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The study reports the evolution of the demyelinization process based on cholesterol ([CHOL]) levels quantified in median nerve samples and collected at different times-from death from both right and left wrists. The statistical data show that the phenomenon evolves differently in the right and left nerves. Such a difference can reasonably be attributed to a different multicenter evolution of the demyelinization. For data analysis, the enrolled subjects were grouped by similar postmortem intervals (PMIs), considering 3 intervals: PMI < 48 hours, 48 hours < PMI < 78 hours, and PMI > 78 hours. Data obtained from tissue dissected within 48 hours of death allowed for a PMI estimation according to the following equations: PMI = 0.000 + 0.7623 [CHOL]right (R = 0.581) for the right wrist and PMI = 0.000 + 0.8911 [CHOL]left (R = 0.794) for the left wrist.At present, this correlation cannot be considered to be definitive because of the limitation of the small size of the samples analyzed, because the differences in the sampling time and the interindividual and intraindividual variation may influence the demyelinization process.

OBJECTIVES: The study aimed to give a first data set of bisphenol A (BPA) levels in the peritoneal fluid of patients suffering from endometriosis and to investigate the relationship between BPA exposure and endometriosis. METHODS: A questionnaire investigating the occupational context, life environment, and habits was administered to 68 patients suffering from endometriosis and 60 endometriosis-free subjects (control group). Urine and peritoneal fluids samples were collected and analysed by GC/MSMS for BPA dosage. RESULTS: Some of the investigated environmental/lifestyle risk factors (closeness to industries/activities at risk) were associated with an increase in endometriosis; smoking resulted as protective factor; others (use of food plastic boxes) did not seem to influence the onset of pathology. The association between the occupational exposure summarising all examined risk factors (working activity, personal protective equipment, seniority) and endometriosis was statistically significant (χ 2 = 5.252, p = 0.02). Contrasting results were obtained when specific activities were examined. Detectable urinary BPA levels were found in all analysed samples (patients: 1.17-12.68 pg/µl; mean ± SD, 5.31 ± 3.36 pg/µl; control group: 1.28-2.35 pg/µl; mean ± SD, 1.64 ± 0.49 pg/µl; median; 1.46 pg/µl), with a statistically significant difference between patients and controls, showing an association between BPA exposure and endometriosis. Only a few subjects from the control group supplied peritoneal fluid; hence, no comparison test with patients (range 0.39-1.46 pg/µl; mean ± SD, 0.67 ± 0.30 pg/µl; median, 0.58 pg/µl) was carried out. CONCLUSIONS: Results highlight the potential association between BPA exposure and endometriosis, as well as the current lack of knowledge regarding occupational exposure to BPA and the need of epidemiological studies focused on single activities/occupations, such as housewives, cleaners, students. Objectives: The study aimed to give a first data set of bisphenol A (BPA) levels in the peritoneal fluid of patients suffering from endometriosis and to investigate the relationship between BPA exposure and endometriosis. Methods: A questionnaire investigating the occupational context, life environment, and habits was administered to 68 patients suffering from endometriosis and 60 endometriosis-free subjects (control group). Urine and peritoneal fluids samples were collected and analysed by GC/MSMS for BPA dosage. Results: Some of the investigated environmental/lifestyle risk factors (closeness to industries/activities at risk) were associated with an increase in endometriosis; smoking resulted as protective factor; others (use of food plastic boxes) did not seem to influence the onset of pathology. The association between the occupational exposure summarising all examined risk factors (working activity, personal protective equipment, seniority) and endometriosis was statistically significant (χ2 = 5.252, p = 0.02). Contrasting results were obtained when specific activities were examined. Detectable urinary BPA levels were found in all analysed samples (patients: 1.17–12.68 pg/µl; mean ± SD, 5.31 ± 3.36 pg/µl; control group: 1.28–2.35 pg/µl; mean ± SD, 1.64 ± 0.49 pg/µl; median; 1.46 pg/µl), with a statistically significant difference between patients and controls, showing an association between BPA exposure and endometriosis. Only a few subjects from the control group supplied peritoneal fluid; hence, no comparison test with patients (range 0.39–1.46 pg/µl; mean ± SD, 0.67 ± 0.30 pg/µl; median, 0.58 pg/µl) was carried out. Conclusions: Results highlight the potential association between BPA exposure and endometriosis, as well as the current lack of knowledge regarding occupational exposure to BPA and the need of epidemiological studies focused on single activities/occupations, such as housewives, cleaners, students. © 2016, Springer-Verlag Berlin Heidelberg.

