Using Macrophage Polarization in Human Platelet Lysate to Test the Immunomodulatory Potential of Cells for Clinical Use.

Macrophage-based co-cultures are used to test the immunomodulatory function of candidate cells for clinical use. This study aimed to characterize a macrophage polarization model using human platelet lysate (hPL) as a GMP-compliant alternative to Fetal Bovine Serum (FBS). Primary human monocytes were differentiated into unpolarized (M0) or polarized (M1, M2a, and M2c) macrophages in an hPL- or FBS-based medium. The protein secretion profiles and expression of phenotypic markers (CD80 for M1, CD206 for M2a, and CD163 for M2c) were analyzed. Subsequently, chondrocytes were tested in an hPL-based co-culture model to assess their immunomodulatory function in view of their possible use in patients with osteoarthritis. The results showed similar marker regulation between hPL and FBS cultures, but lower basal levels of CD206 and CD163 in hPL-cultured macrophages. Functional co-culture experiments with chondrocytes revealed increased CD206 expression both in hPL and in FBS, indicating an interaction between macrophages and chondrocytes. While markers in FBS-cultured macrophages were confirmed in hPL-cultured cells, the interpretation of marker modulation in immunomodulatory assays with hPL-based cultures should be carried out cautiously due to the observed differences in the basal marker levels for CD206 and CD163. This research underscores the utility of hPL as a GMP-compliant alternative to FBS for macrophage-based co-cultures and highlights the importance of understanding marker expressions in different culture conditions.

Complex or not too complex? One size does not fit all in next generation microphysiological systems.

In the attempt to overcome the increasingly recognized shortcomings of existing in vitro and in vivo models, researchers have started to implement alternative models, including microphysiological systems. First examples were represented by 2.5D systems, such as microfluidic channels covered by cell monolayers as blood vessel replicates. In recent years, increasingly complex microphysiological systems have been developed, up to multi-organ on chip systems, connecting different 3D tissues in the same device. However, such an increase in model complexity raises several questions about their exploitation and implementation into industrial and clinical applications, ranging from how to improve their reproducibility, robustness, and reliability to how to meaningfully and efficiently analyze the huge amount of heterogeneous datasets emerging from these devices. Considering the multitude of envisaged applications for microphysiological systems, it appears now necessary to tailor their complexity on the intended purpose, being academic or industrial, and possibly combine results deriving from differently complex stages to increase their predictive power.

Human platelet lysate stimulates neurotrophic properties of human adipose-derived stem cells better than Schwann cell-like cells.

BACKGROUND: Trauma-associated peripheral nerve injury is a widespread clinical problem causing sensory and motor disabilities. Schwann cells (SCs) contribute to nerve regeneration, mainly by secreting nerve growth factor (NGF) and brain-derived neurotrophic factor. In the last years, adipose-derived stem cells (ASCs) differentiated into SCs (SC-ASCs) were considered as promising cell therapy. However, the cell trans-differentiation process has not been effectively showed and presents several drawbacks, thus an alternative approach for increasing ASCs neurotrophic properties is highly demanded. In the context of human cell-based therapies, Good Manufacturing Practice directions indicate that FBS should be substituted with a xenogeneic-free supplement, such as Human Platelet Lysate (HPL). Previously, we demonstrated that neurotrophic properties of HPL-cultured ASCs were superior compared to undifferentiated FBS-cultured ASCs. Therefore, as following step, here we compared the neurotrophic properties of differentiated SC-like ASCs and HPL-cultured ASCs. METHODS: Both cell groups were investigated for gene expression level of neurotrophic factors, their receptors and neuronal markers. Moreover, the expression of nestin was quantitatively evaluated by flow cytometry. The commitment toward the SC phenotype was assessed with immunofluorescence pictures. Proteomics analysis was performed on both cells and their conditioned media to compare the differential protein profile. Finally, neurotrophic abilities of both groups were evaluated with a functional co-culture assay, assessing dorsal root ganglia survival and neurite outgrowth. RESULTS: HPL-cultured ASCs demonstrated higher gene expression of NGF and lower expression of S100B. Moreover, nestin was present in almost all HPL-cultured ASCs and only in one quarter of SC-ASCs. Immunofluorescence confirmed that S100B was not present in HPL-cultured ASCs. Proteomics analysis validated the higher expression of nestin and the increase in cytoskeletal and ECM proteins involved in neural regeneration processes. The co-culture assay highlighted that neurite outgrowth was higher in the presence of HPL-ASCs or their conditioned medium compared to SC-ASCs. CONCLUSIONS: All together, our results show that HPL-ASCs were more neurotrophic than SC-ASCs. We highlighted that the HPL triggers an immature neuro-induction state of ASCs, while keeping their stem properties, paving the way for innovative therapies for nerve regeneration.

