Antonio Osculati

A role for leukocyte-endothelial adhesion mechanisms in epilepsy

The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately one percent of the world population, are not well understood. Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1, encoded by Selplg) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocyte-vascular interactions, either with blocking antibodies or by genetically interfering with PSGL-1 function in mice, markedly reduced seizures. Treatment with blocking antibodies after acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with the potential leukocyte involvement in epilepsy in humans, leukocytes were more abundant in brains of individuals with epilepsy than in controls. Our results suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.

Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands.

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS‐I) and choline‐acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene‐related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS‐I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply.

TWO CASES OF ACCIDENTAL ASPHYXIA BY NECK COMPRESSION BETWEEN BED BARS

Two cases of asphyxia due to compression of the neck between the side bars of a bed in elderly subjects affected by neuropsychiatric pathologies are presented. In both cases no lesions were found on the skin or in the anatomic structures of the neck. The absence of lesions made determining the cause of death difficult. Generic evidence of asphyxia (acute pulmonary emphysema and petechiae) allowed a diagnosis to be formulated. The discovery of the object that caused the death was possible only with information regarding the circumstances and inspection of the scenes of the deaths.

Achalasia and sudden death: a case report

A 23-year-old pregnant woman was found dead at home. The anamnesis was completely negative, except for a slight dysphagia. The most important autopsy finding was a remarkable previously undiagnosed, megaesophagus. Conjunctival and pleural petechiae were also observed. The most likely etiopathogenetic factors are considered and discussed.

All muscarinic acetylcholine receptors (M 1 -M 5 ) are expressed in murine brain microvascular endothelium

Clinical and experimental studies indicate that muscarinic acetylcholine receptors are potential pharmacological targets for the treatment of neurological diseases. Although these receptors have been described in human, bovine and rat cerebral microvascular tissue, a subtype functional characterization in mouse brain endothelium is lacking. Here, we show that all muscarinic acetylcholine receptors (M1-M5) are expressed in mouse brain microvascular endothelial cells. The mRNA expression of M2, M3, and M5 correlates with their respective protein abundance, but a mismatch exists for M1 and M4 mRNA versus protein levels. Acetylcholine activates calcium transients in brain endothelium via muscarinic, but not nicotinic, receptors. Moreover, although M1 and M3 are the most abundant receptors, only a small fraction of M1 is present in the plasma membrane and functions in ACh-induced Ca2+ signaling. Bioinformatic analyses performed on eukaryotic muscarinic receptors demonstrate a high degree of conservation of the orthosteric binding site and a great variability of the allosteric site. In line with previous studies, this result indicates muscarinic acetylcholine receptors as potential pharmacological targets in future translational studies. We argue that research on drug development should especially focus on the allosteric binding sites of the M1 and M3 receptors.

A Fatal Case of Coin Battery Ingestion in an 18-Month-Old Child: Case Report and Literature Review

Abstract The ingestion of extraneous substances is quite common in clinical practice; it usually befalls in the pediatric age, mostly between 6 months and 6 years. In most cases, complications do not emerge, and the prognosis is considered favorable. However, when a case of battery ingestion occurs, serious adverse events may develop. The ingestion of these components is a potential life-threatening event for children. In this article, we report the case of an 18-month-old child who died from hemorrhagic shock due to an aortoesophageal fistula caused by a 20 mm lithium button battery lodged in the esophagus. The child presented vomiting blood, and laboratory results revealed a severe anemization, which later led to death. The autopsy showed a coin battery located in the middle third of the esophagus as well as a transmural erosion of the esophageal wall with fistulization into the aortic wall. The histological examination revealed a severe necrosis of the esophageal and aortic walls in line with the junction between the aortic arch and the descending part.

