Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods.

Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

Synthesis, Molecular Docking, and Preclinical Evaluation of a New Succinimide Derivative for Cardioprotective, Hepatoprotective and Lipid-Lowering Effects.

Cardiac and hepatotoxicities are major concerns in the development of new drugs. Better alternatives to other treatments are being sought to protect these vital organs from the toxicities of these pharmaceuticals. In this regard, a preclinical study is designed to investigate the histopathological effects of a new succinimide derivative (Comp-1) on myocardial and liver tissues, and the biochemical effects on selected cardiac biomarkers, hepatic enzymes, and lipid profiles. For this, an initially lethal/toxic dose was determined, followed by a grouping of selected albino rats into five groups (each group had n = 6). The control group received daily oral saline for 8 days. The 5-FU (5-Fluorouracil) group received oral saline daily for 8 days, added with the administration of a single dose of 5-FU (150 mg/kg I.P.) on day 5 of the study. The atenolol group received oral atenolol (20 mg/kg) for 8 days and 5-FU (150 mg/kg I.P.) on day 5 of the protocol. Similarly, two groups of rats treated with test compound (Comp-1) were administered with 5 mg/kg I.P. and 10 mg/kg I.P. for 8 days, followed by 5-FU (150 mg/kg I.P.) on day 5. Toxicity induced by 5-FU was manifested by increases in the serum creatinine kinase myocardial band (CK-MB), troponin I (cTnI) and lactate dehydrogenase (LDH), lipid profile, and selected liver enzymes, including ALP (alkaline phosphatase), ALT (alanine transaminase), AST (aspartate aminotransferase), BT (bilirubin total), and BD (direct bilirubin). These biomarkers were highly significantly decreased after the administration of the mentioned doses of the test compound (5 mg/kg and 10 mg/kg). Similarly, histological examination revealed cardiac and hepatic tissue toxicity by 5-FU. However, those toxic effects were also significantly recovered/improved after the administration of Comp-1 at the said doses. This derivative showed dose-dependent effects and was most effective at a dose of 10 mg/kg body weight. Binding energy data computed via docking simulations revealed that our compound interacts toward the human beta2-adrenergic G protein-coupled receptor (S = -7.89 kcal/mol) with a slight stronger affinity than the calcium channel T-type (S = -7.07 kcal/mol). In conclusion, the histological and biochemical results showed that the test compound (Comp-1) had prominent cardioprotective, hepatoprotective, and lipolytic effects against 5-FU-induced toxicity in the subjected animal model.

Formation of Volatile Heterocyclic Compounds and Open-Chain Amides of Theanine in Model Systems with Glucose, Tea Leaves, and Tea Extract under Tea-Roasting Conditions.

Theanine is a non-proteinogenic amino acid found in the tea plant Camellia sinensis. At an elevated temperature (>90 °C), it released two major volatile compounds 1-ethyl-1,5-dihydro-2H-pyrrol-2-one and N-ethylsuccinimide. Other products were identified, including 10 pyrroles and 12 amides/imides. The formation of the two major compounds was proposed to be initiated by the deamination of theanine and through the intermediate α-keto acid. In the presence of glucose, the two major products and many other volatiles from theanine thermal degradation were accelerated and further Maillard reactions occurred. A total of 56 compounds were identified in the model system of theanine and glucose, including 12 amides/imides, 16 pyrazines, 16 pyrroles and other N-heterocycles, and 12 furans and other O-heterocycles. Although most of the reaction products were detected in tea leaves and in their aqueous extract with or without the addition of theanine under the same experiment conditions, imides and amides were considerably suppressed, left only minute amounts, or were even no longer detectable. Pyrazines and pyrroles were also shown at reduced concentrations as a result of the interaction with tea components but to a lesser extent. A total of 16 and 12 pyrazines were identified in the theanine/glucose reaction system and tea leaves/aqueous extract after roasting, respectively. The results indicated that pyrazines and other main volatiles in roasted tea leaves were formed from the Maillard reactions of the aqueous fraction of tea leaves. Theanine participated in the formation of pyrazines in tea leaves under roasting conditions.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

Extracellular Vesicle-Derived microRNAs of Human Wharton's Jelly Mesenchymal Stromal Cells May Activate Endogenous VEGF-A to Promote Angiogenesis.