COVID-19 and brain-heart-lung microbial fingerprints in Italian cadavers.

Introduction: The fact that SARS-CoV-2, the coronavirus that caused COVID-19, can translocate within days of infection to the brain and heart and that the virus can survive for months is well established. However, studies have not investigated the crosstalk between the brain, heart, and lungs regarding microbiota that simultaneously co-inhabit these organs during COVID-19 illness and subsequent death. Given the significant overlap of cause of death from or with SARS-CoV-2, we investigated the possibility of a microbial fingerprint regarding COVID-19 death. Methods: In the current study, the 16S rRNA V4 region was amplified and sequenced from 20 COVID-19-positive and 20 non-COVID-19 cases. Nonparametric statistics were used to determine the resulting microbiota profile and its association with cadaver characteristics. When comparing non-COVID-19 infected tissues versus those infected by COVID-19, there is statistical differences (p < 0.05) between organs from the infected group only. Results: When comparing the three organs, microbial richness was significantly higher in non-COVID-19-infected tissues than infected. Unifrac distance metrics showed more variance between control and COVID-19 groups in weighted analysis than unweighted; both were statistically different. Unweighted Bray-Curtis principal coordinate analyses revealed a near distinct two-community structure: one for the control and the other for the infected group. Both unweighted and weighted Bray-Curtis showed statistical differences. Deblur analyses demonstrated Firmicutes in all organs from both groups. Discussion: Data obtained from these studies facilitated the defining of microbiome signatures in COVID-19 decedents that could be identified as taxonomic biomarkers effective for predicting the occurrence, the co-infections involved in its dysbiosis, and the evolution of the virus.

In vitro models of breast cancer bone metastasis: analyzing drug resistance through the lens of the microenvironment.

Even though breast cancers usually have a good outcome compared to other tumors, the cancer can progress and create metastases in different parts of the organism, the bone being a predilection locus. These metastases are usually the cause of death, as they are mostly resistant to treatments. This resistance can be caused by intrinsic properties of the tumor, such as its heterogeneity, but it can also be due to the protective role of the microenvironment. By activating signaling pathways protecting cancer cells when exposed to chemotherapy, contributing to their ability to reach dormancy, or even reducing the amount of drug able to reach the metastases, among other mechanisms, the specificities of the bone tissue are being investigated as important players of drug resistance. To this date, most mechanisms of this resistance are yet to be discovered, and many researchers are implementing in vitro models to study the interaction between the tumor cells and their microenvironment. Here, we will review what is known about breast cancer drug resistance in bone metastasis due to the microenvironment and we will use those observations to highlight which features in vitro models should include to properly recapitulate these biological aspects in vitro. We will also detail which elements advanced in vitro models should implement in order to better recapitulate in vivo physiopathology and drug resistance.

A case of intrauterine fetal death related to furcate cord insertion: a brief review of the literature and professional liability considerations.

The furcate insertion of the umbilical cord is an uncommon abnormality, often asymptomatic, potentially dangerous, or lethal for the fetus and the mother. This report shows the case of a healthy 29-year-old patient, at 37 weeks of gestation, admitted to the hospital two days before the due date because of the appearance of uterine contractions; clinical exams were regular. The following day, no fetal movements were perceived, a cardiotocography was performed, showing the absence of fetal heartbeat. A dead fetus was delivered. Autopsy showed furcate insertion of the umbilical cord and the rupture of the umbilical vessel, which caused fetal hemorrhagic shock. Furcate insertion still remains mostly undiagnosed and rarely it can be identified prenatally (only three cases are reported in literature). Future research, mainly in forensic fields, could improve the knowledge about this condition, helping prenatal diagnosis and providing warnings that can prevent similar deaths in the future.

