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Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1 . Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

The complex pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP) generates a spectrum of phenotypes with a wide variety of inflammatory states. We enrolled 44 very-likely-to-be type 2 CRSwNP patients in order to evaluate the load of inflammation and to analyze human interleukins in nasal secretion. Clinical data were collected to evaluate the severity of the disease. High levels of IL-5, IL-4, IL-6, and IL-33 were detected in all type 2 CRSwNP patients. By analyzing type 2 cytokine profiles and local eosinophil count, we identified two coherent clusters: the first was characterized by high levels of IL-4, IL-5, IL-6, and a high-grade eosinophil count (type 2-high); the second had lower levels of cytokines and poor or absent eosinophilic inflammation (type-2 low). IL-5 levels were significantly higher within the type 2 cytokine and it was the most reliable biomarker for differentiating the two clusters. In type 2-high inflammatory profile clinical scores, the mean number of previous surgeries and need for systemic corticosteroids were significantly higher compared to type 2-low. Our research demonstrated the potential role of type 2 biomarkers, and in particular, of IL-5 in identifying patients with a more severe phenotype based on a high inflammatory load.

Depression is commonly associated with psoriatic arthritis (PsA), but its risk factors in these patients are largely unrecognized. Pro-inflammatory cytokines involved in the pathogenesis of PsA have been associated with depression in patients without autoimmune diseases. The aim of this study was to establish whether PsA patients with and without depressive symptoms differed for general or clinical variables and serum cytokines milieu.One hundred and fifty consecutive patients with PsA were screened for depressive symptoms with Hospital Anxiety and Depression Scale (HADS-D). Patients with and without depressive symptoms were compared according to the prevalence of general risk factors for depression, comorbidities, PsA features and serum IL-6, TNF-α, and IL-17A.Fifty-eight patient (38.7%) had a depressive mood. Depressive symptoms were associated with female sex (p = 0.03) and current smoking (p = 0.05). Patients with and without depressive symptoms did not differ for general risk factors for depression and comorbidities. Depressed patients had more frequently psoriatic nail disease (p = 0.02) and significant physical disability (HAQ-DI ≥ 0.5) (p0.01) and were more frequently in moderate or high disease activity according to DAPSA score (p = 0.01). Depressed patients had higher serum IL-6 (p0.01) and comparable serum IL-17A and TNF-α. A cutoff of 2.27 pg/ml of serum IL-6 had the best ability to predict an HADS-D ≥ 8 (AUC 0.666 ± 0.044; p0.01). Multivariate logistic regression analysis confirmed that serum IL-6 ≥ 2.27 pg/ml was independently associated with depressive symptoms (OR 3.5; CI 1.6-7.8; p0.01).Higher serum Il-6 is associated with depressive symptoms. This association suggests a direct role of systemic inflammation in the modulation of mood in PsA patients. Key Points • High PsA disease activity and physical disability are associated with depression. • Higher serum levels of IL-6 are independently associated with depression in PsA. • IL-6 might play a direct role in the development of depression in PsA patients.

Marker di staminalità nei carcinomi squamocellulari del cavo orale e orofaringe in relazione al sito di origine e a infezione da HPV: implicazioni cliniche.In questo studio è stata esaminata l’espressione di potenziali marcatori di staminalità nei carcinomi della testa e collo (HNSCC) per valutarne il loro possibile ruolo clinico. Sono stati arruolati 69 carcinomi squamocellulari del cavo orale (OSCC) e dell’orofaringe (OPSCC) primitivi e non precedentemente sottoposti a trattamento, raccogliendo i dati anagrafici, clinici e sul follow up. Abbiamo valutato l’eventuale infezione da HPV e l’espressione di 5 potenziali marker di staminalità (CD44, CD133, Oct-4, Nanog, and Sox-2). Gli OPSCC positivi per HPV hanno mostrato minor espressione di Nanog, mentre la sua espressione citoplasmatica è stata associata con una prognosi significativamente peggiore negli OPSCC ma non in OSCC. La colorazione di Sox-2 si è rivelata più intensa tra gli OPSCC, e la sua espressione nucleare è associata con una peggiore prognosi. L’espressione di Nanog è associata a OPSCC HPV-negativi e può avere un ruolo come marker diagnostico surrogato. In conclusione il profilo di espressione di alcuni marker di cellule staminali nei HNSCC sembra essere differente a seconda del sito di origine del tumore e dell’infezione da HPV. Inoltre Nanog e Sox-2 potrebbero presentare un significato prognostico.The expression of potential stem cell markers in HNSCCs was investigated to assess their potential clinical role. 69 primary, previously untreated oral (OSCC) and oropharyngeal squamous cell carcinomas (OPSCC) were enrolled; personal, clinical and follow-up data were collected. HPV infection and expression of 5 potential stem cell markers (CD44, CD133, Oct-4, Nanog, and Sox-2) were evaluated. HPV+ OPSCC showed lower expression of Nanog. The cytoplasmic expression of Nanog was associated with significantly worse prognosis in OPSCC, but not in OSCC. Sox-2 staining was more intense among OPSCCs. Sox-2 nuclear staining was associated with worse prognosis. Nanog expression was associated with HPV- OPSCC and may have a role as a surrogate diagnostic marker. In general, the expression profile of some stem cell markers in HNSCC seems to vary according to the site of origin and HPV infection. Nanog and Sox-2 may also have prognostic value.