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton's jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.

The Recurring Roles of Chlorine in Synthetic and Biological Studies of the Lissoclimides.

Halogenated natural products number in the thousands, but only in rare cases are the evolutionary advantages conferred by the halogens understood. We set out to investigate the lissoclimide family of cytotoxins, which includes several chlorinated members, because of our long-standing interest in the synthesis of chlorinated secondary metabolites.Our initial success in this endeavor was a semisynthesis of chlorolissoclimide (CL) from the commercially available sesquiterpenoid sclareolide. Featuring a highly selective and efficient-and plausibly biomimetic-C-H chlorination, we were able to access enough CL for collaborative studies, including X-ray cocrystallography with the eukaryotic ribosome. Through this experiment, we learned that CL's chlorine atom engages in a novel halogen-π dispersion interaction with a neighboring nucleobase in the ribosome E-site.Owing to the limitations of our semisynthesis approach, we established an analogue-oriented approach to access numerous lissoclimide compounds to both improve our understanding of structure-activity relationships and to learn more about the halogen-π interaction. In the course of these studies, we made over a dozen lissoclimide-like compounds, the most interesting of which contained chlorine-bearing carbons with unnatural configurations. Rationalizing the retained potency of these compounds that appeared to be a poor fit for the lissoclimide binding pocket, we came to realize that the chlorine atoms would engage in these same halogen-π interactions even at the expense of a chair to twist-boat conformational change, which also permitted the compounds to fit in the binding site.Finally, because neither of the first two approaches could easily access the most potent natural lissoclimides, we designed a synthesis that took advantage of rarely used terminal epoxides to initiate polyene cyclizations. In this case, the chlorine atom was incorporated early and helped control the stereochemical outcome of the key step.Over the course of this project, three different synthesis approaches were designed and executed, and our ability to access numerous lissoclimides fueled a range of collaborative biological studies. Further, chlorine played impactful roles throughout various aspects of both synthesis and biology. We remain inspired to learn more about the mechanism of action of these compounds and to deeply investigate the potentially valuable halogen-π dispersion interaction in the context of small molecule/nucleic acid binding. In that context, our work offers an instance wherein we might have gained a rudimentary understanding of the evolutionary importance of the halogen in a halogenated natural product.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

Bis(sulfosuccinimidyl)suberate-Based Helix-Shaped Microchannels as Enhancers of Biomolecule Isolation from Liquid Biopsies.

Most studies of ultrasensitive diagnosis of biomolecules from liquid specimens are limited by problems during sample preparation steps, including enrichment and isolation of biomolecules. Here we report a novel platform combining bis(sulfosuccinimidyl)suberate (BS3) and helix-shaped microchannels (BSH) to change the sample preparation paradigm. This BSH system is composed of BS3 for pathogen enrichment and nucleic acid isolation by electrostatic and covalent interaction, and helix-shaped microchannels to minimize sample loss and remove bubbles in large liquid specimens without pH change. The system detected Mycobacterium tuberculosis following enrichment and isolation of 10 mL of liquefied sputum from 11 patients with tuberculosis. Moreover, the system identified KRAS mutations following cell-free DNA isolation of blood plasma from 10 patients with colorectal cancer. This system allows ultrasensitive diagnosis in various disease applications with large volumes of liquid samples.

Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides.

Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l-isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10-3 /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10-3 /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10-3 /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.

NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain.

Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting K562 cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using K562-G*01:03 or K562-G*01:01 cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.

Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS.

AIM: To demonstrate the capabilities of our new capillary electrophoresis - mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. BACKGROUND: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. OBJECTIVE: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. MATERIALS AND METHODS: Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates. RESULTS: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. CONCLUSION: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

New Chlorinated 2,5-Diketopiperazines from Marine-Derived Bacteria Isolated from Sediments of the Eastern Mediterranean Sea.