Differential angiogenesis of bone and muscle endothelium in aging and inflammatory processes.

Different tissues have different endothelial features, however, the implications of this heterogeneity in pathological responses are not clear yet. "Inflamm-aging" has been hypothesized as a possible trigger of diseases, including osteoarthritis (OA) and sarcopenia, often present in the same patient. To highlight a possible contribution of organ-specific endothelial cells (ECs), we compare ECs derived from bone and skeletal muscle of the same OA patients. OA bone ECs show a pro-inflammatory signature and higher angiogenic sprouting as compared to muscle ECs, in control conditions and stimulated with TNFα. Furthermore, growth of muscle but not bone ECs decreases with increasing patient age and systemic inflammation. Overall, our data demonstrate that inflammatory conditions in OA patients differently affect bone and muscle ECs, suggesting that inflammatory processes increase angiogenesis in subchondral bone while associated systemic low-grade inflammation impairs angiogenesis in muscle, possibly highlighting a vascular trigger linking OA and sarcopenia.

Assessing the response of human primary macrophages to defined fibrous architectures fabricated by melt electrowriting.

The dual role of macrophages in the healing process depends on macrophage ability to polarize into phenotypes that can propagate inflammation or exert anti-inflammatory and tissue-remodeling functions. Controlling scaffold geometry has been proposed as a strategy to influence macrophage behavior and favor the positive host response to implants. Here, we fabricated Polycaprolactone (PCL) scaffolds by Melt Electrowriting (MEW) to investigate the ability of scaffold architecture to modulate macrophage polarization. Primary human macrophages unpolarized (M0) or polarized into M1, M2a, and M2c phenotypes were cultured on PCL films and MEW scaffolds with pore geometries (square, triangle, and rhombus grid) characterized by different angles. M0, M2a, and M2c macrophages wrapped along the fibers, while M1 macrophages formed clusters with rounded cells. Cell bridges were formed only for angles up to 90°. No relevant differences were found among PCL films and 3D scaffolds in terms of surface markers. CD206 and CD163 were highly expressed by M2a and M2c macrophages, with M2a macrophages presenting also high levels of CD86. M1 macrophages expressed moderate levels of all markers. The rhombus architecture promoted an increased release by M2a macrophages of IL10, IL13, and sCD163 compared to PCL films. The proangiogenic factor IL18 was also upregulated by the rhombus configuration in M0 and M2a macrophages compared to PCL films. The interesting findings obtained for the rhombus architecture represent a starting point for the design of scaffolds able to modulate macrophage phenotype, prompting investigations addressed to verify their ability to facilitate the healing process in vivo.

Inflammatory priming with IL-1ß promotes the immunomodulatory behavior of adipose derived stem cells.

Inflammatory processes contribute to osteoarthritis (OA) severity and progression. Mesenchymal stem cells, particularly those derived from adipose tissue (ASCs), are able to sense and control the inflammatory environment. This immunomodulatory potential can be boosted by different priming strategies based on inflammatory stimulation. The aim of the present study is to investigate the transcriptional modulation of a huge panel of genes and functionally verify the predicted immunomodulatory ability of ASCs after interleukin one beta (IL-1ß) priming. ASCs were isolated from adipose tissue obtained from three donors and expanded. After stimulation with 1 ng/ml of IL-1ß for 48 h, cells were collected for gene array and functional tests. Pooled cells from three donors were used for RNA extraction and gene array analysis. Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA) were performed to assess the involvement of the modulated genes after priming in specific biological processes and pathways. Functional co-culture tests of ASCs with T cells and macrophages were performed to assess the ability of primed ASCs to modulate immune cell phenotype. Among the overall genes analyzed in the gene array, about the 18% were up- or down-regulated in ASCs after IL-1ß priming. GO enrichment analysis of up- or down-regulated genes in ASCs after IL-1ß priming allowed identifying specific pathways involved in the modulation of inflammation and extracellular matrix remodeling. The main processes enriched according to the GSEA are related to the inflammatory response and cell proliferative processes. Functional tests on immune cells showed that primed and non-primed ASCs induced a decrease in the CD3+ T lymphocytes survival rate and an anti-inflammatory macrophage polarization. In conclusion, IL-1ß priming represents a tailored strategy to enhance the ability of ASCs to direct macrophages towards an anti-inflammatory phenotype and, consequently, improve the efficacy of ASCs in counteracting the OA inflammatory component.

