess eventual fetal exposure to cannabis. Within positive samples, THC-COOH and THC-OH (range: 33.7 to 182.1 and 20.7 to 493.3 μg/g, respectively) were both present in the majority of cases with only 1 specimen with THC-OH as the most abundant metabolite and 2 with THC only.

A survey of Italian and Spanish neonatologists and paediatricians regarding awareness of the diagnosis of FAS and FASD and maternal ethanol use during pregnancy

BackgroundEthanol is the most widely used drug in the world and a human teratogen whose consumption among women of childbearing age has been steadily increasing. There are no Italian or Spanish statistics on ethanol consumption during pregnancy nor any information regarding prevalence of fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). There is also a reasonable suspicion that these two diseases are underdiagnosed by professionals from the above-reported countries. The objectives of this study were: 1) to evaluate the experience, knowledge and confidence of Italian and Spanish neonatologists and paediatricians with respect to the diagnosis of FAS and FASD, and 2) to evaluate professionals awareness of maternal drinking patterns during pregnancy.MethodsA multiple-choice anonymous questionnaire was e-mailed to Italian neonatologists registered in the mailing list of the corresponding Society and administered to Italian and Spanish paediatricians during their National Congress.ResultsThe response rate was 16% (63/400) for the Italian neonatologists of the National Society while a total of 152 Spanish and 41 Italian paediatricians agreed to complete the questionnaire during National Congress. Over 90% of the surveyed physicians declared that FAS is an identifiable syndrome and over 60% of them identified at least one of the most important features of FAS. Although over 60% Italian responders and around 80% Spanish responders were aware that ethanol use in pregnancy is dangerous, approximately 50% Italian responders and 40% Spanish ones allowed women to drink sometimes a glass of wine or beer during pregnancy.Neonatologists and paediatricians rated confidence in the ability to diagnosis FAS and FASD as low, with over 50% responders feeling they needed more information regarding FAS and FASD identification in newborn and child.ConclusionsItalian and Spanish neonatologists and paediatricians do not feel confident about diagnosing FAS and FASD. More training is needed in order to accurately diagnose ethanol use during pregnancy and correctly inform pregnant women on the consequences on the newborn.

Immunoassays for the measurement of IGF-II, IGFBP-2 and -3, and ICTP as indirect biomarkers of recombinant human growth hormone misuse in sport Values in selected population of athletes

Insulin-like growth factor-II (IGF-II), insulin-like growth factor binding proteins (IGFBPs) -2 and -3 and C-terminal telopeptide of type I collagen (ICTP) have been proposed, among others, as indirect biomarkers of the recombinant human growth hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays for biomarkers detection was performed. ELISA assays for total IGF-II, IGFBP-2 and IGFBP-3 (IGF-II/ELISA1: DSLabs, IGFBP-2/ELISA2: Biosource, and IGFBP-3/ELISA3: BioSource) and an EIA assay for ICTP (ICTP/EIA: Orion Diagnostica) were evaluated. The inter- and intra-laboratory precision values were acceptable for all evaluated assays (maximum imprecision of 30% and 66% were found only for the lowest quality control samples of IGF-II and IGFBP-3). Correct accuracy was obtained for all inter-laboratory immunoassays and for IGFBP-2 intra-laboratory immunoassay. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, 11% and 15% of the samples felt below the estimated LOQ for IGF-II and ICTP, respectively, in the zone where lower precision was obtained. Although the majority of evaluated assays showed an overall reliability not always suitable for antidoping control analysis, relatively high concordances between laboratory results were obtained for all assays. Evaluated immunoassays were used to measure serum concentrations of IGF-II, IGFBP-2 and -3 and ICTP in elite athletes of various sport disciplines at different moments of the training season; in recreational athletes at baseline conditions and finally in sedentary individuals. Serum IGF-II was statistically higher both in recreational and elite athletes compared to sedentary individuals. Elite athletes showed lower IGFBP-2 and higher IGFBP-3 concentration with respect to recreational athletes and sedentary people. Among elite athletes, serum IGFBP-3 (synchronized swimming), and ICTP (rhythmic gymnastics) concentrations were sport-dependent. Over the training season, within athlete variability was observed for IGFBP-2 in case of taekwondo and IGFBP-2 and -3 in case of weightlifting. Variations due to those aspects should be taken in careful consideration in the hypothesis of setting reference concentration ranges for doping detection.

