Clodronate Reduces ATP-Containing Microvesicle Releasing Induced by Nociceptive Stimuli in Human Keratinocytes.

Clodronate (Clod), a first-generation bisphosphonate, acts as a natural analgesic inhibiting vesicular storage of the nociception mediator ATP by vesicular nucleotide transporter (VNUT). Epidermal keratinocytes participate in cutaneous nociception, accumulating ATP within vesicles, which are released following different stimulations. Under stress conditions, keratinocytes produce microvesicles (MVs) by shedding from plasma membrane evagination. MV secretion has been identified as a novel and universal mode of intercellular communication between cells. The aim of this project was to evaluate if two nociceptive stimuli, Capsaicin and Potassium Hydroxide (KOH), could stimulate MV shedding from human keratinocytes, if these MVs could contain ATP, and if Clod could inhibit this phenomenon. In our cellular model, the HaCaT keratinocyte monolayer, both Capsaicin and KOH stimulated MV release after 3 h incubation, and the released MVs contained ATP. Moreover, Clod (5 µM) was able to reduce Caps-induced MV release and abolish the one KOH induced, while the Dansylcadaverine, an endocytosis inhibitor of Clod uptake, partially failed to block the bisphosphonate activity. Based on these new data and given the role of the activation of ATP release by keratinocytes as a vehicle for nociception and pain, the "old" bisphosphonate Clodronate could provide the pharmacological basis to develop new local analgesic drugs.

Liquid Biopsy in Glioblastoma Management: From Current Research to Future Perspectives.

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. Arising from neuroepithelial glial cells, GBM is characterized by invasive behavior, extensive angiogenesis, and genetic heterogeneity that contributes to poor prognosis and treatment failure. Currently, there are several molecular biomarkers available to aid in diagnosis, prognosis, and predicting treatment outcomes; however, all require the biopsy of tumor tissue. Nevertheless, a tissue sample from a single location has its own limitations, including the risk related to the procedure and the difficulty of obtaining longitudinal samples to monitor treatment response and to fully capture the intratumoral heterogeneity of GBM. To date, there are no biomarkers in blood or cerebrospinal fluid for detection, follow-up, or prognostication of GBM. Liquid biopsy offers an attractive and minimally invasive solution to support different stages of GBM management, assess the molecular biology of the tumor, identify early recurrence and longitudinal genomic evolution, predict both prognosis and potential resistance to chemotherapy or radiotherapy, and allow patient selection for targeted therapies. The aim of this review is to describe the current knowledge regarding the application of liquid biopsy in glioblastoma, highlighting both benefits and obstacles to translation into clinical care. IMPLICATIONS FOR PRACTICE: To translate liquid biopsy into clinical practice, further prospective studies are required with larger cohorts to increase specificity and sensitivity. With the ever-growing interest in RNA nanotechnology, microRNAs may have a therapeutic role in brain tumors.

Global transcriptomic changes occur in uterine fluid-derived extracellular vesicles during the endometrial window for embryo implantation.