From the organic extracts of five bacterial strains isolated from marine sediments collected in the East Mediterranean Sea, three new (15, 16, 31) and twenty-nine previously reported (1-14, 17-30, 32) metabolites bearing the 2,5-diketopiperazine skeleton were isolated. The structures of the chlorinated compounds 15, 16, and 31 were elucidated by extensive analysis of their spectroscopic data (NMR, MS, UV, IR). Compounds 15 and 16 were evaluated for their antifungal activity against Candida albicans and Aspergillus niger but were proven inactive. The relevant literature is supplemented with complete NMR assignments and revisions for the 29 previously reported compounds.

Fluorescence-based method is more accurate than counting-based methods for plotting growth curves of adherent cells.

OBJECTIVE: Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. RESULTS: The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.

Labeling Antibodies with N-Hydroxysuccinimide-Long Chain (NHS-LC)-Biotin.

Labeling antibodies with biotin (biotinylation) is a useful and simple technique. Biotin's small size (244 Da) usually has little effect on the biological activity of the protein target. The most common way to biotinylate an antibody is to cross-link a biotin succinimidyl ester to a primary amine. There are many commercially available types of biotin analogs that can be used for labeling. They vary in reactive group chemistry as well as spacer length. For example, a common analog used for biotinylation is the succinimidyl ester of biotin with an aminohexanoic acid spacer (Long Chain or LC-Biotin), utilized here. A PEG spacer of varying length can also be used.

CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea.

AIM: To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS: Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS: CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION: CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.

Promotion Effect of Succinimide on Amyloid Fibrillation of Hen Egg-White Lysozyme.

Amyloid fibrillation is closely associated with a series of neurodegenerative diseases. According to that, the intermediate soluble oligomers and protofibrils are more toxic; reducing their concentrations in protein solutions by accelerating fibrillation is believed as a feasible strategy for treatment or remission of the diseases. Using hen egg-white lysozyme (HEWL) as a model protein, the promotion effect of succinimide was revealed by a series of experiments, e.g., atomic force microscopy (AFM), thioflavin T (ThT) fluorescence assay, Far-UV circular dichroism (CD) and Raman spectroscopy, and modeling the effect of succinimide-like derivative intermediates of intramolecular deamidation of the backbone during amyloid fibrillation. The AFM measurement confirmed that succinimide effectively accelerated the morphological changes of HEWL, while at the molecular level, the accelerative transformation of protein secondary structures was also clarified by ThT fluorescence assay and Far-UV CD spectroscopy. The incubation time-dependent Raman spectroscopy further revealed that the direct transformation from α-helices to organized ß-sheets occurred upon skipping the intermediate random coils under the action of succinimide. This "bridge" effect of succinimide was attributed to its special influence on disulfide bonds. In the presence of succinimide in protein solutions, the native disulfide bonds of lysozyme could be broken more efficiently and quickly within hydrolysis, resulting in exposure of the buried hydrophobic residues and accelerating the formation of cross ß-sheet structures. The present investigation provides very useful information for understanding the effect of intramolecular deamidation on the whole amyloid fibrillation.

Conjugation of Antibodies to Horseradish Peroxidase.

Antibodies conjugated with horseradish peroxidase (HRP) are one of the most widely used bioreagents in the biological sciences. This protocol is a basic method for adding HRP to a thiolated antibody and can be adapted for use with different cross-linkers. Conjugation methods usually focus on linking through the lysines on HRP because there are only six of them and their modification does not adversely affect enzyme activity.

Synthesis, in-vitro α-glucosidase inhibition, antioxidant, in-vivo antidiabetic and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives.

