Effect of 5-Aminolevulinic Acid (5-ALA) in "ALADENT" Gel Formulation and Photodynamic Therapy (PDT) against Human Oral and Pancreatic Cancers.

Oral squamous-cell and pancreatic carcinomas are aggressive cancers with a poor outcome. Photodynamic therapy (PDT) consists of the use of photosensitizer-induced cell and tissue damage that is activated by exposure to visible light. PDT selectively acts on cancer cells, which have an accumulation of photosensitizer superior to that of the normal surrounding tissues. 5-aminolevulinic acid (5-ALA) induces the production of protoporphyrin IX (PpIX), an endogenous photosensitizer activated in PDT. This study aimed to test the effect of a new gel containing 5% v/v 5-ALA (ALAD-PDT) on human oral CAL-27 and pancreatic CAPAN-2 cancer cell lines. The cell lines were incubated in low concentrations of ALAD-PDT (0.05%, 0.10%, 0.20%, 0.40%, 0.75%, 1.0%) for 4 h or 8 h, and then irradiated for 7 min with 630 nm RED light. The cytotoxic effects of ALAD-PDT were measured using the MTS assay. Apoptosis, cell cycle, and ROS assays were performed using flow cytometry. PpIX accumulation was measured using a spectrofluorometer after 10 min and 24 and 48 h of treatment. The viability was extremely reduced at all concentrations, at 4 h for CAPAN-2 and at 8 h for CAL-27. ALAD-PDT induced marked apoptosis rates in both oral and pancreatic cancer cells. Elevated ROS production and appreciable levels of PpIX were detected in both cell lines. The use of ALA-PDT as a topical or intralesional therapy would permit the use of very low doses to achieve effective results and minimize side effects. ALAD-PDT has the potential to play a significant role in complex oral and pancreatic anticancer therapies.

Exploring the Immunomodulatory Potential of Pancreatic Cancer-Derived Extracellular Vesicles through Proteomic and Functional Analyses.

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network (p = 1 × 10-16) and were involved in the "Immune System" (FDR: 1.10 × 10-24 and 3.69 × 10-19, respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

BACKGROUND: The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. METHODS: We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. RESULTS: All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. CONCLUSIONS: Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Multi-omics staging of locally advanced rectal cancer predicts treatment response: a pilot study.

Treatment response assessment of rectal cancer patients is a critical component of personalized cancer care and it allows to identify suitable candidates for organ-preserving strategies. This pilot study employed a novel multi-omics approach combining MRI-based radiomic features and untargeted metabolomics to infer treatment response at staging. The metabolic signature highlighted how tumor cell viability is predictively down-regulated, while the response to oxidative stress was up-regulated in responder patients, showing significantly reduced oxoproline values at baseline compared to non-responder patients (p-value < 10-4). Tumors with a high degree of texture homogeneity, as assessed by radiomics, were more likely to achieve a major pathological response (p-value < 10-3). A machine learning classifier was implemented to summarize the multi-omics information and discriminate responders and non-responders. Combining all available radiomic and metabolomic features, the classifier delivered an AUC of 0.864 (± 0.083, p-value < 10-3) with a best-point sensitivity of 90.9% and a specificity of 81.8%. Our results suggest that a multi-omics approach, integrating radiomics and metabolomic data, can enhance the predictive value of standard MRI and could help to avoid unnecessary surgical treatments and their associated long-term complications.

Cellular Strategies for Separating GvHD from GvL in Haploidentical Transplantation.

GvHD still remains, despite the continuous improvement of transplantation platforms, a fearful complication of transplantation from allogeneic donors. Being able to separate GvHD from GvL represents the greatest challenge in the allogeneic transplant setting. This may be possible through continuous improvement of cell therapy techniques. In this review, current cell therapies are taken into consideration, which are based on the use of TCR alpha/beta depletion, CD45RA depletion, T regulatory cell enrichment, NK-cell-based immunotherapies, and suicide gene therapies in order to prevent GvHD and maximally amplify the GvL effect in the setting of haploidentical transplantation.

Extracellular vesicles (EVs): A promising therapeutic tool in the heart tissue regeneration.

