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Extracellular vesicles, released by cell pullulation, are surrounded by a phospholipid bilayer and carry proteins as well and genetic material. It has been shown that extracellular vesicles mediate intercellular communication in several conditions, such as inflammation, immunodeficiency, tumor growth, and viral infections. Here, we analyzed circulating levels of extracellular vesicles in order to clarify their role in chronic inflammation mechanisms characterizing HIV patients.We analyzed and subtyped circulating levels of extracellular vesicles, through a recently developed flow cytometry method. In detail, endothelial-derived extracellular vesicles (CD31+/CD41a-/CD45-, EMVs), extracellular vesicles stemming from leukocytes (CD45+, LMVs) and platelets (CD41a+/CD31+) were identified and enumerated. Moreover, we analyzed the extracellular vesicle protein cargo with proteomic analysis.Circulating levels of total extracellular vesicles, EMVs and LMVs were significantly lower in the HIV+ patients than in healthy subjects, whereas platelet-derived extracellular vesicles resulted higher in patients than in the healthy population. Proteomic analysis showed the upregulation of gammaIFN and IL1α, and down-regulation of OSM, NF-kB, LIF, and RXRA signaling resulted activated in this patients.These data demonstrate, for the first time that HIV infection induces the production of extracellular vesicles containing mediators that possibly feed the chronic inflammation and the viral replication. These two effects are connected as the inflammation itself induces the viral replication. We, therefore, hypothesize that HIV infection inhibits the production of extracellular vesicles that carry anti-inflammatory molecules.

Extracellular Vesicles (EVs) are cell-derived particles released during different pathophysiological processes, circulating in many body fluids and mediating the inter-cellular crosstalk. We have analyzed, for the first time, different EV phenotypes and concentrations in the peripheral blood of uncomplicated pregnant women.In this prospective case-control study, uncomplicated singleton pregnant women at term (N = 59) and aged matched non-pregnant women (N = 21) were enrolled. Freshly drowned peripheral blood samples were stained for flow cytometry analyses of EVs.EVs derived from platelets, leukocytes, endothelial and epithelial cells were identified and counted. Platelet-derived EVs were higher in pregnant compared to non-pregnant women, both in terms of absolute counts (2064.4 ± 1156.3 vs 701.1 ± 378.8; p 0.0001) and percentages (27.6 ± 17.2 vs 10.7 ± 5.9; p 0.0001). The opposite pattern was observed both for concentrations of endothelial-EV counts (525.8 ± 499.6 vs 844.7 ± 652.9; p = 0.007) and percentages (6.1 ± 5.5 vs 11.8 ± 8.0; p 0.0001) and leukocyte-derived EV percentages (10.2 ± 7.4 vs 17.9 ± 11.2; p = 0.002) EVs.Uncomplicated pregnancies are characterized by a specific EV signature. These cell-derived particles may therefore represent promising biomarkers of different pathological conditions complicating pregnancies, such as preeclampsia or preterm birth.

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and β-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

Simple Summary The dysregulation of Wnt pathways is involved in colorectal carcinogenesis. H2O2 differentially regulates the Wnt/β-Catenin pathway in colorectal cancer (CRC), but the molecular mechanisms remain unclear. Cellular stress might also stimulate APC protein production retaining some functions in N-terminus and lead to cancer progression. The effect of oxidative distress on Wnt/β-catenin signaling in the light of APC functions is unknown. We exposed starved HCT116, SW480 and SW620 CRC cell lines to H2O2-induced short-term oxidative stress. This treatment promoted the activation of β-Catenin and increased cytoplasmic APC. H2O2 regulated gene expression, related to cellular phenotype and stimulated both Wnt/β-Catenin-dependent and -independent signaling. These findings suggest that oxidative distress may influence APC functions in Wnt signaling and open up new perspectives to develop personalized therapeutic approaches for CRC. Abstract Colorectal cancer (CRC) is a multistep process that arises in the colic tissue microenvironment. Oxidative stress plays a role in mediating CRC cell survival and progression, as well as promoting resistance to therapies. CRC progression is associated with Wnt/β-Catenin signaling dysregulation and loss of proper APC functions. Cancer recurrence/relapse has been attributed to altered ROS levels, produced in a cancerous microenvironment. The effect of oxidative distress on Wnt/β-Catenin signaling in the light of APC functions is unclear. This study evaluated the effect of H2O2-induced short-term oxidative stress in HCT116, SW480 and SW620 cells with different phenotypes of APC and β-Catenin. The modulation and relationship of APC with characteristic molecules of Wnt/β-Catenin were assessed in gene and protein expression. Results indicated that CRC cells, even when deprived of growth factors, under acute oxidative distress conditions by H2O2 promote β-Catenin expression and modulate cytoplasmic APC protein. Furthermore, H2O2 induces differential gene expression depending on the cellular phenotype and leading to favor both Wnt/Catenin-dependent and -independent signaling. The exact mechanism by which oxidative distress can affect Wnt signaling functions will require further investigation to reveal new scenarios for the development of therapeutic approaches for CRC, in the light of the conserved functions of APC.