The presence of macrophages and their activation on the pulmonary tissues of 21 subjects deceased after CO intoxication has been studied. A notable number of activated macrophages, especially in the interstitial level, have been evidenced, and such phenomenon supports the hypothesis of a possible association between CO intoxication and pulmonary macrophages activity. The highlighted association could be mediated by changes of the surfactant, by impairing of mitochondrial respiration and by release of pro-inflammatory cytokines.

A long-term psychiatric 40 years-old male patient was found dead at 9:00 a.m. in the clinic where he lived. Death was caused by traumatic injuries, which the sanitary staff imputed to a fall. Nurses declared that the patient refused having breakfast, whereas at autopsy the stomach contained 350 g of whitish semifluid material. Using both shotgun and gel-based proteomics, we demonstrated that the chyme contained partly digested milk- and bread-derived proteins, eaten during a recent breakfast. The conflict between evidence and assertions of the attending sanitary staff prompted the Legal Authority to undertake detailed investigations to ascertain facts and possible responsibilities. The herein characterization provides insights in the in vivo mechanisms of gastric breakdown of food proteins in a real meal. β-lactoglobulin was partially resistant to gastric digestion as confirmed by Western blot analysis, in contrast to caseins and wheat gluten proteins, which had been degraded by gastric fluids. In addition to a complex pattern of gastric proteins (e.g., mucin-5AC, pepsin A-3, pepsinogen C, gastric lipase, gastrokine-2, trefoil factors), chyme contained intact proteins and variably sized food-derived polypeptides arising from peptic and nonpeptic proteolytic cleavage as well as heterodimeric disulfide-cross-linked peptides. These findings suggest that the current analytical workflows offer only a partial picture of the real complexity of the human "digestome".

Post-mortem morphological modifications in median nerve samples collected at different times after death were studied in subjects with known circumstances and times of death. Histological analyses were performed by light and electron microscopy on single fiber and on transversal sections. Results of morphological observations evidenced the progressive disappearance of myelin over time according to a process similar to Wallerian degeneration occurring in vivo. Electron microscope analysis of transversal sections evidenced four steps through which post-mortal myelin degradation proceeded, from the initial structure modification (after 12 h from death) to the complete collapse and the simultaneous appearance of dropsy bumps toward the axon (after 48 h from death). The post-mortal demyelinization process can reasonably be thought to have a multifocal origin. Post-mortem alterations of nerve fibers do not evolve simultaneously and in a uniform manner in all the fibers of the same nerve, where different factors come into play, particularly fiber gauge.

The measurement of blood concentration of immunosuppressive drugs is strongly recommended because of the narrow therapeutic range. An important aspect in the therapeutic monitoring of a drug is its possible degradation. This paper is aimed at investigating the stability of two widely-used immunosuppressants, sirolimus and everolimus. Short (storage at 30 degrees C for 3 or 7 days) and long term (storage at -20 degrees C for 0-90 days with a single freeze-thaw cycle) stability of sirolimus and everolimus in whole blood samples from kidney transplant patients were examined by using MEIA and FPIA. Sirolimus and everolimus samples stored at 30 degrees C in light for up to a week showed a decrement in concentration of 5.2 percent and 6.1 percent, respectively. Our findings on long term stability for both sirolimus and everolimus highlight the possibility of storing samples at -20 degrees C for up to 90 days, without the need to use lower storage temperatures. The results have important implications for patients living far from laboratories where drug concentration is measured or when the storing of blood samples is needed for pharmacokinetic studies.