The aetiology of cryptoglandular anal fistula (AF) is poorly understood. Evidence suggests that persistence and/or recurrence of the disease is more related to inflammatory than infectious factors. The aim of this study was to investigate the immune profile of cryptoglandular AF and to perform a histopathological characterization.Fistulectomy was performed in all patients; healthy ischioanal fat from the same patients was used as a control. Samples were evaluated by the Luminex xMAP system for the detection of 27 analytes. AF tissues were analysed using immunofluorescence. Staining was performed using primary antibodies to identify M1 inflammatory and M2 anti-inflammatory macrophages. Selective staining of total T lymphocytes and different T lymphocyte subsets was performed.Twenty patients with AF underwent a fistulectomy. Specific cytokine pathways differentiated AF from healthy tissue: pro-inflammatory cytokines interleukin (IL)-1β, IL-4, IL-8 and IL-17 and the anti-inflammatory cytokine IL-10 were overexpressed in AF compared with controls. Chemokines involved in macrophage recruitment (CCL2, CCL3, CCL4) were higher in AF than in healthy fatty tissue. Moreover, we showed that Tc17 cells characterize AF patients, thus confirming the enzyme-linked immunosorbent assay data. Furthermore, elevated infiltration of CD68+ myeloid cells and a reduction of the M1/M2 ratio characterize AF patients.A combination of inflammatory cytokines, chemokines and growth factors reside in the wound microenvironment of AF patients. For the first time an important prevalence of Tc17 cells and a reduction in the M1/M2 ratio was observed, thus suggesting new insights into the immunological characterization of AF patients.

Simple Summary Intra-cranial ependymoma (EPN) accounts for approximately 10% of pediatric brain tumors. The current therapeutic strategies have not significantly improved prognosis, which is still dismal in nearly 40% of patients. Major challenges for treatment are chemorefractoriness of EPN, tendency to recur, and high intra-tumoral heterogeneity (ITH). It is increasingly emerging that stalled neurodevelopmental programs driven by cancer stem cells (CSCs)/progenitor cells are at the root of oncogenesis and ITH of pediatric brain tumors, including EPN. This is the first review that examines how genetic and heritable epigenetic alterations and environmental selection forces drive ITH of pediatric intra-cranial EPN in the perspective of the CSC model. This review also summarizes how improvement in the single-cell technology has deepened the comprehension of the complexity, cell-of-origin, and developmental trajectories of EPN, paving the way for novel therapeutic options. Abstract Intra-tumoral heterogeneity (ITH) is a complex multifaceted phenomenon that posits major challenges for the clinical management of cancer patients. Genetic, epigenetic, and microenvironmental factors are concurrent drivers of diversity among the distinct populations of cancer cells. ITH may also be installed by cancer stem cells (CSCs), that foster unidirectional hierarchy of cellular phenotypes or, alternatively, shift dynamically between distinct cellular states. Ependymoma (EPN), a molecularly heterogeneous group of tumors, shows a specific spatiotemporal distribution that suggests a link between ependymomagenesis and alterations of the biological processes involved in embryonic brain development. In children, EPN most often arises intra-cranially and is associated with an adverse outcome. Emerging evidence shows that EPN displays large intra-patient heterogeneity. In this review, after touching on EPN inter-tumoral heterogeneity, we focus on the sources of ITH in pediatric intra-cranial EPN in the framework of the CSC paradigm. We also examine how single-cell technology has shed new light on the complexity and developmental origins of EPN and the potential impact that this understanding may have on the therapeutic strategies against this deadly pediatric malignancy.

Liquid biopsy has become a useful alternative in metastatic colorectal cancer (mCRC) patients when tissue biopsy of metastatic sites is not feasible. In this study we aimed to investigate the clinical utility of circulating exosomes DNA in the management of mCRC patients. Exosomes level and KRAS mutational status in exosomal DNA was assesed in 70 mCRC patients and 29 CRC primary tumor and were analysed at different disease steps evaluating serial blood samples (240 blood samples). There was a significant correlation between the extension of disease and exosomes level and the resection of primary localized tumor was correlated with a decrease of KRAS G12V/ D copies and fractional abundance in metastatic disease. CEA expression and liver metastasis correlated with a higher number of KRAS G12V/D copies/ml and a higher fractional abundance; in the subgroup of mCRC patients eligible for surgery, the size of tumor and the radiological response were related to exosomes level but only the size was related to the number of KRAS WT copies; both KRAS wild-type and mutated levels were identified as a prognostic factor related to OS. Finally, we found that 91% of mutated mCRC patients became wild type after the first line chemotherapy but this status reverted in mutated one at progression in 80% of cases. In a prospective cohort of mCRC patients, we show how longitudinal monitoring using exosome-based liquid biopsy provides clinical information relevant to therapeutic stratification.