COVID-19 Pathology in the Lung, Kidney, Heart and Brain: The Different Roles of T-Cells, Macrophages, and Microthrombosis.

Here, we aim to describe COVID-19 pathology across different tissues to clarify the disease's pathophysiology. Lungs, kidneys, hearts, and brains from nine COVID-19 autopsies were compared by using antibodies against SARS-CoV-2, macrophages-microglia, T-lymphocytes, B-lymphocytes, and activated platelets. Alzheimer's Disease pathology was also assessed. PCR techniques were used to verify the presence of viral RNA. COVID-19 cases had a short clinical course (0-32 days) and their mean age was 77.4 y/o. Hypoxic changes and inflammatory infiltrates were present across all tissues. The lymphocytic component in the lungs and kidneys was predominant over that of other tissues (p < 0.001), with a significantly greater presence of T-lymphocytes in the lungs (p = 0.020), which showed the greatest presence of viral antigens. The heart showed scant SARS-CoV-2 traces in the endothelium-endocardium, foci of activated macrophages, and rare lymphocytes. The brain showed scarce SARS-CoV-2 traces, prominent microglial activation, and rare lymphocytes. The pons exhibited the highest microglial activation (p = 0.017). Microthrombosis was significantly higher in COVID-19 lungs (p = 0.023) compared with controls. The most characteristic pathological features of COVID-19 were an abundance of T-lymphocytes and microthrombosis in the lung and relevant microglial hyperactivation in the brainstem. This study suggests that the long-term sequelae of COVID-19 derive from persistent inflammation, rather than persistent viral replication.

A microfluidic model of human vascularized breast cancer metastasis to bone for the study of neutrophil-cancer cell interactions.

The organ-specific metastatization of breast cancer to bone is driven by specific interactions between the host microenvironment and cancer cells (CCs). However, it is still unclear the role that circulating immune cells, including neutrophils, play during bone colonization (i.e. pro-tumoral vs. anti-tumoral). Here, we aimed at analyzing the migratory behavior of neutrophils when exposed to breast CCs colonizing the bone and their contribution to the growth of breast cancer micrometastases. Based on our previous bone metastasis models, we designed a microfluidic system that allows to independently introduce human vascularized breast cancer metastatic seeds within a bone-mimicking microenvironment containing osteo-differentiated mesenchymal stromal cells and endothelial cells (ECs). ECs self-assembled into microvascular networks and connected the bone-mimicking microenvironment with the metastatic seed. Compared to controls without CCs, metastatic seeds compromised the architecture of microvascular networks resulting in a lower number of junctions (5.7 â€‹± â€‹1.2 vs. 18.8 â€‹± â€‹4.5, p â€‹= â€‹0.025) and shorter network length (10.5 â€‹± â€‹1.0 vs. 13.4 â€‹± â€‹0.8 [mm], p â€‹= â€‹0.042). Further, vascular permeability was significantly higher with CCs (2.60 â€‹× â€‹10-8 â€‹± â€‹3.59 â€‹× â€‹10-8 â€‹vs. 0.53 â€‹× â€‹10-8 â€‹± â€‹0.44 â€‹× â€‹10-8 [cm/s], p â€‹= â€‹0.05). Following metastatic seed maturation, neutrophils were injected into microvascular networks resulting in a higher extravasation rate when CCs were present (27.9 â€‹± â€‹13.7 vs. 14.7 â€‹± â€‹12.4 [%], p â€‹= â€‹0.01). Strikingly, the percentage of dying CCs increased in presence of neutrophils, as confirmed by confocal imaging and flow cytometry on isolated cells from the metastatic seeds. The biofabricated metastatic niche represents a powerful tool to analyze the mechanisms of interaction between circulating immune cells and organ-specific micrometastases and to test novel drug combinations targeting the metastatic microenvironment.

