Serum Interleukin-36 α as a Candidate Biomarker to Distinguish Behçet's Syndrome and Psoriatic Arthritis.

Behçet's syndrome (BS) is a rare systemic vasculitis characterized by different clinical manifestations. As no specific laboratory tests exist, the diagnosis relies on clinical criteria, and the differential diagnosis with other inflammatory diseases can be challenging. Indeed, in a relatively small proportion of patients, BS symptoms include only mucocutaneous, articular, gastrointestinal, and non-typical ocular manifestations, which are frequently found also in psoriatic arthritis (PsA). We investigate the ability of serum interleukin (IL)-36α-a pro-inflammatory cytokine involved in cutaneous and articular inflammatory diseases-to differentiate BS from PsA. A cross-sectional study was performed on 90 patients with BS, 80 with PsA and 80 healthy controls. Significantly lower IL-36α concentrations were found in patients with BS as compared to PsA, although in both groups IL-36α was significantly increased compared to healthy controls. An empirical cut-off of 420.6 pg/mL displayed a specificity of 0.93, with a sensitivity of 0.70 (AUC 0.82) in discriminating PsA from BS. This cut-off displayed a good diagnostic performance also in BS patients lacking highly specific BS manifestations. Our results indicate that IL-36α might be involved in the pathogenesis of both BS and PsA, and might be a candidate biomarker to support the differential diagnosis of BS.

Erythrocyte oxidative stress and thrombosis.

Thrombosis is a common disorder with a relevant burden of morbidity and mortality worldwide, particularly among elderly patients. Growing evidence demonstrated a direct role of oxidative stress in thrombosis, with various cell types contributing to this process. Among them, erythrocytes produce high quantities of intracellular reactive oxygen species (ROS) by NADPH oxidase activation and haemoglobin autoxidation. Concomitantly, extracellular ROS released by other cells in the blood flow can be uptaken and accumulate within erythrocytes. This oxidative milieu can alter erythrocyte membrane structure, leading to an impaired erythrocyte function, and promoting erythrocytes lysis, binding to endothelial cells, activation of platelet and of coagulation factors, phosphatidylserine exposure and release of microvesicles. Moreover, these abnormal erythrocytes are able to adhere to the vessel wall, contributing to thrombin generation within the thrombus. This process results in accelerated haemolysis and in a hypercoagulable state, in which structurally impaired erythrocytes contribute to increase thrombus size, to reduce its permeability and susceptibility to lysis. However, the wide plethora of mechanisms by which oxidised erythrocytes contribute to thrombosis is not completely elucidated. This review discusses the main biochemical aspects linking erythrocytes, oxidative stress and thrombosis, addressing their potential implication for clinical and therapeutic management.

A unique circulating miRNA profile highlights thrombo-inflammation in Behçet's syndrome.

OBJECTIVES: Behçet's syndrome (BS) is a rare systemic vasculitis often complicated by thrombotic events. Given the lack of validated biomarkers, BS diagnosis relies on clinical criteria.In search of novel biomarkers for BS diagnosis, we determined the profile of plasmatic circulating microRNAs (ci-miRNAs) in patients with BS compared with healthy controls (HCs). METHODS: ci-miRNA profile was evaluated by microarray in a screening cohort (16 patients with BS and 18 HCs) and then validated by poly(T) adaptor PCR (PTA-PCR) in a validation cohort (30 patients with BS and 30 HCs). Two disease control groups (30 patients with systemic lupus erythematosus (SLE) and 30 patients with giant cell arteritis (GCA) were also analysed. RESULTS: From the microarray screening, 29 deregulated (differentially expressed (DE)) human ci-miRNAs emerged. A hierarchical cluster analysis indicated that DE ci-miRNAs clearly segregated patients from controls, independently of clinical features. PTA-PCR analysis on the validation cohort confirmed the deregulation of miR-224-5p, miR-206 and miR-653-5p. The combined receiver operating characteristic (ROC) curve analyses showed that such ci-miRNAs discriminate BS from HCs (and BS with active vs inactive disease), as well as BS from patients with SLE and GCA.The functional annotation analyses (FAAs) showed that the most enriched pathways affected by DE ci-miRNAs (ie, cell-matrix interaction, oxidative stress and blood coagulation) are related to thrombo-inflammatory mechanisms. Accordingly, the expression of the three ci-miRNAs from the validation cohort significantly correlated with leucocyte reactive oxygen species production and plasma lipid peroxidation. CONCLUSIONS: The ci-miRNA profile identified in this study may represent a novel, poorly invasive BS biomarker, while suggesting an epigenetic control of BS-related thrombo-inflammation.

The Transcriptional Landscape of BRAF Wild Type Metastatic Melanoma: A Pilot Study.

