Potential Biomarkers for Therapeutic Monitoring and Clinical Outcome in Breast Cancer

Non-coding RNAs are a species of RNA that are not translated to proteins. These include transfer RNAs and ribosomal RNAs, microRNAs, transfer RNA-derived fragments, and long non-coding RNA. It is known that expression levels of some non-coding RNAs included microRNAs are altered in cancer cells or tumor tissues. Moreover, expression profiles of such non-coding RNAs correlate between tissues and body fluids. Therefore, several non-coding RNAs are being used as diagnostic/prognosis biomarkers or therapeutic targets in cancer. In this chapter, we review about representative non-coding RNAs and introduce especially microRNA as diagnosis/prognosis biomarkers and therapeutic targets

Pathological Proteins Are Transported by Extracellular Vesicles of Sporadic Amyotrophic Lateral Sclerosis Patients

Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset neurodegenerative disease, that affects cortical, bulbar and spinal motor neurons, and it is considered a proteinopathy, in which pathological proteins (SOD1, TDP-43, and FUS) may accumulate and interfere with neuronal functions eventually leading to cell death. These proteins can be released from cells and transported in the body fluids by extracellular vesicles (EVs). EVs are spherical vesicles, which are classified mainly in microvesicles (MVs) and exosomes (EXOs) based on their biogenesis, size and surface markers. In this study we characterized MVs and EXOs isolated from plasma of sporadic ALS patients and healthy controls and determined their number, size and SOD1, TDP-43, and FUS protein composition. No variation was found in the number of EVs between ALS patients and controls. However, the mean size both for MVs and for EXOs resulted increased in ALS patients compared to controls. MVs derived from ALS patients were enriched in SOD1, TDP-43, phospho-TDP-43, and FUS proteins compared to CTRLs. SOD1 was generally more concentrated in EXOs than in MVs, while TDP-43 and FUS protein levels were slightly higher in MVs than in EXOs. We demonstrated that MVs and EXOs size were increased in ALS patients compared to controls and that MVs of ALS patients were enriched with toxic proteins compared to CTRLs. EXOs did not show any protein changes. These data may suggest that MVs can transport toxic proteins and might play a role in prion-like propagation of ALS disease

Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients (r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression

Isolation and Characterization of Several Extracellular Vesicles Subtypes in Amyotrophic Lateral Sclerosis