STUDY QUESTION: Are uterine fluid-derived extracellular vesicles (UF-EVs) a 'liquid biopsy' reservoir of biomarkers for real-time monitoring of endometrial status? SUMMARY ANSWER: The transcriptomic cargo of UF-EVs reflects the RNA profile of the endometrial tissue as well as changes between the non-receptive and the receptive phase, possibly supporting its use for a novel endometrial receptivity test. WHAT IS KNOWN ALREADY: EVs have been previously isolated from uterine fluid, where they likely contribute to the embryo-endometrium crosstalk during implantation. Based on a meta-analysis of studies on endometrial tissue implantation-associated genes and the human exosomes database, 28 of the 57 transcripts considered as receptivity markers refer to proteins present in human exosomes. However, the specific transcriptomic content of receptive phase UF-EVs has yet to be defined. STUDY DESIGN, SIZE, DURATION: Two experimental series were set up. First, we simultaneously sequenced RNA species derived from paired UF-EVs and endometrial tissue samples collected from physiologically cycling women. Second, we analyzed RNA species of UF-EVs collected during the non-receptive (LH + 2) and receptive (LH + 7) phase of proven fertile women and from the receptive (LH + 7) phase of a population of women undergoing ART and transfer of euploid blastocysts. PARTICIPANTS/MATERIALS, SETTING, METHODS: For paired UF-endometrial tissue sampling, endometrial tissue biopsies were obtained with the use of a Pipelle immediately after UF collection performed by lavage of the endometrial cavity. Overall, n = 87 UF samples were collected and fresh-processed for EV isolation and total RNA extraction, while western blotting was used to confirm the expression of EV protein markers of the isolated vesicles. Physical characterization of UF-EVs was performed by Nanoparticle Tracking Analysis. To define the transcriptomic cargo of UF-EV samples, RNA-seq libraries were successfully prepared from n = 83 UF-EVs samples and analyzed by RNA-seq analysis. Differential gene expression (DGE) analysis was used to compare RNA-seq results between different groups of samples. Functional enrichment analysis was performed by gene set enrichment analysis with g:Profiler. Pre-ranked gene set enrichment analysis (GSEA) with WebGestalt was used to compare RNA-seq results with the gene-set evaluated in a commercially available endometrial receptivity array. MAIN RESULTS AND THE ROLE OF CHANCE: A highly significant correlation was found between transcriptional profiles of endometrial biopsies and pairwise UF-EV samples (Pearson's r = 0.70 P < 0.0001; Spearman's ρ = 0.65 P < 0.0001). In UF-EVs from fertile controls, 942 gene transcripts were more abundant and 1305 transcripts less abundant in the LH + 7 receptive versus the LH + 2 non-receptive phase. GSEA performed to evaluate concordance in transcriptional profile between the n = 238 genes included in the commercially available endometrial receptivity array and the LH + 7 versus LH + 2 UF-EV comparison demonstrated an extremely significant and consistent enrichment, with a normalized enrichment score (NES)=9.38 (P < 0.001) for transcripts up-regulated in LH + 7 in the commercial array and enriched in LH + 7 UF-EVs, and a NES = -5.40 (P < 0.001) for transcripts down-regulated in LH + 7 in the commercial array and depleted in LH + 7 UF-EVs. When analyzing LH + 7 UF-EVs of patients with successful versus failed implantation after transfer of one euploid blastocyst in the following cycle, we found 97 genes whose transcript levels were increased and 64 genes whose transcript levels were decreased in the group of women who achieved a pregnancy. GSEA performed to evaluate concordance in transcriptional profile between the commercially available endometrial receptivity array genes and the comparison of LH + 7 UF-EVs of women with successful versus failed implantation, demonstrated a significant enrichment with a NES = 2.14 (P = 0.001) for transcripts up-regulated in the commercial array in the receptive phase and enriched in UF-EVs of women who conceived, and a not significant NES = -1.18 (P = 0.3) for transcripts down-regulated in the commercial array and depleted in UF-EVs. In terms of physical features, UF-EVs showed a homogeneity among the different groups analyzed except for a slight but significant difference in EV size, being smaller in women with a successful implantation compared to patients who failed to conceive after euploid blastocyst transfer (mean diameter ± SD 205.5± 22.97 nm vs 221.5 ± 20.57 nm, respectively, P = 0.014). LARGE SCALE DATA: Transcriptomic data were deposited in NCBI Gene Expression Omnibus (GEO) and can be retrieved using GEO series accession number: GSE158958. LIMITATIONS, REASONS FOR CAUTION: Separation of RNA species associated with EV membranes might have been incomplete, and membrane-bound RNA species-rather than the internal RNA content of EVs-might have contributed to our RNA-seq results. Also, we cannot definitely distinguish the relative contribution of exosomes, microvesicles and apoptotic bodies to our findings. When considering patients undergoing ART, we did not collect UFs in the same cycle of the euploid embryo transfer but in the one immediately preceding. We considered this approach as the most appropriate in relation to the novel, explorative nature of our study. Based on our results, a validation of UF-EV RNA-seq analyses in the same cycle in which embryo transfer is performed could be hypothesized. WIDER IMPLICATIONS OF THE FINDINGS: On the largest sample size of human EVs ever analyzed with RNA-seq, this study establishes a gene signature to use for less-invasive endometrial receptivity tests. This report is indeed the first to show that the transcriptome of UF-EVs correlates with the endometrial tissue transcriptome, that RNA signatures in UF-EVs change with endometrial status, and that UF-EVs could serve as a reservoir for potential less-invasive collection of receptivity markers. This article thus represents a step forward in the design of less-invasive approaches for real-time monitoring of endometrial status, necessary for advancing the field of reproductive medicine. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a competitive grant from European Society of Human Reproduction and Embryology (ESHRE Research Grant 2016-1). The authors have no financial or non-financial competing interests to disclose. TRIAL REGISTRATION NUMBER: NA.