Mesenchymal stem cells (MSCs) treatment has been widely explored as a therapy for myocardial infarction, peripheral ischemic vascular diseases, dilated cardiomyopathy, and pulmonary hypertension. Latest in vitro studies suggest that MSCs can differentiate into contractile cardiomyocytes. One of the best-characterized MSCs products are MSCs-derived extracellular vesicles (EVs). EVs are crucial paracrine effectors of MSCs. Based on previous works, paracrine effects of MSCs play a primary role in the regenerative ability. Hence, in the current paper, we focused our attention on an alternative approach, exploiting products derived from human dental pulp stem cells (hDPSCs) rather than MSCs themselves, which may denote a cost-effective and safer approach. The focus has been on EVs and the bioactive molecules they contain to evaluate their ability to influence the differentiation process toward cardiomyogenic lineage. The expression of GATA4, ACTC1, CX43, and Nkx2.5 was evaluated using Immunofluorescence, real time-PCR, and Western blotting analyses. Furthermore, the expression profiling analysis of the microRNA hsa-miR-200c-3p, targeting the GATA4 gene, was studied. The hsa-miR-200c-3p was found significantly down-regulated in both c-hDPSCs + EVs-hDPSCs and c-hDPSCs + EVs-HL-1 compared to untreated c-hDPSCs underlying a possible epigenetic mechanism behind the prevalent up-regulation of its targeted GATA4 gene. The aim of the present work was to develop an in vitro model of hDPSCs able to differentiate into cardiomyocytes in order to investigate the role of EVs derived from hDPSCs and derived from HL-1 cardiomyocyte cell line in modulating the differentiation process toward cardiomyogenic lineage.

Quantum Molecular Resonance Inhibits NLRP3 Inflammasome/Nitrosative Stress and Promotes M1 to M2 Macrophage Polarization: Potential Therapeutic Effect in Osteoarthritis Model In Vitro.

This study aimed to investigate the anti-inflammatory effects of Quantum Molecular Resonance (QMR) technology in an in vitro model of osteoarthritis-related inflammation. The study used THP-1-derived macrophages stimulated with lipopolysaccharide and hyaluronic acid fragments to induce the expression of inflammatory cytokines and nitrosative stress. QMR treatment inhibited COX-2 and iNOS protein expression and activity and reduced NF-κB activity. Furthermore, QMR treatment led to a significant reduction in peroxynitrite levels, reactive nitrogen species that can form during inflammatory conditions, and restored tyrosine nitration values to those similar to sham-exposed control cells. We also investigated the effect of QMR treatment on inflammasome activation and macrophage polarization in THP-1-derived macrophages. Results showed that QMR treatment significantly decreased NLRP3 and activated caspase-1 protein expression levels and downregulated IL-18 and IL-1ß protein expression and secretion. Finally, our findings indicate that QMR treatment induces a switch in macrophage polarization from the M1 phenotype to the M2 phenotype.

Extracellular vesicles during the three trimesters of pregnancy.

OBJECTIVES: Extracellular vesicles (EVs) are cell-derived particles released during different pathophysiological processes and emerging as relevant players in inter-cellular crosstalk. Previous studies have highlighted the role of EVs as potential biomarkers for several pregnancy complications, including miscarriage, pre-eclampsia and gestational diabetes. Despite that, the actual distribution of EVs through gestation has not been reported yet. The aim of this study was to report the concentration of different sub-types of EVs in the first, second and third trimester of pregnancy and to correlate them with different pregnancy and ultrasound characteristics. STUDY DESIGNS: Prospective observational study including uncomplicated pregnancies in the first, second and third trimester of pregnancy. The first aim of the study was to report the concentration of the EVs derived from endothelial, epithelial, platelet and leukocyte cells of maternal peripheral blood samples in the first, second and third trimester pregnancy using polychromatic flow cytometry. The secondary aim was to correlate EVs with neonatal birthweight and fetal Dopplers, including uterine and umbilical arteries. Un and multivariate analyses were used to compute the data. RESULTS: 64 women (20 in the first, 22 in the second and 22 in the third trimester of pregnancies) were included in the analysis. There was no difference in the median concentration of either platelet, leukocyte and endothelial EVs between the first, second and third trimester of pregnancy. The concentration of epithelial derived EVs was higher in the third compared to first and second trimester of pregnancy. When analyzing the percentage of EV vesicles through gestation, there was no difference in the percentage of either leukocyte or endothelial EVs through gestation. Conversely, the median percentage of platelet derived vesicles was higher in the first (48.7 %, IQR 34.1-58.5) compared to second (34.0 %, IQR 22.7-44.9) and third (9.13 %, IQR 5.01-12.1) trimester of pregnancy, while the median percentage of third trimester (6.01, IQR 2.42-7.34) epithelial derived vesicles was higher than that of the second (1.53 %, IQR 0.65-2.98), but not of the first (4.45 %, IQR 1.44-6.07) trimester. Finally, we found no association between the median concentration or percentage of endothelial, epithelial, leukocyte vesicles, neonatal birthweight and fetal or maternal Dopplers. CONCLUSIONS: Distribution of EVs examined does not change during the three trimesters of pregnancy and is not influenced by neonatal birthweight or maternal and fetal Dopplers. The findings from this study allows a more objective interpretation of studies comparing EVs in pregnancies with compared to those without obstetric complication. EVs in future can be used for "liquid biopsy" for the early diagnosis of pathological pregnancies up to the development of possible screening protocols.