The causal connection between serum biomarkers and COVID-19 severity or pathogenicity in children is unclear. The aim of this study was to describe clinical and immunological features of children affected by COVID-19. The secondary aim was to evaluate whether these cytokines could predict severity of COVID-19. All children (aged 0−18) admitted to the Pediatric Emergency Department and tested with nasopharyngeal swab for SARS-CoV-2 were recruited and assigned to three groups: COVID-19, other infections, control group. Clinical and laboratory data of these patients, including circulating cytokine levels, were analyzed in three groups. Fever was the most frequent symptom in COVID-19 (67.3%). Neutropenia was found in the COVID-19 group (p &lt, 0.05), no difference was observed for lymphocyte counts in the three groups. Higher levels of IL-6 and TNF-alpha were found in the COVID-19 group compared to other infections and control groups (p = 0.014 and p = 0.001, respectively). Whereas, in the COVID-19 group, no difference was observed as for the same cytokines among sub-groups of different disease severity (p = 0.7 and p = 0.8). Serum levels of IL-6 and TNF-alpha were higher in COVID-19 children than in children with other infectious diseases, but those levels did not correlate with disease severity. Clinical studies in a large pediatric population are necessary to better define the role of the immune-mediated response in SARS-CoV-2 infections in children.

Background: Cardiovascular events (CVEs) are the first cause of death in patients with psoriatic arthritis (PsA). Depression is a recognized risk factor in cardiovascular events and is frequently associated with PsA. Flow-mediated dilatation (FMD) is a widely used method for assessing endothelial dysfunction, a parameter with strong prognostic implications for CVEs. The study aims to explore the relationship between FMD, depressive symptoms and serum cytokines in a cohort of patients with PsA.Patients and Methods: FMD was assessed in 50 consecutive PsA patients aged between 30 and 75 years without known cerebrovascular and coronary heart disease or diabetes. Depressive symptoms were reported using the related subscale of the Hospital Anxiety and Depression Scale (HDS). Disease features, activity indexes, and adjusted Framingham risk score (aFRS) were calculated. Serum level of IL-6, TNF-α, and IL-17A were also assessed.Results: In PsA patients (age 50.7 ± 10.2 years, male 42%, disease duration 5.9 ± 3.3 years, Disease Activity in PSoriatic Arthritis (DAPSA) score 14.0 ± 9.4) FMD inversely correlated with the severity of depressive symptoms according to HDS (ρ = −0.339, p = 0.016), age (ρ = −0.507, p = 0.001), aFRS (rs = −0.453, p < 0.001), duration of PsA (ρ = −0.507, p = 0.001), intensity of pain (ρ = −0.507, p = 0.001), and DAPSA (ρ = −0.507, p = 0.001). No statistically significant correlation was found between FMD or HDS and serum cytokines concentrations. HDS predicted FMD in a model adjusted for age, aFRS, PsA duration, and pain intensity (β = −0.271, p = 0.008), with depressive symptoms contributing directly to 6.4% of the variance.Conclusions: Depressive symptoms correlate with endothelial dysfunction with an exposure-response pattern in our cohort of PsA patients.

Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.

Extracellular vesicles (EVs), nano-sized (20–1000 nm) membranous structures released from cells, play critical roles in both physiological and pathological processes [1]. EVs can be classified based on their size into small (sEVs 200 nm EVs, also known as microvesicles) [1]. EVs might be a target for personalised medicine, given their content and biological origin [2]. Indeed, EVs detected in various biological fluids, including sputum supernatants, mucus, epithelial lining fluid, the pulmonary circulation and bronchoalveolar lavage fluid, represent a unique tool for both investigating the pathophysiology of respiratory disease and for biomarker discovery [2, 3]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Lucchetti has nothing to disclose. Conflict of interest: Dr. Santini has nothing to disclose. Conflict of interest: Dr. Perelli has nothing to disclose. Conflict of interest: Dr. Ricciardi-Tenore has nothing to disclose. Conflict of interest: Dr. Colella has nothing to disclose. Conflict of interest: Dr. Mores has nothing to disclose. Conflict of interest: Dr. Macis has nothing to disclose. Conflict of interest: Dr. Bush has nothing to disclose. Conflict of interest: Dr. Sgambato has nothing to disclose. Conflict of interest: Dr. Montuschi has nothing to disclose.

Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate, both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

A better understanding of the mechanisms of cell communication between cancer cells and the tumor microenvironment is crucial to develop personalized therapies. It has been known for a while that cancer cells are metabolically distinct from other non-transformed cells. This metabolic phenotype is not peculiar to cancer cells but reflects the characteristics of the tumor microenvironment. Recently, it has been shown that extracellular vesicles are involved in the metabolic switch occurring in cancer and tumor-stroma cells. Moreover, in an immune system, the metabolic programs of different cell subsets are distinctly associated with their immunological function, and extracellular vesicles could be a key factor in the shift of cell fate modulating cancer immunity. Indeed, during tumor progression, tumor-associated immune cells and fibroblasts acquire a tumor-supportive and anti-inflammatory phenotype due to their interaction with tumor cells and several findings suggest a role of extracellular vesicles in this phenomenon. This review aims to collect all the available evidence so far obtained on the role of extracellular vesicles in the modulation of cell metabolism and immunity. Moreover, we discuss the possibility for extracellular vesicles of being involved in drug resistance mechanisms, cancer progression and metastasis by inducing immune-metabolic effects on surrounding cells.