The efficacy of SARS-CoV-2 mRNA-based vaccines in preventing COVID-19 disease has been extensively demonstrated; however, it is of uttermost importance to acquire knowledge on the persistence of immune-protection both in terms of levels of neutralizing antibodies and specialized memory cells. This can provide important scientific basis for decisions on the need of additional vaccine doses and on when these should be administered thus resulting in an improvement in vaccination schedules. Here, we briefly report the changes in antibody levels and cellular immunity following BNT162b2 administration. We show an important fall in anti S1-Spike antibodies in BNT162b2 vaccinated subjects overtime, paralleled by a contextual consolidation of specific spike (S) T-cells, mainly of the CD8+ compartment. Contrariwise, CD4+ S-specific response shows a considerable interindividual variability. These data suggest that the well-known antibody drop in vaccinated subjects is replaced by memory cell consolidation that can protect from severe adverse effects of SARS-CoV-2 infection.

COVID-19 pandemic has hit people’s health, economy, and society worldwide. Great confidence in returning to normality has been placed in the vaccination campaign. The knowledge of individual immune profiles and the time required to achieve immunological protection is crucial to choose the best vaccination strategy. We compared anti-S1 antibody levels produced over time by BNT162b2 and AZD1222 vaccines and evaluated the induction of antigen-specific T-cells. A total of 2569 anti-SARS-CoV-2 IgG determination on dried blood spot samples were carried out, firstly in a cohort of 1181 individuals at random time-points, and subsequently, in an independent cohort of 88 vaccinated subjects, up to the seventeenth week from the first dose administration. Spike-specific T-cells were analysed in seronegative subjects between the two doses. AZD1222 induced lower anti-S1 IgG levels as compared to BNT162b2. Moreover, 40% of AZD1222 vaccinated subjects and 3% of BNT162b2 individuals resulted in seronegative during all the time-points, between the two doses. All these subjects developed antigen-specific T cells, already after the first dose. These results suggest that this test represents an excellent tool for a wide sero-surveillance. Both vaccines induce a favourable immune profile guaranteeing efficacy against severe adverse effects of SARS-CoV-2 infection, already after the first dose administration.

Protease proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of LDL cholesterol clearance and has been associated with cardiovascular risk. PCSK9 inhibitors increase in vivo circulating endothelial progenitor cells (EPCs), a subtype of immature cells involved in ongoing endothelial repair. We hypothesized that the effect of PCSK9 on vascular homeostasis may be mediated by EPCs in patients with or without type 2 diabetes mellitus (T2DM). Eighty-two patients (45 with, 37 without T2DM) at high cardiovascular risk were enrolled in this observational study. Statin treatment was associated with higher circulating levels of PCSK9 in patients with and without T2DM (p neg/CD34bright (total EPC compartment) (p = 0.016) and CD45neg/CD34bright/CD146neg (early EPC) (p = 0.040) only among patients with T2DM. In the whole group of patients, statin treatment was the only independent predictor of low number of CD45neg/CD34bright (β = − 0.230; p = 0.038, adjusted R2 = 0.041). Among T2DM patients, PCSK9 circulating levels were inversely related and predicted both the number of CD45neg/CD34bright (β = − 0.438; p = 0.003, adjusted R2 = 0.173), and CD45neg/CD34bright/CD146neg (β = − 0.458; p = 0.002, adjusted R2 = 0.191) independently of age, gender, BMI and statin treatment. In high-risk T2DM patients, high endogenous levels of PCSK9 may have a detrimental effect on EPCs by reducing the endothelial repair and worsening the progression of atherothrombosis.

