Insights in opiates toxicity: impairment of human vascular mesenchymal stromal cells.

The most common pulmonary findings in opiate-related fatalities are congestion and oedema, as well as acute and/or chronic alveolar haemorrhage, the cause of which is thought to be a damage to the capillary endothelium related to ischemia. Human vascular mesenchymal stromal cells (vMSCs) play a fundamental role in tissue regeneration and repair after endothelial cell injury, and they express opioid receptors. The aim of this study was to assess the effect of in vitro morphine exposure on the physiological activity and maintenance of human vMSCs. vMSCs were obtained from abdominal aorta fragments collected during surgery repair and were exposed to incremental doses (0.1 mM, 0.4 mM, 0.8 mM and 1 mM) of morphine sulphate for 7 days. The effect was investigated through cell viability assessment, proliferation assay, reactive oxygen species (ROS) detection assay, senescence-associated ß-galactosidase assay, senescent-related markers (p21WAF1/CIP1 and p16INK4) and the apoptosis-related marker caspase 3. Moreover, an ultrastructural analysis by transmission electron microscopy and in vitro vascular differentiation were evaluated. Results showed a decrease of the cellular metabolic activity, a pro-oxidant and pro-senescence effect, an increase in intracellular ROS and the activation of the apoptosis signalling, as well as ultrastructural modifications and impairment of vascular differentiation after morphine treatment of vMSC. Although confirmation studies are required on real fatal opiate intoxications, the approach based on morphological and immunofluorescence methodologies may have a high potential also as a useful tool or as a complementary method in forensic pathology. The application of these techniques in the future may lead to the identification of new markers and morphological parameters useful as complementary investigations for drug-related deaths.

Assessment of the structural and functional characteristics of human mesenchymal stem cells associated with a prolonged exposure of morphine.

The discovery of the expression of opioid receptors in the skin and their role in orchestrating the process of tissue repair gave rise to questions regarding the potential effects of clinical morphine treatment in wound healing. Although short term treatment was reported to improve tissue regeneration, in vivo chronic administration was associated to an impairment of the physiological healing process and systemic fibrosis. Human mesenchymal stem cells (hMSCs) play a fundamental role in tissue regeneration. In this regard, acute morphine exposition was recently reported to impact negatively on the functional characteristics of hMSCs, but little is currently known about its long-term effects. To determine how a prolonged treatment could impair their functional characteristics, we exposed hMSCs to increasing morphine concentrations respectively for nine and eighteen days, evaluating in particular the fibrogenic potential exerted by the long-term exposition. Our results showed a time dependent cell viability decline, and conditions compatible with a cellular senescent state. Ultrastructural and protein expression analysis were indicative of increased autophagy, suggesting a relation to a detoxification activity. In addition, the enhanced transcription observed for the genes involved in the synthesis and regulation of type I collagen suggested the possibility that a prolonged morphine treatment might exert its fibrotic potential risk, even involving the hMSCs.

Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm.

Cellular senescence is a hallmark of ageing and it plays a key role in the development of age-related diseases. Abdominal aortic aneurysm (AAA) is an age related degenerative vascular disorder, characterized by a progressive dilatation of the vascular wall and high risk of rupture over time. Nowadays, no pharmacological therapies are available and the understanding of the molecular mechanisms that lead to AAA onset and development are poorly defined. In this study we investigated the cellular features of senescence in vascular mesenchymal stromal cells, isolated from pathological (AAA - MSCs) and healthy (h - MSCs) segments of human abdominal aorta and their implication in impairing the vascular repair ability of MSCs. Cell proliferation, ROS production, cell surface area, the expression of cyclin dependent kinase inhibitors p21CIP1 and p16INK4a, the activation of the DNA damage response and a dysregulated autophagy showed a senescent state in AAA - MSCs compared to h-MSCs. Moreover, a reduced ability to differentiate toward endothelial cells was observed in AAA - MSCs. All these data suggest that the accumulation of senescent vascular MSCs over time impairs their remodeling ability during ageing. This condition could support the onset and development of AAA.

Mitochondrial DNA Footprints from Western Eurasia in Modern Mongolia.