Gender differences in substance use/abuse have been the focus of research in the last 15 years. Initiation, use patterns, acceleration of disease course, and help-seeking patterns are known to be influenced by gender differences with regard to biological, psychological, cultural and socioeconomic factors. This paper presents a systematic review of published data on gender differences in the use/abuse of psychoactive and psychotic drugs, focusing on the importance of a multidisciplinary approach. The basis for this paper was obtained by Medline searches using the search terms "human" and "gender", combined with individual drug names or "drugs of abuse". The reference lists of these papers were further checked for other relevant studies. The gender difference in drug abuse is more evident in adults than in adolescents (13-19 years): adult men are 2-3 times more likely than women to develop drug abuse/dependence disorders and approximately 4 times as likely to have an alcohol use disorder. Such prevalence rates have not been observed in adolescents. Differences between men and women involve: (i) the biological response to the drug, (ii) the progression to drug dependence, and (iii) the comorbid psychiatric diagnoses, which may be due to both sociocultural factors and innate biological differences. A crucial role played by ovarian hormones (oestrogens and progesterone) has been documented in both human and animal model studies. Epidemiological data on how particular psychobiological and physiological characteristics in females influence vulnerability to both drug addiction and toxicological consequences of drugs are still in their infancy. Significant gaps remain in our knowledge, which are primarily attributable to the lack of empirical data that only a systematic and multidisciplinary approach to the topic can generate. The introduction of gender into forensic toxicological evaluations may help elucidate the relationship between the body's absorption of abused drugs (alone or in combination) and the onset of intoxications, both lethal and none.

Cholesterol levels in the median nerve were studied at various post-mortem intervals (PMIs). Single median nerve samples were collected from the wrists of 36 subjects during forensic autopsies of subjects with known circumstances and times of death. Although the absolute values varied, increments in cholesterol concentration were recorded. Subsequently, 16 subjects who did not suffer of any neurological and/or metabolic diseases with known times and circumstances of death were enrolled. For each enrolled subject, two samples were collected from the wrist at an interval of approximately two hours (t1 and t2). The obtained results revealed a gradual increase in cholesterol level with increasing time since death. The cholesterol concentration data obtained for each subject at t1 and t2 were correlated with the time since death, a linear interpolation was applied, and the PMI was back-calculated. Similar trends were obtained for the samples collected at similar PMIs; thus, three groups were considered: PMI78h. Good correlation coefficients were obtained, especially for the first group (R(2)=0.9362) for which the PMI could be calculated with an error that ranged from -4 to 5.9h. Although it requires further confirmation via analyses of larger numbers of samples, the method proposed here can currently be applied to PMI determinations.

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

OBJECTIVES Occupational exposure to antineoplastic drugs can represent a potential health risk for hospital staff. Assessing exposure is the first step in providing a safe work environment; the present study aimed to perform a biological monitoring (BM) of nurses exposed to doxorubicin and epirubicin. In order to assure data accuracy and reproducibility, the high-performance liquid chromatography with fluorescence detection method was validated. METHODS Validation experiments were carried out according to the Food and Drug Administration guidelines. A detailed questionnaire about workplace practices and work organization was administered to 56 nurses of oncology department of two hospitals (A and B) located in southern Italy. End-shift urine samples were collected. Amounts of drugs handled were registered. RESULTS The quantification and detection limits were 1.1 and 0.6 pg microl(-1) (doxorubicin) and 2.0 and 1.2 pg microl(-1) (epirubicin); moreover, the analytical method fulfilled all guidelines requirements. Questionnaire information evidenced that vertical laminar flow hoods were present in both hospitals, surfaces were cleaned with inappropriate detergents, no antispilling devices were adopted, and gloves were not changed during the work shift. A lower percentage of positive samples was found in the hospital where higher amounts of anthracyclines were handled (3.4% in A and 14.8% in B), suggesting individual incorrect working/cleaning practices in hospital A and overall hygienic standards to be improved in hospital B, where 'critical practices' were carried out. CONCLUSIONS Results showed the crucial role of adopting effective safety precautions and handling practices to reduce exposure. Environmental and BM should be performed to discriminate between incorrect personal working modalities and general hygienic standards.