Trans-kingdom small RNA transfer during host-pathogen interactions: The case of P. falciparum and erythrocytes.

This review focuses on the role of trans-kingdom movement of small RNA (sRNA) molecules between parasites, particularly Plasmodium falciparum, and their respective host cells. While the intercellular transfer of sRNAs within organisms is well recognized, recent studies illustrate many examples of trans-kingdom sRNA exchange within the context of host-parasite interactions. These interactions are predominantly found in the transfer of host sRNAs between erythrocytes and the invading P. falciparum, as well as other host cell types. In addition, parasite-encoded sRNAs can also be transferred to host cells to evade the immune system. The transport of these parasite sRNAs in the body fluids of the host may also offer means to detect and monitor the parasite infection. These isolated examples may only represent the tip of the iceberg in which the transfer of sRNA between host and parasites is a critical aspect of host-pathogen interactions. In addition, the levels of these sRNAs and their speed of transfer may vary dramatically under different contexts to push the biologic equilibrium toward the benefit of hosts vs. parasites. Therefore, these sRNA transfers may offer potential strategies to detect, prevent or treat parasite infections. Here, we review a brief history of the discovery of host erythrocyte sRNAs, their transfers and interactions in the context of P. falciparum infection. We also provide examples and discuss the functional significance of the reciprocal transfer of parasite-encoded sRNAs into hosts. These understandings of sRNA exchanges are put in the context of their implications for parasite pathogenesis, host defenses and the evolution of host polymorphisms driven by host interactions with these parasites.

Smart blood cell and microvesicle-based Trojan horse drug delivery: Merging expertise in blood transfusion and biomedical engineering in the field of nanomedicine.

Therapeutic and diagnostic applications of nanomedicine are playing increasingly important roles in human health. Various types of synthetic nanoparticles, including liposomes, micelles, and other nanotherapeutic platforms and conjugates, are being engineered to encapsulate or carry drugs for treating diseases such as cancer, cardiovascular disorders, neurodegeneration, and inflammations. Nanocarriers are designed to increase the half-life of drugs, decrease their toxicity and, ideally, target pathological sites. Developing smart carriers with the capacity to deliver drugs specifically to the microenvironment of diseased cells with minimum systemic toxicity is the goal. Blood cells, and potentially also the liposome-like micro- and nano-vesicles they generate, may be regarded as ideally suited to perform such specific targeting with minimum immunogenic risks. Blood cell membranes are "decorated" with complex physiological receptors capable of targeting and communicating with other cells and tissues and delivering their content to the surrounding pathological microenvironment. Blood cells, such as erythrocytes, have been developed as permeable carriers to release drugs to diseased tissues or act as biofactory allowing enzymatic degradation of a pathological substrate. Interestingly, attempts are also being made to improve the targeting capacity of synthetic nanoparticles by "decorating" their surface with blood cell membrane receptor-like biochemical structures. Research is needed to further explore the benefits that blood cell-derived microvesicles, as a Trojan horse delivery systems, can bring to the arsenal of therapeutic micro- and nanotechnologies. This short review focuses on the therapeutic roles that red blood cells and platelets can play as smart drug-delivery systems, and highlights the benefits that blood transfusion expertise can bring to this exciting and novel biomedical engineering field.