Mongolia is located in a strategic position at the eastern edge of the Eurasian Steppe. Nomadic populations moved across this wide area for millennia before developing more sedentary communities, extended empires, and complex trading networks, which connected western Eurasia and eastern Asia until the late Medieval period. We provided a fine-grained portrait of the mitochondrial DNA (mtDNA) variation observed in present-day Mongolians and capable of revealing gene flows and other demographic processes that took place in Inner Asia, as well as in western Eurasia. The analyses of a novel dataset (N = 2,420) of mtDNAs highlighted a clear matrilineal differentiation within the country due to a mixture of haplotypes with eastern Asian (EAs) and western Eurasian (WEu) origins, which were differentially lost and preserved. In a wider genetic context, the prevalent EAs contribution, larger in eastern and central Mongolian regions, revealed continuous connections with neighboring Asian populations until recent times, as attested by the geographically restricted haplotype-sharing likely facilitated by the Genghis Khan's so-called Pax Mongolica. The genetic history beyond the WEu haplogroups, notably detectable on both sides of Mongolia, was more difficult to explain. For this reason, we moved to the analysis of entire mitogenomes (N = 147). Although it was not completely possible to identify specific lineages that evolved in situ, two major changes in the effective (female) population size were reconstructed. The more recent one, which began during the late Pleistocene glacial period and became steeper in the early Holocene, was probably the outcome of demographic events connected to western Eurasia. The Neolithic growth could be easily explained by the diffusion of dairy pastoralism, as already proposed, while the late glacial increase indicates, for the first time, a genetic connection with western Eurasian refuges, as supported by the unusual high frequency and internal sub-structure in Mongolia of haplogroup H1, a well-known post-glacial marker in Europe. Bronze Age events, without a significant demographic impact, might explain the age of some mtDNA haplogroups. Finally, a diachronic comparison with available ancient mtDNAs made it possible to link six mitochondrial lineages of present-day Mongolians to the timeframe and geographic path of the Silk Route.

Genetic variability of CYP2D6, CYP2B6, CYP2C9 and CYP2C19 genes across the Italian Peninsula.

BACKGROUND: Environmental conditions and past migratory events may have shaped genetic heterogeneity of clinically relevant enzymes involved in the phase I metabolism of the most common therapeutic drugs. AIM: To investigate the genetic variability of CYP2D6, CYP2B6, CYP2C19 and CYP2C9 across the Italian Peninsula, by sampling only ancestrally and geographically homogeneous individuals from northern, central and southern Italy. SUBJECTS AND METHODS: A total of 25 SNPs were genotyped in 174 unrelated Italian individuals by means of multiplex PCR and minisequencing reactions. CYP2D6 genotypic data were used to predict phenotypes and the phylogenetic relationships among reconstructed haplotypes were represented by means of a Median Joining Network. RESULTS: Pairwise Fisher Exact tests revealed significant differences between northern and southern Italy in the distribution of CYP2C19 genotypes, with the CYP2C19*2 allele appearing over-represented in northern Italy. Likewise, significant differences in the distribution of CYP2D6 genotypes (*4/*3, *4/*4 and *6/*4) responsible for the poor metabolizer phenotype were observed in northern with respect to both central and southern Italy. CONCLUSIONS: The north-south structuring pattern showed by CYP2D6 and CYP2C19 underline how a deeper knowledge of the geographic distribution of alleles may improve clinical practice and help to avoid hypothetical bias in drug trials.

Expanding X-chromosomal forensic haplotype frequencies database: Italian population data of four linkage groups.

Requests for solving complex kinship casework involving at least one female are increasing and in these circumstances the analysis of X-chromosomal STR markers plays a relevant role. Actually, it is well known the superior statistical power of X-STRs compared to autosomal markers in solving relationship when two sisters or half-sisters are involved and none of parents is available, in maternity testing or in cases involving close relatives as alternative putative fathers. In addition, the possibility to amplify more loci simultaneously and the strategy based on the analysis of four linkage groups to obtain the X-haplotype provide a powerful and validated tool. Nevertheless, haplotypes frequency distribution in different populations is still needed for calculation of probabilities in relationship testing. Published haplotype frequencies from German population data are available, but in different caseworks we found unreported X-haplotypes. To enlarge the forensic X-chromosome database, we present haplotype frequencies and other parameter of forensic interest obtained from 200 anonymous DNA samples of unrelated Italian males for the four linkage groups included in the Investigator Argus X-12 kit. From the comparison of the Italian sample haplotype frequencies with other populations, significant genetic distances were found with Asian and African populations, but not with Europeans. Finally, casework examples of complex kinship analysis are presented.

CYP2B6 gene single-nucleotide polymorphisms in an Italian population sample and relationship with nicotine dependence.

The extensively polymorphic CYP2B6 gene metabolizes endogenous and exogenous compounds, among which are nicotine and bupropion, although its contribution to the systemic metabolism of nicotine still remains controversial. In the present study, the distribution of the CYP2B6 variant and genotype frequencies were analyzed in a sample of 202 Italian individuals who were also invited to answer the Fagerström test for nicotine dependence (FTND), in an effort to assess the involvement of CYP2B6 polymorphisms in nicotine dependence. Eight single-nucleotide polymorphisms of CYP2B6 were tested and seven different variants were identified showing frequencies similar to the European population. The reduced activity of the CYP2B6*6 variant was significantly (p=0.025) distributed among the nicotine-dependent individuals compared to non-nicotine dependents. Also, the CYP2B6*1/*6 genotype achieved statistical significance (p=0.016) within the nicotine-dependent individuals. The high occurrence of CYP2B6*6 carriers among nicotine-dependent individuals may suggest a possible involvement in nicotine dependence, with a potential impact on smoking cessation treatments tailored to the individual smoker's genotype.