Regulatory T Cells (Tregs) are a T-lymphocyte subset involved in the maintenance of immune peripheral tolerance. Despite evidence of the adaptive immune system’s role in Alzheimer’s Disease (AD), the involvement of Tregs is still not clear. We focused on the Flow-Cytometry analysis of the Treg frequencies and phenotypes in the AD. The aim of the study is to analyse similarities and differences in Tregs profile between Alzheimer’s Disease and Multiple Sclerosis. Regulatory T Cells (CD4+/CD25high/CD127low-neg) were identified using an innovative Flow Cytometry method and subtyped as Resting (analysed CD45RApos/CD25dim), Activated (CD45RAneg/CD25bright) and Secreting (CD45RAneg/CD25dim) cells. Our data demonstrate a significant decrease in the total and Resting Tregs in AD patients when compared to healthy subjects. The percentage of the results of the Resting Tregs were also reduced in MS patients together with a parallel frequency increase of Activated Tregs. Our data suggest that altered Treg phenotypes observed in both diseases could play a role in the impairment of the Treg-mediated immunological tolerance, recalling a possible link between the two pathologies. Given that this study was conducted on a restricted population, if confirmed by a further and enlarged study, the implications of the autoimmune mechanisms in AD pathophysiology could open new immunotherapeutic perspectives based on Treg modulation.

Simple Summary Purpose of this study was to investigate the prognostic and predictive role of blood circulating extracellular vesicles (EVs) in patients with advanced non-small cell lung cancer treated with immunotherapy. A newly optimized flow cytometry protocol was applied for identification and subtyping of blood circulating EVs in a total cohort of 59 NSCLC patients, which included 31 patients treated with anti-PD-1/PD-L1 agents and 28 patients treated with traditional chemotherapy. Our results show that pre-treatment concentration of blood circulating endothelial-derived EVs was correlated with overall survival and clinical response in patients treated with immunotherapy. Additionally, proteomic analysis of purified blood circulating EVs indicated differences in EV protein cargo between responders and non-responders to immunotherapy. These findings may pave the way to the identification of novel immunotherapy biomarkers in patients with advanced NSCLC. Abstract Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

Extracellular vesicles (EVs) actively participate in inter-cellular crosstalk and have progressively emerged as key players of organized communities of cells within multicellular organisms in health and disease. For these reasons, EVs are attracting the attention of many investigators across different biomedical fields. In this scenario, the possibility to study specific placental-derived EVs in the maternal peripheral blood may open novel perspectives in the development of new early biomarkers for major obstetric pathological conditions. Here we reviewed the involvement of EVs in feto–maternal crosstalk mechanisms, both in physiological and pathological conditions (preeclampsia, fetal growth restriction, preterm labor, gestational diabetes mellitus), also underlining the usefulness of EV characterization in maternal–fetal medicine.

Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the &ldquo, tip of the iceberg&rdquo, of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV &lt, 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

The recent introduction of the “precision medicine” concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326− EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

In the pathophysiology of neurodegenerative disorders, the role of misfolded protein deposition leading to neurodegeneration has been primarily discussed. In the last decade, however, it has been proposed a parallel involvement of innate immune activation, chronic inflammation and adaptive immunity in the neurodegeneration mechanisms triggered by proteinopathies. New insights in the neurodegenerative field strongly suggest a role for the immune system in the pathophysiology of neurodegenerative disorders. Therefore, the hypothesis underlining the modulation of the innate and the adaptive immune system in the events linked to brain deposition of misfolded proteins could open new perspectives in the setting of specific immunotherapeutic strategies for the treatment of neurodegenerative diseases. Therefore, we have reviewed the pathogenic hypothesis in neurodegenerative pathologies, underling the links between the deposition of misfolded protein mechanisms and the immune activation.