Extracellular LGALS3BP: a potential disease marker and actionable target for antibody-drug conjugate therapy in glioblastoma.

Glioblastoma multiforme (GBM) is a lethal disease characterized by an overall survival of about 1 year, making it one of the most aggressive tumours, with very limited therapeutic possibilities. Specific biomarkers for early diagnosis as well as innovative therapeutic strategies are urgently needed to improve the management of this deadly disease. In this work, we demonstrated that vesicular galectin-3-binding protein (LGALS3BP), a glycosylated protein overexpressed in a variety of human malignancies, is a potential GBM disease marker and can be efficiently targeted by a specific antibody-drug conjugate (ADC). Immunohistochemical analysis on patient tissues showed that LGALS3BP is highly expressed in GBM and, compared with healthy donors, the amount of vesicular but not total circulating protein is increased. Moreover, analysis of plasma-derived extracellular vesicles from mice harbouring human GBM revealed that LGALS3BP can be used for liquid biopsy as a marker of disease. Finally, an ADC targeting LGALS3BP, named 1959-sss/DM4, specifically accumulates in tumour tissue, producing a potent and dose-dependent antitumor activity. In conclusion, our work provides evidence that vesicular LGALS3BP is a potential novel GBM diagnostic biomarker and therapeutic target deserving further preclinical and clinical validation.

Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

CAR-T-Derived Extracellular Vesicles: A Promising Development of CAR-T Anti-Tumor Therapy.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples.

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Kinetics of Circulating Extracellular Vesicles Over the 24-Hour Dosing Interval After Low-Dose Aspirin Administration in Patients at Cardiovascular Risk.

Extracellular vesicles (EVs) are small vesicles deriving from all cell types during cell activation, involved in transcellular communication, and regarded as predictors of vascular damage and of cardiovascular events. We tested the hypothesis that, in patients on chronic low-dose aspirin treatment for cardiovascular prevention, aspirin may affect the release of EVs within the 24-hour interval. We enrolled 84 patients, mostly at high or very high cardiovascular risk, on chronic low-dose aspirin treatment. The numbers of circulating EVs (cEVs) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leucocyte-derived) were assessed immediately before, and after 10 and 24 hours of a witnessed aspirin administration. Platelet cyclooxygenase 1 (COX-1) recovery was characterized by measuring serum thromboxane B2 (sTXB2 ) at the same timepoints. Nine healthy participants were also enrolled. In patients, daily aspirin administration acutely inhibited after 10 hours following aspirin administrations the release of cEVs (total and leukocyte-derived) and annexinV+ cEVs (total, platelet-derived, endothelial-derived, and leukocyte-derived), with a rapid recovery at 24 hours. The inhibition after 10 hours suggests a COX-1-dependent mechanism. Interestingly, the slope of platelet-derived and of annexinV+ platelet-derived cEVs were both directly related to sTXB2 slope and COX-1 messenger RNA, raising the hypothesis that vice versa, cEVs may affect the rate of COX-1 recovery and the subsequent duration of aspirin effect. In healthy participants, no circadian difference was observed, except for leukocyte-derived cEVs. Our findings suggest a previously unappreciated effect of aspirin on the kinetics of a subset of cEVs possibly contributing to the cardioprotective effects of this drug.

Resveratrol Derivative Exhibits Marked Antiproliferative Actions, Affecting Stemness in Pancreatic Cancer Cells.