Cancer cells secrete small extracellular vesicles (sEVs) that are involved in the remodeling of tumor microenvironment (TME) and can promote tumor progression. The role of sEVs and their molecular key players in colon cancer stem cells differentiation are poorly understood. This study aimed to analyze the role and content of sEVs released during the differentiation of colorectal cancer stem cells. Here we show that sEVs secretion during colon cancer stem cells differentiation is partially controlled by CD147, a well-known player involved in colon cancer tumorigenesis. CD147 + sEVs activate a signaling cascade in recipient cells inducing molecular invasive features in colon cancer cells. CD147 knockdown as well as anti-CD147 antibodies impaired sEVs release and downstream effects on recipient cells and blocking multivesicular body maturation prevented sEVs release during the differentiation. Our findings reveal a functional role of CD147 in promoting sEVs release during the differentiation of colon cancer stem cells and in triggering cellular changes in recipient cells.

Background Probiotics are defined as live, nonpathogenic bacteria that confer health benefits beyond their nutritional value. In particular, VSL#3 exhibits demonstrated efficacy in the management of diseases characterized by an increased intestinal permeability. Our study aimed to understand how VSL#3 promotes gut health by secreting bioactive factors and identify which human pathways are modulated by secretome derived from the VSL#3 formula. Methods Two different lots of VSL#3 were used, and Caco-2 cell line was treated with conditioned media (CM) prepared using 1 g of the probiotic formula. We evaluated the effects of the probiotics on cellular proliferation and apoptosis by cytometry and the expression of tight junction proteins by western blotting. A proteomics analysis of both culture media and the whole proteome of Caco-2 cells treated with VSL#3-CM was performed by nano-ultra performance liquid chromatography - tandem mass (nUPLC MS/MS) spectrometry. Results The probiotic formula increased cell proliferation, decreased cellular apoptosis cells, and increased re-epithelialization in the scratch assay. Several peptides specifically synthetized by all the species within the probiotic preparation were recognized in the proteomics analysis. Human proteins synthesized by CaCo-2 cells were also identified. Conclusions To our knowledge, this manuscript describes the first evaluation of the probiotic secretome, and the results showed that the improvement in intestinal barrier functions induced by probiotics seems to be accompanied by the modulation of some human cellular pathways.

Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management.

Background:Cardiovascular complications are the leading cause of death in patients with psoriatic arthritis (PsA), but current strategies for reducing cardiovascular risk are still inadequate. Depression is a common comorbidity in PsA patients and it is recognized as an independent cardiovascular risk factor in the general population. Endothelial dysfunction, assessed as a reduction in brachial artery Flow Mediated Dilation (FMD), is a predictor of major cardiovascular events in high and low risk populations.Objectives:To investigate the relationship between endothelial function and depressive symptoms in a cohort of patients with PsA.Methods:Sixty consecutive patients with PsA, aged between 30 and 79 years, with no history of major cardiovascular events, were characterized for traditional cardiovascular risk factors and features of psoriatic disease. The risk of cardiovascular events according to traditional risk factors was calculated using the Framingham Risk Score (FRS) and the presence of depressive symptoms was defined through the Hospital Anxiety and Depression Scale (HDS) using the validated cut-off of 8. Endothelial function was assessed by FMD. Serum IL-6 was quantified by ELISA, IL-17 and TNF-α levels by Luminex method.Results:Patients had an average age of 52.1±11.0 years, 43.3% of them were male, 23.3% obese and 25.0% active smokers; 38.3%, 25.0% and 11.7% were treated for high blood pressure, dyslipidemia and diabetes mellitus, respectively. The 10-year risk of major cardiovascular events estimated by FRS was 10.4%. The mean duration of PsA was 9.4 years, 30.0% of patients were in minimal disease activity (MDA) and 61.7% and 46.7% were treated with conventional and biotechnological DMARDs, respectively. The mean HDS value was 6.9±3.2 and 43.4% of patients had significant depressive symptoms. The severity of depressive symptoms according to HDS correlated with disease activity according to DAPSA (r=0.449, p=0.001). The mean FMD was 7.8±3.8%, this value correlated inversely with age (r=-0.408,p2=0.23) and FRS normalized through logarithmic transformation (β=-0.32, p=0.009, R2=0.22).Conclusion:The degree of endothelial dysfunction quantified by FRS correlates with the severity of the depressive symptoms in patients with PsA, independently of the cardiovascular risk attributable to classical risk factors. The weak relationship between FRS and serum levels of IL-6, IL-17 and TNF-alpha suggests a role of factors independent of inflammation in the regulation of endothelial function in patients with PsA. Systematic research and treatment of depressive symptoms could contribute to a more complete stratification and a better management of cardiovascular risk in patients with PsA.Disclosure of Interests:Enrico De Lorenzis: None declared, Angela Di Giorgio: None declared, Gerlando Natalello: None declared, Antonio Nesci: None declared, Dario Bruno: None declared, Donatella Lucchetti: None declared, Giacomo Tanti: None declared, Clara Di Mario: None declared, Pietro Rubortone: None declared, Maria Rosaria Magurano: None declared, Barbara Tolusso: None declared, Angelo Santoliquido: None declared, Giusy Peluso: None declared, Elisa Gremese Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Janssen, Merck Sharp & Dohme, Novartis, Sanofi, UCB, Roche, Pfizer, Speakers bureau: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly, Janssen, Merck Sharp & Dohme, Novartis, Sanofi, UCB, Roche, Pfizer

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6−/−-MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6−/−-MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.