Evaluation of immunoassays for the measurement of insulin and C-peptide as indirect biomarkers of insulin misuse in sport: values in selected population of athletes.

Insulin and C-peptide have been proposed as possible biomarkers of human insulin hormone misuse in sport. An extended intra- and inter-laboratory validation of commercially available immunoassays was performed. Enzyme Amplified Sensitivity Immunoassay (EASIA) assays (Human Insulin-EASIA and C-peptide EASIA kits from BioSource) were evaluated for insulin and C-peptide in serum. The intra- and inter-laboratory precision and accuracy values were good for the evaluated assays with maximum imprecision and inaccuracy of 16% and 23%, respectively, obtained just for one day C-peptide assay evaluation. The range of concentrations found in serum samples under investigation was always covered by the calibration curves of the studied immunoassays. However, a 19.7% of the samples felt below the estimated insulin limit of quantification. High concordance between laboratory results was obtained for insulin assay (intraclass correlation coefficient -ICC=0.857), whereas that for C-peptide was lower (ICC=0.539). Evaluated immunoassays were used to measure serum concentrations of insulin and C-peptide in elite athletes of various sport disciplines at different moment of training season, in recreational athletes at baseline conditions and finally in sedentary individuals. Serum insulin was statistically lower both in recreational and elite athletes when compared to sedentary individuals. Among elite athletes, the specific sport affected serum insulin (e.g., weightlifting) and C-peptide (e.g., triathlon) concentrations. Over the training season, a within athletes variability was observed for taekwondo, swimming and weightlifting athletes. Variations due to those aspects should be taken in careful consideration in the hypothesis of setting reference concentration ranges for doping detection.

DETERMINATION OF FAT-SOLUBLE NUTRIENTS IN SERUM BY LIQUID CHROMATOGRAPHY AND MULTIWAVELENGTH DETECTION

Preanalytical and analytical requirements to attain a precise and accurate determination of fat-soluble nutrients in serum by reversed-phase liquid chromatography and multiwavelength detection are described. Retinol, α-tocopherol, α and β-carotene and lycopene were extracted from serum samples with two volumes of hexane-ethanol (5 : 1 v/v). Analytical recovery of retinol and α-tocopherol ranged from 80–90%, while those of αand β-carotene and lycopene never exceed 50%. Chromatography was performed using a reversed-phase column with a mobile phase consisting of ethanol-acetonitrile 1 : 1 with 0.1 mL diethylamine per liter of solvent. Limits of quantitation were 5 ng/mL for retinol, 200 ng/mL for α-tocopherol, and 5 ng/mL for α, β-carotene, and lycopene, respectively. This method has been used in large-scale determinations of fat-soluble nutrients, never needing dilution of samples or being under limit of quantitation.

The NASOROSSO (Rednose) Project: An Italian Study on Alcohol Consumption in Recreational Places

The Nasorosso project of the Italian Youth Department and the National Institute of Health, aimed to raise awareness about drinking and driving under the influence of alcohol among club goers with a series of initiatives. Within the framework of the project, blood alcohol concentration (BAC) was measured on 106,406 individuals before and after clubbing in 66 different recreational sites from 11 Italian provinces, over 16 months. Participating individuals were interviewed regarding sociodemographic and environmental characteristics and alcohol intoxicated people were offered to be taken home. The BAC median at the club entry was 0.26 g/L with 65.3% subjects showing a BAC value under the driving legal limit of 0.5g/L. At the exit from clubs, BAC median value rose to 0.44 g/L and subjects with BAC value under the legal limit decreased to 54.9%. Being male, aged between 18 and 34 years with a diploma, being a drinker and entering the disco with a BAC already beyond the legal limit predicted a BAC value beyond 0.5 g/L at exit from the recreational place. Conversely, being a driver, being a student and exiting from the disco before 4 a.m. reduced the probability of having a BAC higher than 0.5 g/L at the end of the night. Health policies to prevent harmful use of alcohol in young people should continue to offer targeted information/ prevention; in order to steadily increase the awareness of the dangers and the damages of excessive use of alcohol.