α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus (DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-dione derivatives and evaluated for their ability to inhibit α-Glucosidase. Pyrrolidine-2,5-dione derivatives (11a-o) showed moderate to poor α-glucosidase inhibition. Compound 11o with the IC50 value of 28.3 ±â€¯0.28 µM emerged as a good inhibitor of α-glucosidase. Thiazolidine-2,4-dione and dihydropyrimidine (TZD-DHPM) hybrids (22a-c) showed excellent inhibitory activities. The most active compound 22a displayed IC50 value of 0.98 ±â€¯0.008 µM. Other two compounds of this series also showed activity in low micromolar range. The in-vivo antidiabetic study of three compounds 11n, 11o and 22a were also determined using alloxan induced diabetes mice model. Compounds 11o and 22a showed significant hypoglycemic effect compared to the reference drug. In-vivo acute toxicity study showed the safety of these selected compounds. In-silico docking studies were carried out to rationalize the in-vitro results. The binding modes and bioassay results of TZD-DHPM hybrids showed that interactions with important residues appeared significant for high potency.

Interleukin-17A affects extracellular vesicles release and cargo in human keratinocytes.

Psoriasis is a chronic inflammatory systemic disease caused by deregulation of the interleukin-23/-17 axis that allows the activation of Th17 lymphocytes and the reprogramming of keratinocytes proliferative response, thereby inducing the secretion of cyto-/chemokines and antimicrobial peptides. Beside cell-to-cell contacts and release of cytokines, hormones and second messengers, cells communicate each other through the release of extracellular vesicles containing DNA, RNA, microRNAs and proteins. It has been reported the alteration of extracellular vesicles trafficking in several diseases, but there is scarce evidence of the involvement of extracellular vesicles trafficking in the pathogenesis of psoriasis. The main goal of the study was to characterize the release, the cargo content and the capacity to transfer bioactive molecules of extracellular vesicles produced by keratinocytes following recombinant IL-17A treatment if compared to untreated keratinocytes. A combined approach of standard ultracentrifugation, RNA isolation and real-time RT-PCR techniques was used to characterize extracellular vesicles cargo. Flow cytometry was used to quantitatively and qualitatively analyse extracellular vesicles and to evaluate cell-to-cell extracellular vesicles transfer. We report that the treatment of human keratinocytes with IL-17A significantly modifies the extracellular vesicles cargo and release. Vesicles from IL-17A-treated cells display a specific pattern of mRNA which is undid by IL-17A neutralization. Extracellular vesicles are taken up by acceptor cells irrespective of their content but only those derived from IL-17A-treated cells enable recipient cells to express psoriasis-associated mRNA. The results imply a role of extracellular vesicles in amplifying the pro-inflammatory cascade induced in keratinocyte by pro-psoriatic cytokines.

Tracking Dye-Independent Approach to Identify and Isolate In Vitro Expanded T Cells.

T cell proliferation is routinely identified in vitro using tracking dyes or through detecting intracellular upregulation of the nuclear protein, Ki-67. However, labeling with tracking dyes is cumbersome, associated with cellular toxicity, while Ki-67 cannot be used to identify and isolate viable T cells, and both techniques are incompatible with MACS technology. Here, we introduce a simple tool to identify and isolate in vitro T cell expansion that is tracking dye-independent and allows for sorting of viable T cells. We show that CD71, a transferrin receptor, and CD98, a heterodimer glycoprotein involved in both integrin signaling and amino-acid transport, are both highly upregulated on proliferating T cells upon in vitro stimulation, and that CD71 expression is maximal on the more recent progeny T cells, while CD98 upregulation remains stable across different generations of progeny T cells. Moreover, we demonstrate that the upregulation of CD71 and CD98 identifies CFSElow T cells and provides further proof of the antigen-specificity of T cells identified by CD71 and CD98 dual upregulation based on tetramer staining. We further show that CD71 can be used to enrich for in vitro expanding T cells using MACS technology. In conclusion, we show that CD71 and CD98 can be used to identify and isolate expanded T cells following in vitro stimulation and that CD71 is an MACS-compatible alternative to tracking dyes or Ki-67 detection. © 2019 International Society for Advancement of Cytometry.