Le vescicole extracellulari sono particelle delle dimensioni dei nanometri rilasciate nei principali biofluidi del nostro corpo tra cui sangue, urina e liquido cerebrospinale come regolatori fisiologici, e classificate secondo la loro biogenesi, le loro dimensioni e la loro densità in esosomi (EXOs), microvescicole (MVs) e corpi apoptotici. Queste vescicole sono carrier di numerose molecole tra cui proteine, DNA e RNA che possono avere sulla cellula target numerosi effetti, tra cui il coinvolgimento nella regolazione genica e nell’espressione proteica, fondamentali per le funzioni della cellula stessa. Tuttavia, il loro ruolo nei diversi compartimenti in cui vengono secrete non è ancora del tutto chiaro. A seconda del tipo di cellula da cui vengono rilasciate e dallo stato biologico a cui si trovano esposte, esercitano sia una funzione protettiva che patogenica. In questo lavoro di tesi abbiamo analizzato le MVs rilasciate nel plasma di pazienti affetti da sclerosi laterale amiotrofica (SLA) sporadica, malattia neurodegenerativa del motoneurone a esito fatale. La messa a punto del protocollo per separare MVs ed EXOs è stato particolarmente difficile a causa delle loro piccole dimensioni. Le MVs hanno dimensioni comprese tra i 100 nm e i 1.000 nm, invece gli EXOs tra i 40 nm e i 150 nm. Abbiamo separato con successo le due popolazioni con un protocollo di ultracentrifugazione e filtrazione e, successivamente, abbiamo verificato la purezza con tecniche di NanoTracking Analysis and Western Blotting. Mediante un’analisi citofluorimetrica abbiamo suddiviso le vescicole sia in base alle loro dimensioni, avvalendoci dell’uso di specifiche biglie calibrate e fluorescenti, sia in base alla positività all’Annessina V (che riconosce la fosfatidilserina). In seguito, abbiamo investigato la derivazione delle MVs positive all’Annessina V, ovvero abbiamo analizzato quali fossero di derivazione leucocitaria (CD45), eritrocitaria (CD235a), endoteliale (CD31) e piastrinica (CD61).Successivamente l’analisi è passata ad identificare mediante IFC quelle vescicole marcate per il CD4,CD8,CD25,CD45RA E CD45RO.Abbiamo quindi osservato che nei pazienti SLA, le MVs Annessina V+ erano caratterizzate prevalentemente dalla positività al marcatore leucocitario CD45. Beers et al. (2008) ha dimostrato che i pazienti SLA caratterizzati da una più rapida progressione della malattia presentano una minore concentrazione di CD4+ CD25High T regolatori, ci siamo quindi chiesti se un’elevata espressione di MVs CD45+ potesse inoltre essere associata con un più alto tasso di infiammazione legato alla formazione di complessi immunitari capaci di favorire la propagazione di tipo prionico di proteine misfoldate nelle cellule nervose. Tuttavia abbiamo osservato alti livelli di CD4 + / CD25 + / + Annv MV in campioni di plasma di pazienti affetti da SLA (MV 2,5 particelle / ul (p = 0.04)) rispetto ai controlli (MVS 0,9 particelle / ul.). Inoltre abbiamo constatato che una differenza tra pazienti affetti da SLA con una progressione veloce della malattia e quelli con una progressione lenta, respittavemante : CD45RA / Annv + (1,0 particelle / ul in fast vs slow 5,4 particelle / ul (p = 0.02)) e CD45RO / Annv + (25,2 particelle / ul in fast vs slow 9,1 particelle / ul (p = 0,036)). Questo ci ha mostrato come i linfociti regolatori T (Tregs) possono essere neuroprotettivi nella SLA (Appel, 2013). Inoltre, i nostri dati indicano che MVs di pazienti affetti da SLA portano marcatori di linfociti T naive (CD45RA) e linfociti T di memoria attivati e (CD45RO).Extracellular vesicles (EVs) are a heterogeneous group of nano-sized membrane vesicles released by all cells in vitro as well as in vivo, classified mainly on their biogenesis, size and buoyant densities in microvesicles (MVs) and exosomes (EXOs). EVs are gaining importance as a mediator of important physiological and pathological intercellular activities possibly through the transfer of their cargo (proteins, DNA and RNA) between target cells. This facilitates the spreading of the disease through the delivery of genetic material and pathogenic proteins. MVs from prion-infected neuronal cells can initiate prion propagation in uninfected cells, underlying a new mechanism of the disease propagation. The aim of this study was to isolate MVs and EXOs in plasma of Amyotrophic Lateral Sclerosis (ALS) patients and characterize them in order to discover new biomarkers of this disease. We isolated and characterized EVs in plasma of ALS patients with the use of Nanoparticle Tracking Analysis (NTA), conventional flow cytometry and image flow cytometry and proteomic technologies (Western Blotting). MVs are shed from cells by budding of a plasma membrane following specific stimulation, so they are surrounded by plasma membrane receptors and they are characterised by translocation of phosphatidylserine (PS). So markers for leukocyte (CD45), endothelial (CD31), platelet (CD61), erythrocyte (CD235a) derivation and Annexin V were investigated on MVs samples from plasma of 32 ALS, 30 Alzheimer’s Disease (AD) and 30 healthy controls by flow cytometry. Interestingly we found two groups of slow progressor ALS patients, one with high expression of CD45+ Annexin V+ MVs and one with low expression of misfolded SOD1. The slow progressor ALS patients with high expression of CD45+ MVs had high expression of misfolded SOD1 protein level compared to healthy controls. We observed high levels of CD4+/CD25+/AnnV+ MVs in plasma samples from ALS patients(MVs 2,5 particles/ul (p=0.04)) compared to controls (Mvs 0,9 particles/ul.) There is also a difference between fast and slow ALS patients using CD45RA/AnnV+(1,0 particles/ul in fast vs slow 5,4 particles/ul(p=0.02)) and CD45RO/AnnV+ (25,2 particles/ul fast vs slow 9,1 particles/ul(p=0.036)). High levels of CD4+/CD25+/AnnV+ (Tregulatory cells) MVs are found in our cohort of ALS patients. This is supporting previous finding that regulatory T-lymphocytes (Tregs) are neuroprotective in ALS (Appel,2013). In addition, our data indicates that MVs of ALS patients carry markers of naive T lymphocytes (CD45RA) and activated and memory T lymphocytes( CD45RO). Further studies are needed to understand this new role of CD45RA/AnnV+ and CD45RO/AnnV+ Mvs in the slow and fast progression group of ALS patients. This finding might indicate different steps of T cell activation

