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. Graphical Abstrac

La promozione della salute nella scuola secondaria superiore della provincia di Milano: stato di attuazione, analisi e valutazione

Dottorato di ricerca in medicina preventiva e di comunita. A.a. 199 4-95. Coordinatore M. T. TenconiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Sphingolipids in Obesity and Correlated Co-Morbidities: The Contribution of Gender, Age and Environment

This paper reviews our present knowledge on the contribution of ceramide (Cer), sphingomyelin (SM), dihydroceramide (DhCer) and sphingosine-1-phosphate (S1P) in obesity and related co-morbidities. Specifically, in this paper, we address the role of acyl chain composition in bodily fluids for monitoring obesity in males and females, in aging persons and in situations of environmental hypoxia adaptation. After a brief introduction on sphingolipid synthesis and compartmentalization, the node of detection methods has been critically revised as the node of the use of animal models. The latter do not recapitulate the human condition, making it difficult to compare levels of sphingolipids found in animal tissues and human bodily fluids, and thus, to find definitive conclusions. In human subjects, the search for putative biomarkers has to be performed on easily accessible material, such as serum. The serum “sphingolipidome” profile indicates that attention should be focused on specific acyl chains associated with obesity, per se, since total Cer and SM levels coupled with dyslipidemia and vitamin D deficiency can be confounding factors. Furthermore, exposure to hypoxia indicates a relationship between dyslipidemia, obesity, oxygen level and aerobic/anaerobic metabolism, thus, opening new research avenues in the role of sphingolipids

Safety of blood reinfusion drains after local infiltration analgesia in total joint replacement

Abstract Background Local infiltration analgesia (LIA) is frequently administered to patient undergoing joint replacement surgical procedures. The aim of the present research was to verify the safety of collected shed blood to be reinfused postoperatively, by measuring levobupivacaine levels in drainage blood in patients undergoing LIA during knee replacement surgery. Patients and Methods 24 patients who underwent total knee arthroplasty (TKA) and 12 scheduled for total hip arthroplasty (THA) who received intraoperative LIA were considered. Blood samples were collected from shed blood which was present in drainage 2 and 5 hours after surgery and serum was analysed by liquid chromatography-tandem mass spectrometry. Results At 2 hours postoperatively, the median levobupivacaine serum concentration in the collected shed blood was 1.2 mg/L (SD: 4.2) for TKA and 17.13 mg/L (SD: 24.4) for THA. At 5 hours, levobupivacaine concentration was 1.84 mg/L (SD: 2.2) for TKA and 17.5 mg/L (SD: 25.2) for THA. Higher values of average serum levobupivacaine concentration were reported in drains collected from patients who had undergone THA compared to TKA (p<0.001). BMI significantly influenced levels of serum drug, that resulted to be higher in patients with BMI<25 (p= 0.01). Conclusion Levobupivacaine from collected shed blood that would have been returned to the patient, was below toxicity level at 2 and 5 hours after LIA during total joint replacement. The average serum levobupivacaine concentration was found to be higher in drains taken from THA patients than TKA patients. Patients with lower BMI demonstrated the highest levels of levobupivacaine in shed blood and a lower blood volume needed for central nervous system toxicity. Therefore, in patients with a lower BMI undergoing THA, anaesthetic dosage should be reduced or autotransfusion should be avoided to prevent potential risks of toxicity

Regulation of Serum Sphingolipids in Andean Children Born and Living at High Altitude (3775 m)

Recent studies on Andean children indicate a prevalence of dyslipidemia and hypertension compared to dwellers at lower altitudes, suggesting that despite similar food intake and daily activities, they undergo different metabolic adaptations. In the present study, the sphingolipid pattern was investigated in serum of 7 underweight (UW), 30 normal weight (NW), 13 overweight (OW), and 9 obese (O) Andean children by liquid chromatography-mass spectrometry (LC-MS). Results indicate that levels of Ceramides (Cers) and sphingomyelins (SMs) correlate positively with biochemical parameters (except for Cers and Vitamin D, which correlate negatively), whereas sphingosine-1-phosphate (S1P) correlates negatively. Correlation results and LC-MS data identify the axis high density lipoprotein-cholesterol (HDL-C), Cers, and S1P as related to hypoxia adaptation. Specifically UW children are characterized by increased levels of S1P compared to O and lower levels of Cers compared to NW children. Furthermore, O children show lower levels of S1P and similar levels of Cers and SMs as NW. In conclusion, our results indicate that S1P is the primary target of hypoxia adaptation in Andean children, and its levels are associated with hypoxia tolerance. Furthermore, S1P can act as marker of increased risk of metabolic syndrome and cardiac dysfunction in young Andeans living at altitude.This research was supported by Italian Space Agency (ASI) project: DC-VUM-2016-068.Peer reviewe

Characterization of Microfragmented Adipose Tissue Architecture, Mesenchymal Stromal Cell Content and Release of Paracrine Mediators