To report a case of acinic cell carcinoma occurred in the lacrimal gland. A 59-year-old man was admitted because of sudden blurring of vision, progressive proptosis of the left eye, and mild double vision in left and down directions of the gaze (Hess-Lancaster test). His medical history detailed controlled bilateral keratoconus and open angle glaucoma. On examination, the best corrected visual acuity decreased from 8/20 till 1/50 in one week. There was a swelling of the left upper eyelid. A hard and tender mass was palpated in the superior temporal left orbit. Ultrasound scan showed an extraconal solid mass, situated in the superior lateral corner of the orbit. Computed tomography and magnetic resonance imaging (MRI) revealed a mass of two centimeters in diameter, with round well-defined outline, within the lacrimal gland. We performed an enucleoresection of the mass, via a coronal approach and a lateral orbitotomy by a piezosurgical device. The lesion appeared nodular, brownish, measuring about 2 × 1.5 cm. Histopathological findings were consistent with acinic cell carcinoma with a microcystic, focally papillary-cystic growth of pattern. Follow-up MRI outcomes led to removal of the residual lacrimal gland for suspicion of recurrence. No tumor recurrences where detected at 7-year follow-up.

Background Multiple sclerosis (MS) is a chronic, immune-mediated, inflammatory, neurodegenerative disorder. Many studies are investigating the potential role of body fluid biomarkers as prognostic factors for early identification of patients presenting with clinical isolated syndrome (CIS) at high risk for conversion to MS or to recognize RRMS patients at high risk for progression. Objectives To evaluate the correlation between levels of BAFF, chitinase 3-like 1 (CHI3L1), sCD163, Osteopontin (OPN), both on serum and cerebral spinal fluid (CSF), and the disease activity and progression. We also want to explore a possible relationship between serological and CSF biomarker's levels. Patients and methods We enrolled 82 patients between June 2014 and June 2016. Seventy-one received a diagnosis of demyelinating disease of CNS (46 RRMS and 25 CIS), while 11 were affected by other neurological diseases. All patients underwent a neural axis MRI, lumbar puncture and blood samples. Levels of BAFF, CHI3L1, sCD163, OPN on serum and CSF were analyzed by Luminex xMAP system, with a kit 11-plex ad hoc. Results The CSF CHI3L1, sCD163 and OPN levels were significantly higher in MS patients than in controls. We did not find significant differences in serum CHI3L1, sCD163 and OPN levels, nor CSF or serum BAFF levels between patient and control groups. We found significantly higher CSF level of sCD163 and CHI3L1 in all patients’ subgroups compared with controls, while OPN was higher in CIS and RR subgroups. We did not find significant differences for serum and CSF levels of all the markers between patients with or without clinical or radiological disease activity. CSF sCD163 and CHI3L1 levels was significant higher in CIS patients who converted to MS (p Using ROC curve analysis, CSF sCD163 resulted the best predictive factor. CSF CHI3L1 and OPN levels resulted useful independent predictors too. Combined ROCs of those three analytes demonstrated a better predictive value, with sCD163 and CHI3L1 resulting as the best combination. Conclusions CSF sCD163 CHI3L1 and OPN levels were higher in MS patients whereas serum CHI3L1, sCD163 and OPN levels did not show differences compared with controls. This finding confirms the high CSF specificity with regards to the analysis of processes, inflammatory and non-inflammatory, that occur within the CNS.

The technologically interesting properties of palladium nanoparticles (Pd-NPs) allowed their widespread industrial application, although concerns emerged on increasing general and occupational levels of exposure. In this context, to assess the toxicological behavior of Pd-NPs, and particularly their endocrine disruptive potential, has become a public health priority. Therefore, we evaluated Pd-NP impact on the female endocrine reproductive system of Wistar rats sub-chronically treated for 90 days with increasing doses of this xenobiotic (0.12, 1.2, and 12 µg/kg, administered at days 1, 30, and 60 for cumulative doses of 0.36, 3.6, and 36 µg/kg) via the intravenous route. In this regard, we investigated potential alterations in different sex hormone, for example, estradiol, follicle-stimulating hormone (FSH), luteinizing hormone, progesterone, and testosterone, serum concentrations. All treated groups showed significantly greater levels of FSH compared to controls, suggesting a possible impact of Pd-NPs on the regulatory system that controls the normal physiology of female reproductive function. Although relevant, since obtained under sub-chronic, low-dose conditions of exposure resembling those encountered in real-world scenarios, the present results are preliminary and require confirmation as well as identification of the possible underlining molecular mechanisms. From a public and occupational health perspective, implications for the reproductive health of exposed subjects and the next generations of women exposed during their childbearing age or pregnancy should be elucidated. This information is essential to elaborate adequate preventive strategies for assessing and controlling possible Pd-NPs adverse effects on the endocrine system.