Pancreatic cancer (PC) is one of the deadliest malignancies, with an increasing incidence and limited response to current therapeutic options. Therefore, more effective and low-toxic agents are needed to improve PC patients' outcomes. Resveratrol (RSV) is a natural polyphenol with multiple biological properties, including anticancer effects. In this study, we explored the antiproliferative activities of newly synthetized RSV analogues in a panel of PC cell lines and evaluated the physicochemical properties of the most active compound. This derivative exhibited marked antiproliferative effects in PC cells through mechanisms involving DNA damage, apoptosis induction, and interference in cell cycle progression, as assessed using flow cytometry and immunoblot analysis of cell cycle proteins, PARP cleavage, and H2AX phosphorylation. Notably, the compound induced a consistent reduction in the PC cell subpopulation with a CD133+EpCAM+ stem-like phenotype, paralleled by dramatic effects on cell clonogenicity. Moreover, the RSV derivative had negligible toxicity against normal HFF-1 cells and, thus, good selectivity index values toward PC cell lines. Remarkably, its higher lipophilicity and stability in human plasma, as compared to RSV, might ensure a better permeation along the gastrointestinal tract. Our results provide insights into the mechanisms of action contributing to the antiproliferative activity of a synthetic RSV analogue, supporting its potential value in the search for effective and safe agents in PC treatment.

Tumor-Educated Platelet Extracellular Vesicles: Proteomic Profiling and Crosstalk with Colorectal Cancer Cells.

BACKGROUND: Platelet-cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. METHODS: We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. RESULTS: The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. CONCLUSIONS: We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.

Reduced platelet glycoprotein Ibα shedding accelerates thrombopoiesis and COX-1 recovery: implications for aspirin dosing regimen.

Cardiovascular (CV) disease prevention with low-dose aspirin can be less effective in patients with a faster recovery of platelet (PLT) cyclooxygenase (COX)-1 activity during the 24-hour dosing interval. We previously showed that incomplete suppression of TXA2 over 24 hours can be rescued by a twice daily aspirin regimen. Here we show that reduced PLT glycoprotein (GP)Ibα shedding characterizes patients with accelerated COX-1 recovery and may contribute to higher thrombopoietin (TPO) production and higher rates of newly formed PLT, escaping aspirin inhibition over 24 hours. Two hundred aspirin-treated patients with high CV risk (100 with type 2 diabetes mellitus) were stratified according to the kinetics of PLT COX-1 activity recovery during the 10- to 24-hour dosing interval. Whole proteome analysis showed that PLT from patients with accelerated COX-1 recovery were enriched in proteins involved in cell survival, inhibition of apoptosis and cellular protrusion formation. In agreement, we documented increased plasma TPO, megakaryocyte maturation and proplatelet formation, and conversely increased PLT galactose and reduced caspase 3, phosphatidylserine exposure and ADAM17 activation, translating into diminished GPIbα cleavage and glycocalicin (GC) release. Treatment of HepG2 cells with recombinant GC led to a dose-dependent reduction of TPO mRNA in the liver, suggesting that reduced GPIbα ectodomain shedding may unleash thrombopoiesis. A cluster of clinical markers, including younger age, non-alcoholic fatty liver disease, visceral obesity and higher TPO/GC ratio, predicted with significant accuracy the likelihood of faster COX-1 recovery and suboptimal aspirin response. Circulating TPO/GC ratio, reflecting a dysregulation of PLT lifespan and production, may provide a simple tool to identify patients amenable to more frequent aspirin daily dosing.

Basophil Activation Test with Different Polyethylene Glycols in Patients with Suspected PEG Hypersensitivity Reactions.

Allergic reactions to COVID-19 vaccine components are rare but should be considered. Polyethylene glycol (PEG) is responsible for anaphylaxis in mRNA vaccines. Skin tests have been used in the allergological work-up programs for COVID-19 vaccine evaluation. However, the reproducibility of the skin prick test is time-dependent and the reactivity declines over time. Therefore, we combined the administration of the skin tests with the basophil activation test (BAT) using PEG2000, PEG4000 and DMG-PEG2000, where the BAT was considered positive when the percentage of activated basophils was higher than 6%, 5% and 6.5%, for PEG 4000, PEG2000 and DMG-PEG2000, respectively. To this end, among the subjects that underwent allergy counseling at the Allergy Unit of our Institution during the 2020/2021 vaccination campaign, 13 patients had a suggested medical history of PEG/drug hypersensitivity and were enrolled together with 10 healthy donors. Among the enrolled patients 2 out of 13 tested patients were positive to the skin test. The BAT was negative in terms of the percentages of activated basophils in all analyzed samples, but the stimulation index (SI) was higher than 2.5 in 4 out of 13 patients. These data evidenced that, when the SI is higher than 2.5, even in the absence of positivity to BAT, the BAT to PEG may be a useful tool to be coupled to skin tests to evidence even low-grade reactions.

