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Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

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BACKGROUND: Celiac disease (CD) is an autoimmune disorder triggered by an abnormal immunological response to gluten ingestion and is associated with deregulated expression of cellular microRNAs (miRNAs) of the gut mucosa. It is frequently misdiagnosed as lactose intolerance (LI) due to symptom resemblance. Microvilli loss may be counteracted by a rigorous gluten-free diet (GFD). AIMS: To identify altered extracellular vesicle miRNAs from plasma among CD patients on GFD (n=34), lactose intolerant individuals on restrictive diet (n=14) and controls (n=23), and to predict biological pathways in which these altered miRNAs may play a part. METHODS: Five different small RNA samples of each group were pooled twice and then screened by new-generation sequencing. Four miRNAs were selected to be quantified by RT-qPCR in the entire sample. RESULTS: The levels of four miRNAs - miR-99b-3p, miR-197-3p, miR-223-3p, and miR-374b-5p - differed between CD patients and controls (P<0.05). Apart from miR-223-3p, all these miRNAs tended to have altered levels also between LI and controls (P<0.10). The results for miR-99b-3p and miR-197-3p between CD and controls were confirmed by RT-qPCR, which also indicated different levels of miR-99b-3p and miR-374b-5p between CD-associated LI and LI (P<0.05). CONCLUSIONS: These miRNAs may have targets that affect cell death, cell communication, adhesion, and inflammation modulation pathways. Hence, altered miRNA levels could be associated with CD-related aspects and gut mucosa recovery.

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A Disintegrin And Metalloprotease 23 (ADAM23) is a member of the ADAMs family of transmembrane proteins, mostly expressed in nervous system, and involved in traffic and stabilization of Kv1-potassium channels, synaptic transmission, neurite outgrowth, neuronal morphology and cell adhesion. Also, ADAM23 has been linked to human pathological conditions, such as epilepsy, cancer metastasis and cardiomyopathy. ADAM23 functionality depends on the molecule presence at the cell surface and along the secretory pathway, as expected for a cell surface receptor. Because endocytosis is an important functional regulatory mechanism of plasma membrane receptors and no information is available about the traffic or turnover of non-catalytic ADAMs, we investigated ADAM23 internalization, recycling and half-life properties. Here, we show that ADAM23 undergoes constitutive internalization from the plasma membrane, a process that depends on lipid raft integrity, and is redistributed to intracellular vesicles, especially early and recycling endosomes. Furthermore, we observed that ADAM23 is recycled from intracellular compartments back to the plasma membrane and thus has longer half-life and higher cell surface stability compared with other ADAMs. Our findings suggest that regulation of ADAM23 endocytosis/stability could be exploited therapeutically in diseases in which ADAM23 is directly involved, such as epilepsy, cancer progression and cardiac hypertrophy.

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MicroRNAs derived from extracellular vesicles (EV-miRNAs) are circulating miRNAs considered as potential new diagnostic markers for cancer that can be easily detected in liquid biopsies. In this study, we performed RNA sequencing analysis as a screening strategy to identify EV-miRNAs derived from serum of clinically well-annotated breast cancer (BC) patients from the south of Brazil. EVs from three groups of samples (healthy controls (CT), luminal A (LA), and triple-negative (TNBC)) were isolated from serum using a precipitation method and analyzed by RNA-seq (screening phase). Subsequently, four EV-miRNAs (miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p) were selected to be quantified by quantitative real-time PCR (RT-qPCR) in individual samples (test phase). A panel composed of miR-142-5p, miR-320a, and miR-4433b-5p distinguished BC patients from CT with an area under the curve (AUC) of 0.8387 (93.33% sensitivity, 68.75% specificity). The combination of miR-142-5p and miR-320a distinguished LA patients from CT with an AUC of 0.9410 (100% sensitivity, 93.80% specificity). Interestingly, decreased expression of miR-142-5p and miR-150-5p were significantly associated with more advanced tumor grades (grade III), while the decreased expression of miR-142-5p and miR-320a was associated with a larger tumor size. These results provide insights into the potential application of EVs-miRNAs from serum as novel specific markers for early diagnosis of BC.

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AIMS: Obesity is associated with comorbidities such as diabetes and hepatic steatosis. ß-Glucans have been described as effective in treating conditions including dyslipidaemia and diabetes. Thus, the objective of this study was to evaluate the effects of botryosphaeran [(1 → 3)(1 → 6)-ß-D-glucan] on obesity and its comorbidities, and understand its mechanism of action. MAIN METHODS: Obesity was induced in adult male Wistar rats by ingestion of a high-fat diet and water with sucrose (300 g/L) for 8 weeks. Control rats received standard diet. After six weeks, treatment commenced with botryosphaeran (12 mg/kg.b.w., via gavage, 15 days), respective controls received water. Rats were divided into 3 groups: control (C), obese (O), and obese + botryosphaeran (OB). In the 8th week, obesity was characterized. Feed-intake, glucose and lipid profiles, glucose tolerance, and concentrations of glycogen and lipids in liver were analyzed. Protein expression was determined by Western blotting. KEY FINDINGS: Obese rats showed significant increases in weight gain and adipose tissue, presented glucose intolerance, dyslipidaemia, and hepatic steatosis. Botryosphaeran significantly reduced feed intake, weight gain, periepididymal and mesenteric fat, and improved glucose tolerance. Botryosphaeran also reduced triglyceride and VLDL, and increased HDL levels. Furthermore, botryosphaeran increased glycogen and reduced total lipids, triglycerides and cholesterol in liver, and increased AMP-activated protein kinase(AMPK) activity and Forkhead transcription factor 3a(FOXO3a) protein expression in adipose tissue. SIGNIFICANCE: This study demonstrated that botryosphaeran was effective in reducing obesity, hepatic steatosis, dyslipidaemia insulin resistance and glucose intolerance in diet-induced obese rats, and these effects were, at least in part, associated with reduced feed intake, and AMPK and FOXO3a activities.

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A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100 kDa) and mature (70 kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23100 kDa. Furthermore, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM2370 kDa partitions between raft and non-raft membrane domains, while the pro-protein ADAM23100 kDa is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neurons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component.

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