Bacterial infection is a frequent event in renal transplant recipients and often requires the use of antimicrobial agents. In this paper it is reported an evidence of pharmacokinetic interaction between clarithromycin and sirolimus in a kidney transplanted woman, suffering from pulmonary infection sustained by a bacterial pathogen, in particular Hemophilus Influenzae. In the present case report, the concomitant administration of clarithromycin and sirolimus determined impressive increase of sirolimus trough blood concentrations from 6.2 up to 54 ng/mL and this increase was associated with an acute impairment of renal function, almost completely reversed upon both drugs discontinuation. This drug-drug interaction is due to a likely inhibition of activity of both cytochrome P450 3A4 and P-glycoprotein. Although this interaction could be predicted, it represents the first reported clinical evidence.

Immunochemical assays represent a promising tool for quantification of immunosuppressants in organ transplanted patients, because they require small sample volumes and minimum sample pre-treatment; nevertheless considerations about method specificity, sensitivity and reproducibility cannot be overlooked. The present paper investigates the reliability of using the immunoparticle enzyme immunoassay (MEIA) for the quantification of blood rapamycin (RAPA) levels in therapeutic drug monitoring of renal transplanted patients with respect to a validated liquid chromatography tandem mass spectrometric (LC/ESI-MSMS) method, used as reference. Linearity of MEIA was tested over the range 0.0-30.0 ng/mL, with accuracy and precision within acceptable limits. Fifty-two blood samples were collected from 42 renal transplanted patients and analyzed simultaneously by both methods. The Pearson's regression analysis gave the following parameters: correlation equation [RAPA](MEIA) = 1.330 + 0.776 [RAPA](LC/ESI-MSMS), r = 0.8526, SD = 1.778, p < 0.0001. The obtained average rapamycin concentration was 8.8 +/- 3.4 ng/mL using MEIA and 9.6 +/- 3.7 ng/mL for LC/ESI-MSMS, with an overall underestimation of about 6% of the immunoenzymatic test. Accuracy of MEIA ranged from -33% to 36% with respect to the reference mass spectrometric method. Although immunoenzymatic test represents a fast and sufficiently accurate method for its use in clinical practice, specificity of the assay is still not sufficiently investigated and reference methods and/or Proficiency Testing Scheme should be used as external control.

The possible cross-reactivity of immunoassays with structurally-related drugs was investigated. Innofluor® Certican® (FPIA) calibrators were measured by using IMx® Sirolimus assay (MEIA) and MEIA Sirolimus calibrators were analysed by using FPIA Certican® assay. Drug concentrations were measured in 95 and 100 samples from renal transplanted patients (RTP) on sirolimus or everolimus treatment by using immunoassays and LC/ESI-MSMS. A high cross-reactivity was found both for MEIA and FPIA. High correlation degrees, confirmed by the Bland-Altman and the Eksborg tests, were found between drug concentrations measured in real samples by both immunoassays (r=0.909 and r=0.970, respectively). LC/ESI-MSMS analysis of samples containing sirolimus showed no positivity for everolimus. Similarly, samples from patients on treatment with everolimus resulted negative as far as regards sirolimus. MEIA and FPIA could be considered mutually reliable and accurate alternatives for the specific-drug immunoassay. It should be noticed that in patients switching from one drug to the other unreal overestimation of the blood levels of the current administered immunosuppressant can occur.