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Abstract Background Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in the pediatric population worldwide and an important cause of death in developing countries. It has been demonstrated that the balance between oxidant and antioxidant systems is disrupted in children with bronchiolitis and that oxidative stress contributes to the pathogenesis of this disease. Platelets play an important role in antimicrobial host defenses and contribute to pulmonary vascular repair being either targets or source of reactive oxidizing species. The main purpose of this study was to assessing sex differences in clinical characteristics and platelets activation during RSV bronchiolitis in infancy. Methods In this retrospective study a total of 203 patients (112 boys and 91 girls) with bronchiolitis, aged 12 months or less, admitted to the Bambino Gesù Pediatric Hospital of Rome (Italy) in the period from January to December 2017, were enrolled. Moreover, in a select group of patients (15 boys and 12 girls) with diagnosis of moderate bronchiolitis from RSV, a pilot study on oxidative stress and platelet characteristics was carried out by electron paramagnetic resonance and flow cytometry respectively. Age-matched healthy control subjects (10 boys and 10 girls) were chosen as controls. Data were analyzed using Student’ T test, Chi Squared test and one-way ANOVA test. Results This study highlights the influence of sex in the clinical course of bronchiolitis. In particular we found: i) a higher incidence of bronchiolitis in boys than in girls (55% vs 45%); ii) higher C reactive protein values in girls than boys (1.11 mg/dL vs 0.92 mg/dL respectively; p

Abstract Background Development of resistance to inhibitors of BRAF (BRAFi) and MEK (MEKi) remains a great challenge for targeted therapy in patients with BRAF-mutant melanoma. Here, we explored the role of miRNAs in melanoma acquired resistance to BRAFi. Methods miRNA expression in two BRAF-mutant melanoma cell lines and their dabrafenib-resistant sublines was determined using Affymetrix GeneChip® miRNA 3.1 microarrays and/or qRT-PCR. The effects of miR-126-3p re-expression on proliferation, apoptosis, cell cycle, ERK1/2 and AKT phosphorylation, dabrafenib sensitivity, invasiveness and VEGF-A secretion were evaluated in the dabrafenib-resistant sublines using MTT assays, flow cytometry, immunoblotting, invasion assays in Boyden chambers and ELISA. ADAM9, PIK3R2, MMP7 and CXCR4 expression in the sensitive and dabrafenib-resistant cells was determined by immunoblotting. Small RNA interference was performed to investigate the consequence of VEGFA or ADAM9 silencing on proliferation, invasiveness or dabrafenib sensitivity of the resistant sublines. Long-term proliferation assays were carried out in dabrafenib-sensitive cells to assess the effects of enforced miR-126-3p expression or ADAM9 silencing on resistance development. VEGF-A serum levels in melanoma patients treated with BRAFi or BRAFi+MEKi were evaluated at baseline (T0), after two months of treatment (T2) and at progression (TP) by ELISA. Results miR-126-3p was significantly down-regulated in the dabrafenib-resistant sublines as compared with their parental counterparts. miR-126-3p replacement in the drug-resistant cells inhibited proliferation, cell cycle progression, phosphorylation of ERK1/2 and/or AKT, invasiveness, VEGF-A and ADAM9 expression, and increased dabrafenib sensitivity. VEGFA or ADAM9 silencing impaired proliferation and invasiveness of the drug-resistant sublines. ADAM9 knock-down in the resistant cells increased dabrafenib sensitivity, whereas miR-126-3p enforced expression or ADAM9 silencing in the drug-sensitive cells delayed the development of resistance. At T0 and T2, statistically significant differences were observed in VEGF-A serum levels between patients who responded to therapy and patients who did not. In responder patients, a significant increase of VEGF-A levels was observed at TP versus T2. Conclusions Strategies restoring miR-126-3p expression or targeting VEGF-A or ADAM9 could restrain growth and metastasis of dabrafenib-resistant melanomas and increase their drug sensitivity. Circulating VEGF-A is a promising biomarker for predicting patients’ response to BRAFi or BRAFi+MEKi and for monitoring the onset of resistance.

Abstract Background Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.