Tumours can be viewed as aberrant tissues or organs sustained by tumorigenic stem-like cells that engage into dysregulated histo/organogenetic processes. Paragangliomas, prototypical organoid tumours constituted by dysmorphic variants of the vascular and neural tissues found in normal paraganglia, provide a model to test this hypothesis. To understand the origin of paragangliomas, we built a biobank comprising 77 cases, 18 primary cultures, 4 derived cell lines, 80 patient-derived xenografts and 11 cell-derived xenografts. We comparatively investigated these unique complementary materials using morphofunctional, ultrastructural and flow cytometric assays accompanied by microRNA studies. We found that paragangliomas contain stem-like cells with hybrid mesenchymal/vasculoneural phenotype, stabilized and expanded in the derived cultures. The viability and growth of such cultures depended on the downregulation of the miR-200 and miR-34 families, which allowed high PDGFRA and ZEB1 protein expression levels. Both tumour tissue- and cell culture-derived xenografts recapitulated the vasculoneural paraganglioma structure and arose from mesenchymal-like cells through a fixed developmental sequence. First, vasculoangiogenesis organized the microenvironment, building a perivascular niche which in turn supported neurogenesis. Neuroepithelial differentiation was associated with severe mitochondrial dysfunction, not present in cultured paraganglioma cells, but acquired in vivo during xenograft formation. Vasculogenesis was the Achilles’ heel of xenograft development. In fact, imatinib, that targets endothelial-mural signalling, blocked paraganglioma xenograft formation (11 xenografts from 12 cell transplants in the control group versus 2 out of 10 in the treated group, P = 0.0015). Overall our key results were unaffected by the SDHx gene carrier status of the patient, characterized for 70 out of 77 cases. In conclusion, we explain the biphasic vasculoneural structure of paragangliomas and identify an early and pharmacologically actionable phase of paraganglioma organization. Electronic supplementary material The online version of this article (10.1007/s00401-017-1799-2) contains supplementary material, which is available to authorized users.

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.

Childhood obesity is characterized by the loss of vascular insulin sensitivity along with altered oxidant-antioxidant state and chronic inflammation, which play a key role in the onset of endothelial dysfunction. We previously demonstrated a reduced insulin-stimulated Nitric Oxide (NO) bioavailability in Human Umbilical Vein Endothelial cells (HUVECs) cultured with plasma from obese pre-pubertal children (OB) compared to those cultured with plasma of normal-weight children (CTRL). However, mechanisms underlying endothelial dysfunction in childhood obesity remains poorly understood. Hence, the present study aimed to better investigate these mechanisms, also considering a potential involvement of mammalian Target Of Rapamycin Complex1 (mTORC1)-ribosomal protein S6 Kinase beta1 (S6K1) pathway. OB-children (N = 32, age: 9.2 ± 1.7; BMI z-score: 2.72 ± 0.31) had higher fasting insulin levels and increased HOMA-IR than CTRL-children (N = 32, age: 8.8 ± 1.2; BMI z-score: 0.33 ± 0.75). In vitro, HUVECs exposed to OB-plasma exhibited significant increase in Reactive Oxygen Species (ROS) levels, higher vascular and intercellular adhesion molecules exposure, together with increased monocytes-endothelial interaction. This was associated with unbalanced pro- and anti-atherogenic endothelial insulin stimulated signaling pathways, as measured by increased Mitogen Activated Protein Kinase (MAPK) and decreased Insulin Receptor Substrate-1 (IRS-1)/protein kinase B (Akt)/ endothelial NO Synthase (eNOS) phosphorylation levels, together with augmented S6K1 activation. Interestingly, inhibition of mTORC1-S6K1 pathway using rapamycin significantly restored the IRS-1/Akt/eNOS activation, suggesting a feedback regulation of IRS-1/Akt signal through S6K1. Overall, our in vitro data shed light on new mechanisms underlying the onset of endothelial dysfunction in childhood obesity.

Childhood obesity is commonly associated with early signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular Nitric Oxide (NO) availability. However, the underlying mechanisms remain to be established. Hence, we tested the hypothesis that endothelial insulin-stimulated NO production and availability was impaired and related to Endoplasmic Reticulum (ER) in human umbilical vein endothelial cells (HUVECs) cultured with plasma obtained from pre-pubertal obese (OB) children. OB children (N = 28, age: 8.8 ± 2.2; BMI z-score: 2.15 ± 0.39) showed impaired fasting glucose, insulin and HOMA-IR than normal weight children (CTRL; N = 28, age: 8.8 ± 1.7; BMI z-score: 0.17 ± 0.96). The in vitro experiments showed that OB-plasma significantly impaired endothelial insulin-stimulated NO production and bioavailability compared to CTRL-plasma. In parallel, in HUVECs OB-plasma increased GRP78 and activated PERK, eIF2α, IkBα and ATF6 (all ER stress markers). Moreover, OB-plasma increased NF-κB activation and its nuclear translocation. Notably, all these effects proved to be significantly restored by using PBA and TUDCA, known ER stress inhibitors. Our study demonstrate for the first time that plasma from obese children is able to induce in vitro endothelial insulin resistance, which is characterized by reduced insulin-stimulated NO production and bioavailability, endothelial ER stress and increased NF-κB activation., Graphical abstract Image 1, Highlights • Plasma from obese children induce in vitro endothelial insulin resistance (IR). • The mechanism involved is the reduced insulin-stimulated NO bioavailability. • In this experimental condition ER stress is associated to endothelial IR. • The additional effect is the increased endothelial inflammation.