An Oral Glucose Load Decreases Postprandial Extracellular Vesicles in Obese Adults with and without Prediabetes

Although extracellular vesicles (EVs) are a novel biomediator of type 2 diabetes (T2D) and cardiovascular disease (CVD), the effects of hyperglycemia on EVs in humans is unknown. We tested the hypothesis that a 75-g oral glucose tolerance test (OGTT) would promote changes in EVs in relation to CVD risk. Twenty-five obese adults (Age: 52.4 ± 3.2 year, BMI: 32.5 ± 1.2 kg/m2) were screened for normal glucose tolerance (NGT, n = 8) and prediabetes (PD, n = 17) using American Diabetes Association criteria (75 g OGTT and/or HbA1c). Body composition (bioelectrical impedance) and fitness (VO2peak) were measured. Arterial stiffness (augmentation index; AIx) was measured at 0, 60- and 120-min while insulin, glucose, and free fatty acids were evaluated every 30 min during the OGTT to assess CVD risk. Annexin V positive (AV+) and Annexin V negative (AV-) total EVs, platelet EVs (CD31+/CD41+; CD41+), leukocyte EVs (CD45+; CD45+/CD41−), platelet endothelial cell adhesion molecule (PECAM) (CD31+) and endothelial EVs (CD 31+/CD41−; CD105+) were collected at 0 and 120 min. There were no differences in age, BMI, or body fat between NGT and PD (all P > 0.63). Total EVs, AV+ CD31+ (PECAM), and AV+ CD31+/CD41- (endothelial) EVs decreased after the OGTT (P ≤ 0.04). Circulating insulin at 2-h correlated with elevated post-prandial AV- CD45+ (r = 0.48, P = 0.04) while arterial stiffness related to reduced total EVs (r = −0.49, P = 0.03) and AV+CD41+ (platelet) (r = −0.52, P = 0.02). An oral glucose load lowers post-prandial total, platelet, and endothelial EVs in obese adults with NGT and prediabetes in relation to CVD risk

Lymphoblastoid cell lines as a model to understand amyotrophic lateral sclerosis disease mechanisms

In the past, amyotrophic lateral sclerosis (ALS) has been considered a ‘neurocentric’ disease; however, new evidence suggests that it should instead be looked at from a ‘multisystemic’ or ‘non-neurocentric’ point of view. From 2006, we focused on the study of non-neural cells: ALS patients’ peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterize LCLs of sporadic ALS (sALS) and patients carrying SOD1, TARDBP and FUS mutations to identify an ALS biologically relevant molecular signature, and determine whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we found some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations have different effects on ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward increased fusion, whereas sALS and SOD1 mutations mainly affect fission. With regards to protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, whereas FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together, these data indicate that LCLs could be a valid cellular model in ALS research in the identification and study of specific pathological pathways

Lymphoblastoid cell lines as a model to understand amyotrophic lateral sclerosis disease mechanisms

In the past, amyotrophic lateral sclerosis (ALS) has been considered a 'neurocentric' disease; however, new evidence suggests that it should instead be looked at from a 'multisystemic' or 'non-neurocentric' point of view. From 2006, we focused on the study of non-neural cells: ALS patients' peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterize LCLs of sporadic ALS (sALS) and patients carrying SOD1, TARDBP and FUS mutations to identify an ALS biologically relevant molecular signature, and determine whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we found some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations have different effects on ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward increased fusion, whereas sALS and SOD1 mutations mainly affect fission. With regards to protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, whereas FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together, these data indicate that LCLs could be a valid cellular model in ALS research in the identification and study of specific pathological pathways