Death after 25C-NBOMe and 25H-NBOMe consumption

A teenager male was found dead in a waterway after he was spotted jumping off into the water stream. The boy looked agitated and confused after a party with friends. At the gathering place, investigators seized packages of blotter papers. A complete autopsy and a histological evaluation of the main tissues were performed; although the death occurred by drowning, the prosecutor requested toxicological exams, in order to evaluate the potential role of drugs of abuse in the episode. Blood (both peripheral and central) and urine samples as well as seized blotter papers were collected and analyzed as follows. The blotter paper, analyzed through a GC-MS method, revealed the presence of 25-NBOMes. A liquid chromatography tandem mass spectrometric (LC-MS/MS) system was used to identify and quantify 5 different 25-NBOMes (namely 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25H-NBOMe, 25I-NBOMe) in blood and urine. 25E-NBOMe was used as internal standard (IS). 1mL of urine and 1mL of blood (both peripheral and cardiac) were diluted in 2mL phosphate buffer at pH 6.0, containing IS and purified on a solid phase extraction (SPE) cartridge. LOD and LOQ for the five 25-NBOMes were calculated at 0.05 and 0.1ng/mL respectively. Linearity, accuracy, precision, ion suppression, carry over and recovery were tested and all parameters fulfilled the acceptance criteria. Blood and urine provided positive results for 25C-NBOMe and 25H-NBOMe. Eventually, the seized blotter papers were analyzed by means of LC-MS/MS and the presence of the two NBOMes was confirmed: 25C-NBOMe and 25H-NBOMe were measured at the concentration of 2.80 and 0.29ng/mL in peripheral blood, of 1.43 and 0.13ng/mL in central blood and of 0.94 and 0.14ng/mL in urine, respectively. THC and THCCOOH were also detected in biological fluids, at the concentration of 15.5 and 56.0ng/mL in peripheral blood, 9.9 and 8.5ng/mL in central blood, respectively. NBOMes can produce severe hallucination even at very low doses, and the 25C-NBOMe levels measured in the subjects blood are considered potentially toxic.

Variability on ethyl glucuronide concentrations in hair depending on sample pretreatment, using a new developed GC–MS/MS method

HighlightsWe developed and validated a GC–MS/MS method for ethyl glucuronide determination in hair.We compared GC–MS/MS and LC–MS/MS in analysis of real samples.We compared EtG concentration obtained after hair cutting and pulverization.Pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains. ABSTRACT Quantitative determination of ethyl glucuronide in keratin matrix, particularly in hair samples, provides a significant contribution to the evaluation of the extent of ethanol intake. The first‐choice method to carry out this analysis is LC–MS/MS, but other techniques may be used. The aim of this work is: a) to develop and validate a GC–MS/MS method for ethyl glucuronide determination in hair; b) to compare GC–MS/MS and LC–MS/MS in analysis of real samples; c) to compare EtG concentration obtained after hair cutting and pulverization. About 30 mg hair samples were washed, pulverized and soaked in 1 ml deionized water. After incubation, the solution was purified through a SPE anion exchange cartridge; the eluate was dried under nitrogen stream, derivatized with PFPA and reconstituted in n‐hexane. Then, the sample was injected in the GC–MS/MS system, operating in negative chemical ionization mode and in selected reaction monitoring. The two most intense transitions were used to monitor ethyl glucuronide and deuterated internal standard. All the validation parameters fulfilled the international acceptance criteria. LOD and LOQ were set at 2.0 and 3.0 pg/mg respectively. This method was applied to 194 hair samples collected from teetotallers and alcohol consumers and represents a suitable alternative to LC–MS/MS for the determination of EtG in hair samples, in particular when scarce quantity of hair is available. This study confirmed that pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains probably due to the presence of non‐homogeneous material even though pulverization.

Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water

&NA; The study aims the development and validation of a LC–MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long‐term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1–2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam‐D5) and sonicated up to two hours. The solution was then direct injected in the LC–MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7‐aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Distribution of the Synthetic Cathinone α-Pyrrolidinohexiophenone in Biological Specimens

We report the analysis of the synthetic cathinones α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopentiophenone (α-PVP), both pyrovalerone derivatives, in blood, urine, gastric contents, main tissues and hair of a deceased person. Qualitative and quantitative analyses were performed by LC-MS-MS. All the biological samples were collected during autopsy and extracted/purified onto a solid phase extraction cartridge before instrumental analysis. The method was validated for blood and urine and proved to be highly sensitive and specific for both the synthetic cathinones (limit of detection: 0.2 ng/mL and limit of quantification: 0.5 ng/mL). Analyses provided negative results for α-PVP in all biological samples except for the 2-cm proximal hair segment, confirming that the young man had consumed in the last 2 months this compound; instead hair analysis proved that the man was a heavy α-PHP user. α-PHP was identified and quantified in biological fluids and tissues. Interestingly, bile and urine concentrations (1.2 and 5.6 ng/mL, respectively) were fairly lower than blood collected into the thoracic cavity (15.3 ng/mL). The highest concentrations were measured for lung (71.1 ng/mL) and spleen (83.8 ng/mL). Concentrations of 3.5, 7.9, 4.7 and 23.6 ng/mL were measured in liver, kidney, brain and heart, respectively. Even if it is not possible to evaluate the presence of this drug in biological samples as a cause of death, to our knowledge, this is the first case of α-PHP finding in postmortem samples, and its potential toxic effects should be elucidated in the future.