Melanoma is a relatively rare disease worldwide; nevertheless, it has a great relevance in some countries, such as in Europe. In order to shed some light upon the transcriptional profile of skin melanoma, we compared the gene expression of six independent tumours (all progressed towards metastatic disease and with wild type BRAF) to the expression profile of non-dysplastic melanocytes (considered as a healthy control) in a pilot study. Paraffin-embedded samples were manually micro-dissected to obtain enriched samples, and then, RNA was extracted and analysed through a microarray-based approach. An exhaustive bioinformatics analysis was performed to identify differentially expressed transcripts between the two groups, as well as enriched functional terms. Overall, 50 up- and 19 downregulated transcripts were found to be significantly changed in the tumour compared to the control tissue. Among the upregulated transcripts, the majority belonged to the immune response group and to the proteasome, while most of the downregulated genes were related to cytosolic ribosomes. A Gene Set Enrichment Analysis (GSEA), along with the RNA-Seq data retrieved from the TCGA/GTEx databases, confirmed the general trend of downregulation affecting cytoribosome proteins. In contrast, transcripts coding for mitoribosome proteins showed the opposite trend.

Integrins regulate hERG1 dynamics by girdin-dependent Gαi3: signaling and modeling in cancer cells.

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with ß1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, ß1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (Vrest) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/ß1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation-like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/ß1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/ß1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells.

SIRT1 and thrombosis.

Thrombosis is a major cause of morbidity and mortality worldwide, with a complex and multifactorial pathogenesis. Recent studies have shown that SIRT1, a member of the sirtuin family of NAD + -dependent deacetylases, plays a crucial role in regulating thrombosis, modulating key pathways including endothelial activation, platelet aggregation, and coagulation. Furthermore, SIRT1 displays anti-inflammatory activity both in vitro, in vivo and in clinical studies, particularly via the reduction of oxidative stress. On these bases, several studies have investigated the therapeutic potential of targeting SIRT1 for the prevention of thrombosis. This review provides a comprehensive and critical overview of the main preclinical and clinical studies and of the current understanding of the role of SIRT1 in thrombosis.

Circulating miRNome profiling data in Behçet's syndrome.

We conducted a screening analysis to assess the presence of a characteristic extracellular circulating microRNAs (ci-miRNAs) profile in Behçet's syndrome (BS). Total RNA was extracted from platelets-free plasma (PFP) samples obtained from 16 BS patients and 18 healthy controls. Ci-miRNAs profiling was conducted by using dedicated Agilent microarray hybridization and data extraction technology. Statistical analysis of data extracted from microarray scanning revealed the deregulation of 36 ci-miRNAs, which turned out be differentially expressed between BS patients and healthy controls. Detailed experimental methods and data analysis were described here. The raw and normalized microarray data were deposited into Gene Expression Omnibus (GEO) under accession number GSE145191.

Harnessing the hERG1/ß1 Integrin Complex via a Novel Bispecific Single-chain Antibody: An Effective Strategy against Solid Cancers.

mAbs, either mono- or bispecific (bsAb), represent one of the most successful approaches to treat many types of malignancies. However, there are certain limitations to the use of full length mAbs for clinical applications, which can be overcome by engineered antibody fragments. The aim of this study was to develop a small bsAb, in the format of a single-chain diabody (scDb), to efficiently target two proteins, the hERG1 potassium channel and the ß1 subunit of integrin receptors, which specifically form a macromolecular complex in cancer cells. We provide evidence that the scDb we produced binds to the hERG1/ß1 complex in cancer cells and tissues, but does not bind to the hERG1 channel in nonpathologic tissues, in particular the heart. The scDb-hERG1-ß1 (i) downregulates the formation of the hERG1/ß1 complex, (ii) inhibits Akt phosphorylation and HIF-1α expression, and (iii) decreases cell survival, proliferation, and migration in vitro These effects only occur in cancer cells (either colon, pancreatic, or breast), but not in normal cells. In vivo, the scDb-hERG1-ß1 shows a good pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity when injected intravenously up to the dose of 8 mg/kg. The scDb-hERG1-ß1 accumulates into subcutaneous xenografted tumors, arising from either colon or pancreatic human cancer cells, and induces a reduction of tumor growth and vascularization. Overall, the scDb-hERG1-ß1 represents an innovative single-chain bispecific antibody for therapeutic applications in solid cancers that overexpress the hERG1/ß1 integrin signaling complex.

KV11.1 Potassium Channel and the Na+/H+ Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells.

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H+/HCO3 - transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K+ channels, which are central regulators of cell membrane potential (Vm), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and KV11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto ß1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the ß1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na+/H+ antiporter NHE1. Blocking KV11.1 with E4031 shows that channel activity contributed to modulate the ß1 integrin-dependent pHi increase. Interestingly, both NHE1 and KV11.1 modulated the colorectal cancer cell motility triggered by ß1 integrin-dependent adhesion. Finally, the ß1 integrin subunit, KV11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between KV11.1, NHE1, and ß1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.

Correction: Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

The financial support for this Article was not fully acknowledged. The acknowledgements should have included the following: We thank M. Lulli (University of Florence, Italy) for acquiring images of immunofluorescence-labeled cells. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (#15627, #21510 and #19766 to A.A.); PAR FAS-Linea di Azione 1.1-Azione 1.1.2-Bando FAS Salute. 2014 (DD 4042/ 2014) Project OMITERC to A.A.; FAR 2018 to A.B.

Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

We have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K+ channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.