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria–host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

Non-celiac wheat sensitivity (NCWS), also referred to as non-celiac gluten sensitivity, is a recently described disorder triggered by wheat/gluten ingestion. NCWS elicits a wide range of symptoms including diarrhoea, intestinal discomfort, and fatigue in analogy with other wheat/gluten-related disorders and celiac disease in particular. From the pathological standpoint, NCWS patients only have a slight increase of intraepithelial lymphocytes, while antibodies to tissue transglutaminase (tTG) and villous atrophy, otherwise diagnostic features of celiac disease, are absent. To date, the diagnosis of NCWS relies on symptoms and exclusion of confounding diseases, since biomarkers are not yet available. Here, the expression levels of selected miRNAs were examined in duodenal biopsies and peripheral blood leukocytes collected from newly diagnosed patients with NCWS and, as controls, from patients with celiac disease and gluten-independent gastrointestinal problems. We identified a few miRNAs whose expression is higher in the intestinal mucosa of patients affected by NCWS in comparison to control patients affect by gluten-independent dyspeptic symptoms (Helicobacter pylori-negative) and celiac disease. The present study provided the first evidence that NCWS patients have a characteristic miRNA expression patterns, such peculiarity could be exploited as a biomarker to the diagnosis of this disease.

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of β-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD, hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions. EVs mediate the cellular crosstalk, thus contributing to defining the tumor microenvironment, including in epithelial ovarian cancer (EOC). The available literature investigating the role of EVs in EOC has been reviewed following PRISMA guidelines, focusing on the role of EVs in early disease diagnosis, metastatic spread, and the development of chemoresistance in EOC. Data were identified from searches of Medline, Current Contents, PubMed, and from references in relevant articles from 2010 to 1 April 2020. The research yielded 194 results. Of these, a total of 36 papers, 9 reviews, and 27 original types of research were retained and analyzed. The literature findings demonstrate that a panel of EV-derived circulating miRNAs may be useful for early diagnosis of EOC. Furthermore, it appears clear that EVs are involved in mediating two crucial processes for metastatic and chemoresistance development: the epithelial–mesenchymal transition, and tumor escape from the immune system response. Further studies, more focused on in vivo evidence, are urgently needed to clarify the role of EV assessment in the clinical management of EOC patients.

Heart valve diseases are usually treated by surgical intervention addressed for the replacement of the damaged valve with a biosynthetic or mechanical prosthesis. Although this approach guarantees a good quality of life for patients, it is not free from drawbacks (structural deterioration, nonstructural dysfunction, and reintervention). To overcome these limitations, the heart valve tissue engineering (HVTE) is developing new strategies to synthesize novel types of valve substitutes, by identifying efficient sources of both ideal scaffolds and cells. In particular, a natural matrix, able to interact with cellular components, appears to be a suitable solution. On the other hand, the well-known Wharton's jelly mesenchymal stem cells (WJ-MSCs) plasticity, regenerative abilities, and their immunomodulatory capacities make them highly promising for HVTE applications. In the present study, we investigated the possibility to use porcine valve matrix to regenerate in vitro the valve endothelium by WJ-MSCs differentiated along the endothelial lineage, paralleled with human umbilical vein endothelial cells (HUVECs), used as positive control. Here, we were able to successfully decellularize porcine heart valves, which were then recellularized with both differentiated-WJ-MSCs and HUVECs. Data demonstrated that both cell types were able to reconstitute a cellular monolayer. Cells were able to positively interact with the natural matrix and demonstrated the surface expression of typical endothelial markers. Altogether, these data suggest that the interaction between a biological scaffold and WJ-MSCs allows the regeneration of a morphologically well-structured endothelium, opening new perspectives in the field of HVTE.