The widespread industrial application of nanomaterials (NMs) has dramatically increased the likelihood of environmental and occupational exposure of humans to such xenobiotics. This issue, together with the increasing public health interest in understanding the effects of chemicals on endocrine system, encouraged to investigate the disruptive potential of NMs on the endocrine function. Therefore, the aim of this study was to evaluate the effects of palladium nanoparticles (Pd-NPs) on the female reproductive system of Wistar rats, intravenously exposed to different doses (0.12, 1.2, and 12 µg/kg), through the assessment of possible quantitative changes in the serum concentrations of several sex hormones. Our results demonstrated that the highest exposure doses significantly reduced the estradiol and testosterone concentrations, while increased the luteinizing hormone levels in treated animals compared to controls. Such alterations are indicative for an abnormal reproductive axis function. However, further investigations are needed to clarify the role of the different NP physicochemical properties in determining such effects, and possible underlining molecular mechanisms, as well as their relevance for the development of diseases in the female reproductive system. Overall, this may be helpful to define accurate risk assessment and management strategies to protect the health of the general and occupational populations exposed to Pd-NPs.

The topical treatment for oral mucosal diseases is often based on products optimized for dermatologic applications; consequently, a lower therapeutic effect may be present. 18-β-glycyrrhetic acid (GA) is extracted from Glycirrhiza glabra. The first aim of this study was to test the cytotoxicity of GA on PE/CA-PJ15 cells. The second aim was to propose and test two different delivery systems, i.e. nanoparticles and fibers, to guarantee a controlled release of GA in vitro. We used chitosan and poly(lactic-co-glycolic) acid based nanoparticles and polylactic acid fibers. We tested both delivery systems in vitro on PE/CA-PJ15 cells and on normal human gingival fibroblasts (HGFs). The morphology of GA-loaded nanoparticles (GA-NPs) and fibers (GA-FBs) was investigated by electron microscopy and dynamic light scattering; GA release kinetics was studied spectrophotometrically. MTT test was used to assess GA cytotoxicity on both cancer and normal cells. Cells were exposed to different concentrations of GA (20-500 μmol l-1) administered as free GA (GA-f), and to GA-NPs or GA-FBs. ROS production was evaluated using dichlorodihydrofluorescein as a fluorescent probe. Regarding the cytotoxic effect of GA on PE/CA-PJ15 cells, the lowest TC50 value was 200 μmol l-1 when GA was added as GA-NPs. No cytotoxic effects were observed when GA was administered to HGFs. N-acetyl Cysteine reduced mortality induced by GA-f in PE/CA-PJ15 cells. The specific effect of GA on PE/CA-PJ15 cells is mainly due to the different sensitivity of cancer cells to ROS over-production; GA-NPs and GA-FBs formulations increase, in vitro, this toxic effect on oral cancer cells.

Recent progresses in the understanding of facioscapulohumeral muscular dystrophy (FSHD) genetics opened the way to the development of targeted therapies. However, knowledge about pathophysiology of muscle damage is still limited and there is increasing need to identify biomarkers of disease activity in the perspective of clinical trial readiness. We analyzed inflammatory mediators in the interstitial fluid of muscles with different MRI signal in FSHD patients, comparing muscles displaying early lesions on short-tau inversion recovery (STIR) sequences with normal ones. Patients with one T1-weighted normal and STIR hyperintense (STIR+) and contralateral T1-weighted and STIR normal (STIR-) lower limb muscle were asked to enter the study. Twelve consecutive patients, five controls, and one non-penetrant gene carrier underwent prolonged muscle microdialysis with high cut-off membranes. Microdialysates were analyzed using xMAP technology with a wide panel for cytokines, chemokines, and growth factors. A small number of inflammatory mediators were dysregulated in STIR+ versus STIR- and control muscles: CXCL13, upregulated in STIR+ muscles compared with controls (p

The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse-phase protein microarrays, flow-cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy-based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.

Recently, palladium nanoparticles (PdNPs) have been increasingly used in many industrial sectors, and this has led to a significant release of nano-sized palladium particles into the environment. However, despite the increase in occupational and general population exposure, information on the potential adverse effects of these PdNPs is still limited and their impact on the immune system constitutes a major health concern. Therefore, the aim of this study was to investigate the potential adverse effects induced by subchronic intravenous administration of PdNPs on the immune system of female Wistar rats by evaluating alterations in Interleukin (IL)-1α, IL-2, IL-4, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), Interferon (INF)-γ, and Tumor Necrosis Factor (TNF)-α serum levels. Exposed and control animals were randomly divided into five groups (0, 0.012, 0.12, 1.2, and 12 μg PdNPs per kg body weight) which were treated with repeated intravenous injections of vehicle or PdNPs (on day 1, 30, and 60). Subchronic exposure to PdNPs induced a decreasing trend in serum levels in most of the cytokines investigated, with the highest concentration (12 μg/kg) determining significant inhibitory effects. Overall, these results showed that PdNPs are able to alter cytokine serum levels in subchronically treated Wistar rats, suggesting a possible impact of these xenobiotics on the immune system after long-term exposures.