Immunomodulatory potential of secretome from cartilage cells and mesenchymal stromal cells in an arthritic context: From predictive fiction toward reality.

The purpose of the present study is to predict by bioinformatics the activity of the extracellular vesicle (EV)-embedded micro RNA (miRNAs) secreted by cartilage cells (CCs), adipose tissue-derived- (ASCs), and bone marrow-derived stem cells (BMSCs) and verify their immunomodulatory potential supporting our bioinformatics findings to optimize the autologous cell-based therapeutic strategies for osteoarthritis (OA) management. Cells were isolated from surgical waste tissues of three patients who underwent total hip replacement, expanded and the EVs were collected. The expression of EV-embedded miRNA was evaluated with the QuantStudio 12 K Flex OpenArray® platform. Mientournet and ingenuity pathway analysis (IPA) were used for validated target prediction analysis and to identify miRNAs involved in OA and inflammation. Cells shared the expression of 325 miRNAs embedded in EVs and differed for the expression of a small number of them. Mienturnet revealed no results for miRNAs selectively expressed by ASCs, whereas miRNA expressed by CCs and BMSCs were putatively involved in the modulation of cell cycle, senescence, apoptosis, Wingless and Int-1 (Wnt), transforming growth factor beta (TGFß), vascular endothelial growth factor (VEGF), Notch, Hippo, tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1ß), insulin like growth factor 1 (IGF-1), RUNX family transcription factor 2 (RUNX2), and endochondral ossification pathways. Cartilage homeostasis, macrophages and T cells activity and inflammatory mediators were identified by IPA as targets of the miRNAs found in all the cell populations. Co-culture tests on macrophages and T cells confirmed the immuno-modulatory ability of CCs, ASCs, and BMSCs. The study findings support the rationale behind the use of cell-based therapy for the treatment of OA.

Determination of Traditional and Designer Benzodiazepines in Urine through LC-MS/MS.

BACKGROUND: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis. OBJECTIVE: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases. METHODS: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode. RESULTS: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine. CONCLUSION: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

Correlation between postmortem microbial signatures and substance abuse disorders.

The microbiota gut-brain-axis is a bidirectional circuit that links the neural, endocrine, and immunological systems with gut microbial communities. The gut microbiome plays significant roles in human mind and behavior, specifically pain perception, learning capacity, memory, and temperament. Studies have shown that disruptions in the gut microbiota have been associated with substance use disorders. The interplay of gut microbiota in substance abuse disorders has not been elucidated; however, postmortem microbiome profiles may produce promising avenues for future forensic investigations. The goal of the current study was to determine gut microbiome composition in substance abuse disorder cases using transverse colon tissues of 21 drug overdose versus 19 non-overdose-related cases. We hypothesized that postmortem samples of the same cause of death will reveal similar microbial taxonomic relationships. We compared microbial diversity profiles using amplicon-based sequencing of the 16S rRNA gene V4 hypervariable region. The results demonstrated that the microbial abundance in younger-aged cases were found to have significantly more operational taxonomic units than older cases. Using weighted UniFrac analysis, the influence of substances in overdose cases was found to be a significant factor in determining microbiome similarity. The results also revealed that samples of the same cause of death cluster together, showing a high degree of similarity between samples and a low degree of similarity among samples of different causes of death. In conclusion, our examination of human transverse colon microflora in decomposing remains extends emerging literature on postmortem microbial communities, which will ultimately contribute to advanced knowledge of human putrefaction.

Congenital cysts of the lower male genitourinary tract: a disorder with various treatment approaches and pitfalls-case report.

