Tissue distribution and acute toxicity of silver after single intravenous administration in mice: nano-specific and size-dependent effects

Background: Silver nanoparticles (AgNPs) are an important class of nanomaterials used as antimicrobial agents for a wide range of medical and industrial applications. However toxicity of AgNPs and impact of their physicochemical characteristics in in vivo models still need to be comprehensively characterized. The aim of this study was to investigate the effect of size and coating on tissue distribution and toxicity of AgNPs after intravenous administration in mice, and compare the results with those obtained after silver acetate administration. Methods: Male CD-1(ICR) mice were intravenously injected with AgNPs of different sizes (10 nm, 40 nm, 100 nm), citrate-or polyvinylpyrrolidone-coated, at a single dose of 10 mg/kg bw. An equivalent dose of silver ions was administered as silver acetate. Mice were euthanized 24 h after the treatment, and silver quantification by ICP-MS and histopathology were performed on spleen, liver, lungs, kidneys, brain, and blood. Results: For all particle sizes, regardless of their coating, the highest silver concentrations were found in the spleen and liver, followed by lung, kidney, and brain. Silver concentrations were significantly higher in the spleen, lung, kidney, brain, and blood of mice treated with 10 nm AgNPs than those treated with larger particles. Relevant toxic effects (midzonal hepatocellular necrosis, gall bladder hemorrhage) were found in mice treated with 10 nm AgNPs, while in mice treated with 40 nm and 100 nm AgNPs lesions were milder or negligible, respectively. In mice treated with silver acetate, silver concentrations were significantly lower in the spleen and lung, and higher in the kidney than in mice treated with 10 nm AgNPs, and a different target organ of toxicity was identified (kidney). Conclusions: Administration of the smallest (10 nm) nanoparticles resulted in enhanced silver tissue distribution and overt hepatobiliary toxicity compared to larger ones (40 and 100 nm), while coating had no relevant impact. Distinct patterns of tissue distribution and toxicity were observed after silver acetate administration. It is concluded that if AgNPs become systemically available, they behave differently from ionic silver, exerting distinct and size-dependent effects, strictly related to the nanoparticulate form

Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers

IntroductionNew early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients.MethodsFourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples.ResultsWe first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups.DiscussionThese data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease

Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

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

Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration

Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues

Critical role of maternal selenium nutrition in neurodevelopment: Effects on offspring behavior and neuroinflammatory profile

Research in both animals and humans shows that some nutrients are important in pregnancy and during the first years of life to support brain and cognitive development. Our aim was to evaluate the role of selenium (Se) in supporting brain and behavioral plasticity and maturation. Pregnant and lactating female rats and their offspring up to postnatal day 40 were fed isocaloric diets differing in Se content - i.e., Optimal, SubOptimal, and Deficient - and neurodevelopmental, neu-roinflammatory, and anti-oxidant markers were analyzed. We observed early adverse behavioral changes in juvenile rats only in SubOptimal offspring. In addition, SubOptimal, more than Deficient supply reduced basal glial reactivity in sex dimorphic and brain-area specific fashion. In female offspring, Deficient and SubOptimal diets reduced the antioxidant Glutathione peroxidase (GPx) activity in the cortex and in the liver, the latter being the key organ regulating Se metabo-lism and homeostasis. The finding that Se SubOptimal was more detrimental than Se Deficient diet may suggest that maternal Se Deficient diet, leading to a lower Se supply at earlier stages of fetal development, stimulated homeostatic mechanisms in the offspring that were not initiated by SubOptimal Se. Our observations demonstrate that even moderate Se deficiency during early life negatively may affect, in a sex-specific manner, optimal brain development

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage

Gain- and Loss-of-Function CFTR Alleles Are Associated with COVID-19 Clinical Outcomes

Carriers of single pathogenic variants of the CFTR (cystic fibrosis transmembrane conductance regulator) gene have a higher risk of severe COVID-19 and 14-day death. The machine learning post-Mendelian model pinpointed CFTR as a bidirectional modulator of COVID-19 outcomes. Here, we demonstrate that the rare complex allele [G576V;R668C] is associated with a milder disease via a gain-of-function mechanism. Conversely, CFTR ultra-rare alleles with reduced function are associated with disease severity either alone (dominant disorder) or with another hypomorphic allele in the second chromosome (recessive disorder) with a global residual CFTR activity between 50 to 91%. Furthermore, we characterized novel CFTR complex alleles, including [A238V;F508del], [R74W;D1270N;V201M], [I1027T;F508del], [I506V;D1168G], and simple alleles, including R347C, F1052V, Y625N, I328V, K68E, A309D, A252T, G542*, V562I, R1066H, I506V, I807M, which lead to a reduced CFTR function and thus, to more severe COVID-19. In conclusion, CFTR genetic analysis is an important tool in identifying patients at risk of severe COVID-19