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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.

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.

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.

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.

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.

Objective: 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., Study Design: 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., Results: 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., Conclusions: 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., Competing Interests: Declaration of Competing Interest Authors have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 10 5 cells/mL), as well as at lower concentrations (10 3 -10 2 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.

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.

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 (EVs) are circulating particles surrounded by a plasma membrane carrying a cargo consisting of proteins, lipids, RNAs, and DNA fragments, stemming from the cells from which they originated. EV factors (i.e., miRNAs) play relevant roles in intercellular crosstalk, both locally and systemically. As EVs increasingly gained attention as potential carriers for targeted genes, the study of EV effects on the host immune response became more relevant. It has been demonstrated that EVs regulate the host immune response, executing both pro- and anti-inflammatory functions. It is also known that physical exercise triggers anti-inflammatory effects. This review underlines the role of circulating EVs as players in the anti-inflammatory events associated with the regulation of the host's immune response to physical exercise.

Recently, the protective and/or pathological role of virus-specific T cells in SARS-CoV-2 infection has been the focus of many studies. We investigated the anti-spike IgG levels and SARS-CoV-2-specific T cells in 125 donors (90 vaccinated with four different vaccine platforms, 16 individuals with a previous natural infection, and 19 not vaccinated donors who did not report previous SARS-CoV-2 infections). Our data show that anti-spike IgG titers were similar between naturally infected subjects and those vaccinated with adenoviral vector vaccines. Of note, all immunized donors produced memory CD4 + and/or CD8 + T cells. A sustained polyfunctionality of SARS-CoV-2-specific T cells in all immunized donors was also demonstrated. Altogether, our data suggest that the natural infection produces an overall response like that induced by vaccination. Therefore, this detailed immunological evaluation may be relevant for other vaccine efforts especially for the monitoring of novel vaccines effective against emerging virus variants.

Currently, several pathologies have corresponding and specific diagnostic and therapeutic branches of interest focused on early and correct detection, as well as the best therapeutic approach. Radiology never ceases to develop newer technologies in order to give patients a clear, safe, early, and precise diagnosis; furthermore, in the last few years diagnostic imaging panoramas have been extended to the field of artificial intelligence (AI) and machine learning. On the other hand, clinical and laboratory tests, like flow cytometry and the techniques found in the "omics" sciences, aim to detect microscopic elements, like extracellular vesicles, with the highest specificity and sensibility for disease detection. If these scientific branches started to cooperate, playing a conjugated role in pathology diagnosis, what could be the results? Our review seeks to give a quick overview of recent state of the art research which investigates correlations between extracellular vesicles and the known radiological features useful for diagnosis.

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.