BACKGROUND: The cysts of the male pelvic floor represent a rare clinical entity. Their origin is linked to an altered development of paramesonephric and mesonephric ducts during embryogenesis. CASE PRESENTATION: We report our experience regarding two patients presenting cysts of the ejaculatory system treated with open and mini-invasive surgery. The patients referred to our clinic with nonspecific symptoms and the diagnosis was obtained by radiological investigations. The patient treated with an open approach developed a pelvic purulent collection and a fistula of the prostatic urethra, managed with surgical drainage and prolonged bladder catheterization. On the other hand, the patient treated with laparoscopic approach did not develop any complications. No sexual or ejaculatory disorders were reported. CONCLUSIONS: Patients with congenital cysts of the pelvic floor must be adequately informed about the risks and benefits of surgery and a careful counseling is mandatory before surgery. Treatment is recommended for symptomatic patients and an endoscopic approach is associated with a high rate of recurrence. A laparoscopic approach, when possible, is desirable.

Conversion of the Native N-Terminal Domain of TDP-43 into a Monomeric Alternative Fold with Lower Aggregation Propensity.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and ß-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

A quantitative biology approach correlates neuronal toxicity with the largest inclusions of TDP-43.

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com).

Ethyl glucuronide in hair: A 5-year retrospective cohort study in subjects sanctioned for driving under the influence of alcohol and psychoactive substances.

The evaluation of drinking behaviors can help in limiting high-risk situation, such as driving under the influence (DUI). We investigate ethyl glucuronide in hair (hEtG) levels to evaluate alcohol consumption behavior in subjects followed up after having been charged for DUI of psychoactive substances and/or alcohol. We performed a retrospective observational cohort study on 4328 subjects over 18 years old who underwent hEtG analysis in the period 2015-2019 in the Italian Province of Pavia. hEtG level was used as a proxy for the alcohol consumption behavior. Effects of age, sex, and district on alcohol drinking behavior were investigated with an ordinal logit model. A state sequence analysis was used to study people's alcohol consumption behavior over time. hEtG was found ≥7.0 pg/mg in 22.2% of the drivers (of which 7% has an hEtG ≥30.0 pg/mg). Among positive cases, a prevalence of males (96.3%) aged 35-44 (32.6%), coming from main city and hinterland (38.2%), was observed. The propensity to drink was higher for males (odds ratio [OR] ≈ 2.28, p < 0.001) and for subject coming from the district devoted to the cultivation of vineyards. Young age classes have a reduced drinking risk if compared to the drivers over 55 years old (p < 0.001). A general decreasing trend over time in hEtG values was observed. Being male, age ≥ 55 years, and coming from rural areas are potential risk factors related to alcohol drinking habits among drivers. Ethyl glucuronide in hair test in the driving license reissuing protocol contributed to decrease alcohol misuse behaviors.

Genetic individual identification from dried urine spots: A complementary tool to drug monitoring and anti-doping testing.

The collection of liquid biological matrices onto paper cards (dried matrix spots [DMS]) is becoming an alternative sampling strategy. The stability over time of molecules of interest for therapeutic, sport drug monitoring, and forensic toxicology on DMS has been recently investigated representing a reliable alternative to conventional analytical techniques. When a tampering of a urine sample in drug monitoring or doping control cases is suspected, it could be relevant to know whether genetic profiles useful for individual identification could be generated from urine samples spotted onto paper (dried urine spot [DUS]). To understand the influence of sex, storage conditions, and time on the quality and quantity of the DNA, five female and ten male urine samples were dispensed onto Whatman 903 paper and sampled after different storage conditions over time, from 1 to 12 weeks. Direct PCR was performed starting from 2-mm punches collected from each spot amplifying a panel of markers useful for individual identification. The female DUS stored in different conditions produced genetic profiles fully matching the reference samples. The same result was obtained for the male DUS but using urine 30X concentrated by centrifugation instead of the original samples. Our data show that this approach is valid for genetic individual identification of urine samples spotted onto paper cards up to 12 weeks after deposition and could be easily incorporated in anti-doping or drug screening protocols to help on the suspicion of evidence tampering or to solve questions on the reliability of samples collection.