3558 Background: Target therapies and new surgical strategies deeply modify the history of CCLM patients (pts). Several prognostic scoring systems have been developed but no one is able to identify pts who should be excluded from a potentially useless surgery. Currently research is committed in identifying early biomarkers able to discern pts who could benefit from an aggressive approach. Exosomes are arising as promising biomarkers in cancer. The aim of this pivotal study was to analyze the association among exosome levels during CCLM-pts treatment, clinical outcomes and the KRAS status. Methods: We enrolled 22 pts with CCLM candidate to preoperative chemotherapy (pCT) and subsequent liver surgery. A blood sample was collected before pCT, after surgery, monthly during follow-up and at progression (PD). Exosomes were isolated by ultracentrifugation and characterized by standard methods. Exosomes concentration was assessed by Bradford assay. We adopted ddPCR™ KRAS G12/G13 Screening Kit to evaluate the KRAS status in exosomal DNA (e-DNA). Results: 22 CCLM pts received pCT and underwent liver surgery: 5 major hepatectomies and 17 multiple liver resections. Changes in exosomes plasma levels were found to correlate with each treatment step,resulting reduced after pCT and surgery and increased at PD, respectively (p = 0.0026). Pts with higher baseline exosome levels experimented shorter PFS than those with lower levels (p = 0.0033 HR 0.2). No association was found between exosome levels after liver surgery and disease free interval nor overall survival. KRAS status on e-DNA was evaluated on 10 pts in baseline, in pCT, after surgery, and in PD samples. In 8 out of 10 pts e-DNA displayed the same mutational status than the one detected on tumor DNA. Changes in e-DNA KRAS copies were found statistically significant in pCT vs surgery and pCT vs PD (p = 0.039; p = 0.04). Conclusions: Our study suggests a prognostic role of exosome levels in CCLM pts. Moreover, we showed that KRAS mutational status could be monitored during the post-surgery follow up by analyzing e-DNA. Overall, our data confirm the potential role of exosomes in liquid biopsy tool to monitor molecular changes during the treatment of CCLM pts.

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate–induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate–induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate–induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition–related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells.

Palladium nanoparticles have been increasingly used in catalytic processes, wastewater treatment, electronics, and biomedicine. However, recent evidence proved that these nanoparticles are able to induce adverse effects both in in vitro and in vivo models. Nevertheless, molecular mechanisms underlying the toxic effects are still poorly understood. Therefore, this study aimed to investigate the potential toxicological mechanisms of palladium nanoparticles assessing their effects on normal diploid rat fibroblast and lung carcinoma human epithelial cell lines. Several endpoints such as cell growth, cell cycle progression, DNA damage, induction of apoptosis, reactive oxygen species production and expression of cell cycle regulatory proteins were evaluated. Results showed that palladium nanoparticles inhibited cell growth in a dose- and time-dependent manner in both cell lines, although with a more evident action on fibroblasts. Interestingly, inhibition of cell growth was not associated with the induction of apoptosis. Cell cycle progression was arrested in the G0/G1 phase and DNA damage was evident in both cell lines even if only a slight increase in the intracellular reactive oxygen species levels was detected. These findings provide valuable insight into understanding the molecular mechanisms responsible of palladium nanoparticles toxicity whose identification is essential to define an adequate risk assessment process.

Tumour microenvironment contributes importantly to the phenotype development of cancer cells, which receive and adapt to chemical and physical signals. It is hypothesized that remodelling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signalling and permit the acquisition of metastatic properties such as anchorage-independent growth and enhanced cell migration. Among different cancer cell lines, the SW480 and SW620 colon cells, derived from the same patient at different stages of tumour progression, represent a unique model to study the changes in mechanical properties during metastases. The SW480 derive from a primary colon tumour, while the SW620 originate from a population of malignant cells appeared in a lymph node metastases months after the initial surgery. In this work we measured by AFM, combined with Electron and Confocal Microscopy, the mechanic adaptability of cancer cell which reached in vivo the lymph node environment. We demonstrate how to resist to apoptotic stimuli in the lymphatic circulation, where the flow shear stress reduce cell survival, the metastatic cell increase their non-specific adhesion while retaining a flexible and soft cytoskeleton, related to uncontrolled growth properties. These features allow accomplishing the spreading in the circulation aim of metastatic cells.

Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing four strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus, and L. delbrueckii subsp. bulgaricus*), three strains of Bifidobacteria (B. longum**, B. infantis**, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. The aim of the present study was to evaluate the mechanism of action of VSL#3 to reduce intestinal permeability, and its effect in modulating the activity on tight junctions, focusing on the effects of secreted bioactive factors. *Recently reclassified as L. helveticus. **Recently reclassified as B. animalis subsp. lactis. Methods Two different lots of VSL#3 (Manufacturer: Nutrilinea Srl, Gallarate (VA), Italy) were used. Caco2 and HT29 cell lines were treated with a conditioning media (CM) prepared using 1g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. The effects of probiotic on proliferations of cells were evaluated by cell growth curve and MTT assay. The effect on apoptosis will be analysed by cytometry using double staining with Annexin V-FITC and Propidium Iodide, as well as it is well established. Furthermore, the effect on expression of tight junctions, in particular claudin-2, occludin, and ZO-1, will be investigated by western blot analysis. Results The multistrain probiotic formula, in particular the more concentrate CM (1:10) increase Caco2 and HT29 cells proliferation and the percentage of metabolic cell activity (up 20%), together with a decrease of 90% of apoptotic cells in the presence of CM. These trophic effects may have an impact on paracellular permeability. The expression of tight junction proteins increase in 24 h for claudin and ZO-1, and after 48 h of treatment in case of occludin. Also the densitometry and the ratio between of relative density of protein and endogenous control (β-actin) confirms these data. The results are the same using two different lots. Conclusions These preliminary data in vitro could be able to explain how VSL#3 works at intestinal mucosa level, in particular by secretion of factors that enhances barrier integrity. The proliferative stimuli and the increase of expression of tight junction proteins are consistent with an effect on mucosa regeneration and re-epithelization. Other experiments, also on other intestinal cell line, are necessary to deeply understand the mechanism of action of this kind of probiotic formula.

OBJECTIVE This retrospective study aimed to evaluate the role of NFKB1-94 insertion/deletion ATTG (rs28362491) and NFkBIA 2758 A>G (rs696) polymorphisms and HLA-Cw6 allele in predicting the response to etanercept, a TNF-α blocker, in a population of psoriatic patients naive to biologics. METHODS Genomic DNA was extracted from whole blood in a series of 96 psoriatic patients who received etanercept for at least 3 months. Patients were classified as responders if they achieved a Psoriasis Area and Severity Index improvement of at least 75% after 12 weeks of etanercept treatment and as nonresponders if Psoriasis Area and Severity Index improvement was less than 75%. Genotyping was performed using the PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS We did not find any significant role of NFKB1-94 insertion/deletion ATTG (rs28362491) and NFkBIA 2758 A>G (rs696) polymorphisms and the HLA-Cw6 allele in predicting the response to etanercept. CONCLUSION Our findings suggest that NFKB1 and NFkBIA polymorphisms are not related to the response to etanercept. They also indicate that the therapeutic response to etanercept is not influenced by the presence of the HLA-Cw6 allele, in contrast with previous evidence on ustekinumab, suggesting that such an association is related more to drug than to disease characteristics.

Aim The pathogenesis of cryptoglandular anal fistula (AF) is still debated. Tissue inflammation could play a primary role. The pathological process of epithelial mesenchymal transition (EMT) might be involved but has never been investigated. Method In a prospective pilot study, twelve patients with an anal fistula had a fistulectomy. The excised track was divided into proximal (intrasphincteric) and distal (extrasphincteric) parts which were subjected to standard histopathological examination. The cytokines IL-8 and IL-1beta were analyzed as markers of inflammation, while EMT was evaluated by expression of TGF-beta, Vimentin, Zeb-1, Snail and E-Cadherin. Both mRNA and proteins expressions of these molecules were investigated by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry and were compared with the normal adjacent tissue. Results Chronic inflammation and granulation tissue and a stratified epithelium were evident on standard histopathological examination. The cytokine IL-8 was more expressed in the proximal than the distal part of the track (4.34 vs. 3.60), while the reverse was found for IL-1beta (1.33 vs 2.01); both were more intensely expressed compared with the normal anal mucosa. EMT was demonstrated, in both proximal and distal parts of the track, with an increase of TGF-beta, Vimentin, Zeb-1 and Snail and a mean decrease of E-Cadherin. Western-blot analysis and immunohistochemistry confirmed the protein expression. Conclusion The study suggests that chronic inflammation is present in cryptoglandular fistula. The inflammatory pattern might be different in the proximal than in the distal part of the fistula track. The cytokines IL-1beta and IL-8 could play a possible role in fistula formation. The study demonstrates for the first time the potential importance of EMT in the pathogenesis of cryptoglandular anal fistula. This article is protected by copyright. All rights reserved.

SW480 and SW620 colon carcinoma cell lines derive from primary tumour and lymph-node metastasis of the same patient, respectively. For this reason, these cells represent an ideal system to analyse phenotypic variations associated with the metastatic process. In this study we analysed SW480 and SW620 cytoskeleton remodelling by measuring the cells' mechanics and morphological properties using different microscopic techniques. We observed that different specialized functions of cells, i.e. the capacity to metastasize of elongated cells inside the primary tumour and the ability to intravasate and resist shear forces of the stream of cells derived from lymph node metastasis, are reflected in their mechanical properties. We demonstrated that, together with stiffness and adhesion between the AFM tip and the cell surface, cell shape, actin organization and surface roughness are strictly related and are finely modulated by colorectal cancer cells to better accomplish their specific tasks in cancer growth and invasion.