The use of microfragmented adipose tissue (µFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis (OA), is gaining popularity, following positive results reported in recent case series and clinical trials. Although these outcomes were postulated to rely on paracrine signals, to date, a thorough fingerprint of released molecules is largely missing. The purpose of this study was to first characterize both structure and cell content of unprocessed lipoaspirate (LA) and µFAT, and further identify and frame the array of signaling factors in the context of OA disease, by means of high throughput qRT-PCR for extracellular-vesicle (EV) embedded miRNAs and proteomics for tissue and secreted factors. Cell count showed reduction of blood cells in µFAT, confirmed by histological and flow cytometry analyses, that also showed a conserved presence of structural, endothelial and stromal components and pericytes. In the secretome, 376 and 381 EV-miRNAs in LA and µFAT, respectively, were identified. In particular, most abundant and µFAT upregulated EV-miRNAs were mainly recapitulating those already reported as ASC-EVs-specific, with crucial roles in cartilage protection and M2 macrophage polarization, while only a scarce presence of those related to blood cells emerged. Furthermore, secretome proteomic analysis revealed reduction in µFAT of acute phase factors driving OA progression. Taken together, these results suggest that processing of LA into µFAT allows for removal of blood elements and maintenance of tissue structure and stromal cell populations, and possibly the increase of OA-protective molecular features. Thus, microfragmentation represents a safe and efficient method for the application of adipose tissue properties in the frame of musculoskeletal disorders

Molecular Fingerprint of BMD Patients Lacking a Portion in the Rod Domain of Dystrophin

BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance

Effects of Omega-3 and Antioxidant Cocktail Supplement on Prolonged Bed Rest: Results from Serum Proteome and Sphingolipids Analysis

Physical inactivity or prolonged bed rest (BR) induces muscle deconditioning in old and young subjects and can increase the cardiovascular disease risk (CVD) with dysregulation of the lipemic profile. Nutritional interventions, combining molecules such as polyphenols, vitamins and essential fatty acids, can influence some metabolic features associated with physical inactivity and decrease the reactive oxidative and nitrosative stress (RONS). The aim of this study was to detect circulating molecules correlated with BR in serum of healthy male subjects enrolled in a 60-day BR protocol to evaluate a nutritional intervention with an antioxidant cocktail as a disuse countermeasure (Toulouse COCKTAIL study). The serum proteome, sphingolipidome and nitrosoproteome were analyzed adopting different mass spectrometry-based approaches. Results in placebo-treated BR subjects indicated a marked decrease of proteins associated with high-density lipoproteins (HDL) involved in lipemic homeostasis not found in the cocktail-treated BR group. Moreover, long-chain ceramides decreased while sphingomyelin increased in the BR cocktail-treated group. In placebo, the ratio of S-nitrosylated/total protein increased for apolipoprotein D and several proteins were over-nitrosylated. In cocktail-treated BR subjects, the majority of protein showed a pattern of under-nitrosylation, except for ceruloplasmin and hemopexin, which were over-nitrosylated. Collectively, data indicate a positive effect of the cocktail in preserving lipemic and RONS homeostasis in extended disuse conditions

Characterization of Microfragmented Adipose Tissue Architecture, Mesenchymal Stromal Cell Content and Release of Paracrine Mediators

The use of microfragmented adipose tissue (&micro;FAT) for the treatment of musculoskeletal disorders, especially osteoarthritis (OA), is gaining popularity, following positive results reported in recent case series and clinical trials. Although these outcomes were postulated to rely on paracrine signals, to date, a thorough fingerprint of released molecules is largely missing. The purpose of this study was to first characterize both structure and cell content of unprocessed lipoaspirate (LA) and &micro;FAT, and further identify and frame the array of signaling factors in the context of OA disease, by means of high throughput qRT-PCR for extracellular-vesicle (EV) embedded miRNAs and proteomics for tissue and secreted factors. Cell count showed reduction of blood cells in &micro;FAT, confirmed by histological and flow cytometry analyses, that also showed a conserved presence of structural, endothelial and stromal components and pericytes. In the secretome, 376 and 381 EV-miRNAs in LA and &micro;FAT, respectively, were identified. In particular, most abundant and &micro;FAT upregulated EV-miRNAs were mainly recapitulating those already reported as ASC-EVs-specific, with crucial roles in cartilage protection and M2 macrophage polarization, while only a scarce presence of those related to blood cells emerged. Furthermore, secretome proteomic analysis revealed reduction in &micro;FAT of acute phase factors driving OA progression. Taken together, these results suggest that processing of LA into &micro;FAT allows for removal of blood elements and maintenance of tissue structure and stromal cell populations, and possibly the increase of OA-protective molecular features. Thus, microfragmentation represents a safe and efficient method for the application of adipose tissue properties in the frame of musculoskeletal disorders