Circular and Circulating DNA in Inflammatory Bowel Disease: From Pathogenesis to Potential Molecular Therapies.

Inflammatory bowel diseases (IBD), including Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic multifactorial disorders which affect the gastrointestinal tract with variable extent. Despite extensive research, their etiology and exact pathogenesis are still unknown. Cell-free DNAs (cfDNAs) are defined as any DNA fragments which are free from the origin cell and able to circulate into the bloodstream with or without microvescicles. CfDNAs are now being increasingly studied in different human diseases, like cancer or inflammatory diseases. However, to date it is unclear how IBD etiology is linked to cfDNAs in plasma. Extrachromosomal circular DNA (eccDNA) are non-plasmidic, nuclear, circular and closed DNA molecules found in all eukaryotes tested. CfDNAs appear to play an important role in autoimmune diseases, inflammatory processes, and cancer; recently, interest has also grown in IBD, and their role in the pathogenesis of IBD has been suggested. We now suggest that eccDNAs also play a role in IBD. In this review, we have comprehensively collected available knowledge in literature regarding cfDNA, eccDNA, and structures involving them such as neutrophil extracellular traps and exosomes, and their role in IBD. Finally, we focused on old and novel potential molecular therapies and drug delivery systems, such as nanoparticles, for IBD treatment.

Growth Inhibition of Retinoblastoma Cell Line by Exosome-Mediated Transfer of miR-142-3p.

Introduction: Retinoblastoma (Rb) is the most common ocular paediatric malignancy and is caused by a mutation of the two alleles of the tumor suppressor gene, RB1. The tumor microenvironment (TME) represents a complex system whose function is not yet well defined and where microvesicles, such as exosomes, play a key role in intercellular communication. Micro-RNAs (mRNAs) have emerged as important modifiers of biological mechanisms involved in cancer and been able to regulate tumor progression. Methods: Co-culture of monocytes with retinoblastoma cell lines, showed a significant growth decrease. Given the interaction between Rb cells and monocytes, we investigated the role of the supernatant in the cross-talk between cell lines, by taking the product of the co-culture and then using it as a culture medium for Rb cells. Results: miR-142-3p showed to be particularly over-expressed both in the Rb cell line and in the medium used for their culture, comparing to control cell line and the normal supernatant, respectively. Therefore, we provided evidence that miR-142-3p is released by monocytes in the co-culture medium's exosomes and that it is subsequently up-taken by Rb cells, causing the inhibition of proliferation of Rb cell line by affecting cell cycle progression. Conclusion: This study highlights the role of exosomic miR-142-3p in the TME of Rb and identifies new molecular targets, which are able to control tumor growth aiming the development of a forward-looking miR-based strategy.

Extracellular Vesicles: Emerging Roles in Developing Therapeutic Approach and Delivery Tool of Chinese Herbal Medicine for the Treatment of Depressive Disorder.

Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by cells, which play an essential role in intercellular communication by delivering cellular components including DNA, RNA, lipids, metabolites, cytoplasm, and cell surface proteins into recipient cells. EVs play a vital role in the pathogenesis of depression by transporting miRNA and effector molecules such as BDNF, IL34. Considering that some herbal therapies exhibit antidepressant effects, EVs might be a practical delivery approach for herbal medicine. Since EVs can cross the blood-brain barrier (BBB), one of the advantages of EV-mediated herbal drug delivery for treating depression with Chinese herbal medicine (CHM) is that EVs can transfer herbal medicine into the brain cells. This review focuses on discussing the roles of EVs in the pathophysiology of depression and outlines the emerging application of EVs in delivering CHM for the treatment of depression.