Air pollution as a contributor to the inflammatory activity of multiple sclerosis

Objective: Air pollution has been recently identified as a risk factor for multiple sclerosis. Aim of this study was to investigate the immunological mechanism underlying the clinical association between air pollution, namely exposure to particulate matter 10 (PM10), and inflammatory activity of multiple sclerosis (MS) METHODS: Daily recording of PM10 was obtained by monitors depending on the residence of subjects. Expression of molecules involved in activation, adhesion, and migration of T lymphocytes were tested by flow cytometry in 57 MS patients and 19 healthy controls. We next assessed in vitro the effect of PM10 on expression of C-C chemokine receptors 6 (CCR6) by peripheral blood mononuclear cells (PBMCs), on cytokine production by monocyte-derived dendritic cells (mdDC), and on T cell polarization in PBMC/mdDC mixed cultures. Results: We identified a significant correlation between mean PM10 levels and expression of CCR6 CD4+ T circulating cells in MS patients. This was paralleled by the observation in vitro of a higher level of CCR6 expression on PBMC following treatment with increased doses of particulate matter. Moreover, in mdDC cultures, particulate matter induced the secretion by mdDC of Th17 polarizing IL1 beta, IL6, and IL23 and, in mdDC/PBMC mixed cultures, enhanced generation of IL17-producing T cells. Conclusions: Ex vivo and in vitro studies support the pro-inflammatory role of PM in MS, by upregulating expression of CCR6 on circulating CD4+ T cells and inducing in innate immune cells the production of Th17 polarizing cytokines. Therefore, we speculate that in MS respiratory exposure to PM10 may induce the production in the lung of autoreactive Th17 lymphocytes and boost their migratory properties through the blood-brain barrier

Extracellular vesicles and insulin‐mediated vascular function in metabolic syndrome

Abstract Metabolic Syndrome (MetS) raises cardiovascular disease risk. Extracellular vesicles (EVs) have emerged as important mediators of insulin sensitivity, although few studies on vascular function exist in humans. We determined the effect of insulin on EVs in relation to vascular function. Adults with MetS (n = 51, n = 9 M, 54.8 ± 1.0 years, 36.4 ± 0.7 kg/m2, ATPIII: 3.5 ± 0.1 a.u., VO2max: 22.1 ± 0.6 ml/kg/min) were enrolled in this cross‐sectional study. Peripheral insulin sensitivity (M‐value) was determined during a euglycemic clamp (40 mU/m2/min, 90 mg/dl), and blood was collected for EVs (CD105+, CD45+, CD41+, TX+, and CD31+; spectral flow cytometry), inflammation, insulin, and substrates. Central hemodynamics (applanation tonometry) was determined at 0 and 120 min via aortic waveforms. Pressure myography was used to assess insulin‐induced arterial vasodilation from mouse 3rd order mesenteric arteries (100–200 μm in diameter) at 0.2, 2 and 20 nM of insulin with EVs from healthy and MetS adults. Adults with MetS had low peripheral insulin sensitivity (2.6 ± 0.2 mg/kg/min) and high HOMA‐IR (4.7 ± 0.4 a.u.) plus Adipose‐IR (13.0 ± 1.3 a.u.). Insulin decreased total/particle counts (p < 0.001), CD45+ EVs (p = 0.002), AIx75 (p = 0.005) and Pb (p = 0.04), FFA (p < 0.001), total adiponectin (p = 0.006), ICAM (p = 0.002), and VCAM (p = 0.03). Higher M‐value related to lower fasted total EVs (r = −0.40, p = 0.004) while higher Adipose‐IR associated with higher fasted EVs (r = 0.42, p = 0.004) independent of VAT. Fasting CD105+ and CD45+ derived total EVs correlated with fasting AIx75 (r = 0.29, p < 0.05) and Pb (r = 0.30, p < 0.05). EVs from MetS participants blunted insulin‐induced vasodilation in mesenteric arteries compared with increases from healthy controls across insulin doses (all p < 0.005). These data highlight EVs as potentially novel mediators of vascular insulin sensitivity and disease risk