Extracellular Vesicles in Regenerative Processes Associated with Muscle Injury Recovery of Professional Athletes Undergoing Sub Maximal Strength Rehabilitation.

Platelet-rich plasma (PRP) has great potential in regenerative medicine. In addition to the well-known regenerative potential of secreted growth factors, extracellular vesicles (EVs) are emerging as potential key players in the regulation of tissue repair. However, little is known about their therapeutic potential as regenerative agents. In this study, we have identified and subtyped circulating EVs (platelet-, endothelial-, and leukocyte-derived EVs) in the peripheral blood of athletes recovering from recent muscular injuries and undergoing a submaximal strength rehabilitation program. We found a significant increase in circulating platelet-derived EVs at the end of the rehabilitation program. Moreover, EVs from PRP samples were isolated by fluorescence-activated cell sorting and analyzed by label-free proteomics. The proteomic analysis of PRP-EVs revealed that 32% of the identified proteins were associated to "defense and immunity", and altogether these proteins were involved in vesicle-mediated transport (GO: 0016192; FDR = 3.132 × 10-19), as well as in wound healing (GO: 0042060; FDR = 4.252 × 10-13) and in the events regulating such a process (GO: 0061041; FDR = 2.812 × 10-12). Altogether, these data suggest that platelet-derived EVs may significantly contribute to the regeneration potential of PRP preparations.

Rapid Detection of Brettanomyces bruxellensis in Wine by Polychromatic Flow Cytometry.

Brettanomyces bruxellensis is found in several fermented matrices and produces relevant alterations to the wine quality. The methods usually used to identify B. bruxellensis contamination are based on conventional microbiological techniques that require long procedures (15 days), causing the yeast to spread in the meantime. Recently, a flow cytometry kit for the rapid detection (1-2 h) of B. bruxellensis in wine has been developed. The feasibility of the method was assessed in a synthetic medium as well as in wine samples by detecting B. bruxellensis in the presence of other yeast species (Saccharomyces cerevisiae and Pichia spp.) and at the concentrations that produce natural contaminations (up to 105 cells/mL), as well as at lower concentrations (103-102 cells/mL). Wine samples naturally contaminated by B. bruxellensis or inoculated with four different strains of B. bruxellensis species together with Saccharomyces cerevisiae and Pichia spp., were analyzed by flow cytometry. Plate counts were carried out in parallel to flow cytometry. We provide evidence that flow cytometry allows the rapid detection of B. bruxellensis in simple and complex mixtures. Therefore, this technique has great potential for the detection of B. bruxellensis and could allow preventive actions to reduce wine spoilage.

Circulating Extracellular Vesicles: Their Role in Patients with Abdominal Aortic Aneurysm (AAA) Undergoing EndoVascular Aortic Repair (EVAR).

Abdominal aortic aneurysm (AAA) is a frequent aortic disease. If the diameter of the aorta is larger than 5 cm, an open surgical repair (OSR) or an endovascular aortic repair (EVAR) are recommended. To prevent possible complications (i.e., endoleaks), EVAR-treated patients need to be monitored for 5 years following the intervention, using computed tomography angiography (CTA). However, this radiological method involves high radiation exposure in terms of CTA/year. In such a context, the study of peripheral-blood-circulating extracellular vesicles (pbcEVs) has great potential to identify biomarkers for EVAR complications. We analyzed several phenotypes of pbcEVs using polychromatic flow cytometry in 22 patients with AAA eligible for EVAR. From each enrolled patient, peripheral blood samples were collected at AAA diagnosis, and after 1, 6, and 12 months following EVAR implantation, i.e. during the diagnostic follow-up protocol. Patients developing an endoleak displayed a significant decrease in activated-platelet-derived EVs between the baseline condition and 6 months after EVAR intervention. Furthermore, we also observed, that 1 month after EVAR implantation, patients developing an endoleak showed higher concentrations of activated-endothelial-derived EVs than patients who did not develop one, suggesting their great potential as a noninvasive and specific biomarker for early identification of EVAR complications.