Lipidic Profile Changes in Exosomes and Microvesicles Derived From Plasma of Monoclonal Antibody-Treated Psoriatic Patients.

Psoriasis is a chronic immune-mediated inflammatory skin disorder affecting children and adults. To date no approved biomarkers for diagnosis of this disease and follow up of patients have been translated into clinical practice. Recently, extracellular vesicles (EVs) secreted by all cells and present in almost all biological fluids are playing a crucial role in diagnosis and follow up of several diseases, including psoriasis. Since many psoriatic patients show altered plasma lipid profiles and since EVs have been involved in psoriasis pathogenesis, we studied the phospholipid profile of EVs, both microvesicles (MV) or exosomes (Exo), derived from plasma of psoriatic patients undergoing systemic biological treatment (secukinumab, ustekinumab, adalimumab), in comparison with EVs of untreated patients and healthy donors (HD). EVs were evaluated by immune electronmicroscopy for their morphology and by NanoSight for their amount and dimensions. EV phospholipid profiling was performed by High Resolution Liquid Chromatography-Mass Spectrometry and statistical Partial Least Squares Discriminant Analysis. Our results demonstrated that psoriatic patients showed a higher concentration of both MV and Exo in comparison to EVs from HD. The phospholipid profile of Exo from psoriatic patients showed increased levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol and lysoPC compared to Exo from HD. Sphingomyelin (SM) and phosphatidylinositol (PI) are the only phospholipid classes whose levels changed in MV. Moreover, the therapy with ustekinumab seemed to revert the PE and PC lipid composition of circulating Exo towards that of HD and it is the only one of the three biological drugs that did not alter SM expression in MV. Therefore, the determination of lipid alterations of circulating EVs could harbor useful information for the diagnosis and drug response in psoriatic patients.

The role of platelets, neutrophils and endothelium in COVID-19 infection.

INTRODUCTION: COVID-19 is associated to an increased risk of thrombosis, as a result of a complex process that involves the activation of vascular and circulating cells, the release of soluble inflammatory and thrombotic mediators and blood clotting activation. AREAS COVERED: This article reviews the pathophysiological role of platelets, neutrophils, and the endothelium, and of their interactions, in the thrombotic complications of COVID-19 patients, and the current and future therapeutic approaches targeting these cell types. EXPERT OPINION: Virus-induced platelet, neutrophil, and endothelial cell changes are crucial triggers of the thrombotic complications and of the adverse evolution of COVID-19. Both the direct interaction with the virus and the associated cytokine storm concur to trigger cell activation in a classical thromboinflammatory vicious circle. Although heparin has proven to be an effective prophylactic and therapeutic weapon for the prevention and treatment of COVID-19-associated thrombosis, it acts downstream of the cascade of events triggered by SARS-CoV-2. The identification of specific molecular targets interrupting the thromboinflammatory cascade upstream, and more specifically acting either on the interaction of SARS-CoV-2 with blood and vascular cells or on the specific signaling mechanisms associated with their COVID-19-associated activation, might theoretically offer greater protection with potentially lesser side effects.

Extracellular Vesicles and HIV-Associated Neurocognitive Disorders: Implications in Neuropathogenesis and Disease Diagnosis.

Extracellular vesicles are heterogeneous cell-derived membranous structures of nanometer size that carry diverse cargoes including nucleic acids, proteins, and lipids. Their secretion into the extracellular space and delivery of their cargo to recipient cells can alter cellular function and intracellular communication. In this review, we summarize the role of extracellular vesicles in the disease pathogenesis of HIV-associated neurocognitive disorder (HAND) by focusing on their role in viral entry, neuroinflammation, and neuronal degeneration. We also discuss the potential role of extracellular vesicles as biomarkers of HAND. Together, this review aims to convey the importance of extracellular vesicles in the pathogenesis of HAND and foster interest in their role in neuroinflammatory diseases.