Influence of hydroxychloroquine blood levels on adhesion molecules associated with endothelial dysfunction in patients with systemic lupus erythematosus.

BACKGROUND: Patients with SLE have an endothelial dysfunction (ED), which is considered the earliest marker of cardiovascular (CV) disease. Endothelial cell activation induced by proinflammatory cytokines is defined by the endothelial expression of cell-surface adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. The aim of this study was to investigate whether serum endothelial adhesion molecule levels are influenced by blood hydroxychloroquine (HCQ) levels in SLE. METHODS: Consecutive patients with SLE taking a stable dose of HCQ were investigated. At study entry and 6 months later HCQ blood levels were quantified by tandem mass spectrometry. Serum levels of P-selectin, E-selectin, ICAM-1 and VCAM-1 were also measured using a Luminex 200 instrument. Comparison of endothelial soluble adhesion molecules in groups with different HCQ blood levels was performed by t-test. RESULTS: 83 patients with SLE were enrolled. Correlation were demonstrated between mean blood HCQ concentrations and endothelial soluble adhesion molecules (E-selectin, ICAM-1 and VCAM-1). Moreover, serum levels of ICAM-1 and VCAM-1 were significantly lower in the patients with SLE with HCQ blood levels >500 ng/mL (83.67±52.8 ng/mL vs 158.81±125.1 ng/mL and 8.9±2.2 ng/mL vs 10.4±2.3 ng/mL). Serum levels of E-selectin were nearly significantly lower in the patients with SLE with HCQ blood levels >500 ng/mL (64.7±30.2 ng/mL vs 71.6±42.2 ng/mL, p=0.06). No significant difference in concentration of P-selectin was detected. CONCLUSIONS: In the present study, there was a trend towards higher adhesion molecules levels with lower HCQ blood levels in patients with SLE. Further longitudinal studies will determine whether changes in endothelial biomarkers reflect decreased clinical CV events.

T-Cell Proapoptotic and Antifibrotic Activity Against Autologous Skin Fibroblasts in vitro Is Associated With IL-17A Axis Upregulation in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) T cells can induce apoptosis of autologous skin fibroblasts in vitro. Th17 cells have been reported to increase in SSc patients, and interleukin-17A (IL-17A) has a profibrotic function. We used a system based on T-cell-autologous fibroblast co-cultures to further investigate a possible role of IL-17A in SSc. Methods: T cells from diffuse SSc patients were co-cultured with autologous skin fibroblasts. IL17A mRNA was assessed by real-time PCR in co-cultured and control T cells, while IL17RA, CXCL1, CCL2, CCL3, COL1A1, COL3A1, CTGF, TGFBR2, and SMAD3 mRNAs were assessed in co-cultured and control fibroblasts. In subset experiments, co-cultures and control cells were treated with either IL-17A or IL-17A plus anti-IL17 receptor monoclonal antibody (α-IL-17RA mAb). Chemokine and procollagen type I (PCI) production was further investigated at the protein level in cell culture supernatants by multiple suspension immunoassay and sandwich ELISA, respectively. Co-cultured and control fibroblasts were also stained with Annexin V and analyzed by flow cytometry. Results: T cell-fibroblast co-cultures overexpressed IL17A and IL17RA. Furthermore, co-cultured fibroblasts upregulated IL-17A targets CXCL1, CCL2, and CCL3, while COL1A1, COL3A1, CTGF, and two key effectors of the TGF-ß signaling, TGFBR2 and SMAD3, were found downregulated. Consistently, chemokine concentrations were increased in co-culture supernatants, while PCI levels were reduced, especially after stimulation with ectopic IL-17A. Finally, simultaneous α-IL-17RA mAb treatment restored PCI levels and reduced fibroblast apoptosis in IL-17A-stimulated co-cultures. Conclusion: These data suggest that IL-17A upregulation might play a role in modulating T cell-mediated antifibrotic and proapoptotic effects in co-cultured autologous skin fibroblasts.

Biomarker panels may be superior over single molecules in prediction of renal flares in systemic lupus erythematosus: an exploratory study.

OBJECTIVE: Recent evidence suggests that some urinary biomarkers, namely Vascular Cell Adhesion Molecule-1 (VCAM-1), Intercellular Adhesion Molecule-1 (ICAM-1), Monocyte Chemoattractant Protein 1 (MCP-1), Neutrophil Gelatinase Associated Lipocalcin and Lipocalin-type Prostaglandin D-Synthetase (L-PGDS), might discriminate SLE patients with ongoing renal activity from those with stable disease. The objective of this study was to assess the role of these markers in predicting renal flares in comparison with conventional biomarkers and to derive a biomarker panel which may improve diagnostic accuracy. METHODS: Eligible participants were SLE patients prospectively followed at our clinic. Urinary biomarker levels were measured in urinary sample by ELISA assay and were compared by the unpaired Student's t test or the Mann-Whitney U test as appropriate. Receiver operating characteristic analysis was used to calculate the area under the curve. Cox regression was used to identify independent factors associated with disease flares. RESULTS: Urine was collected from 61 patients. During 8 months' follow-up, eight patients experienced a renal flare. Urinary L-PGDS, ICAM-1 and VCAM-1 levels were significantly increased in the patients who subsequently experienced a renal flare with respect to the remaining 53. At Cox regression analysis, L-PGDS, ICAM-1, VCAM-1, hypocomplementemia and anti-dsDNA antibodies were factors associated with renal flares. Based on receiver operating characteristic analysis, a combination of novel and conventional biomarkers demonstrated an excellent ability for accurately identifying a flare. CONCLUSION: This study might suggest the usefulness of a novel biomarker panel in predicting a renal flare in SLE.

Performance of a new quantitative computed tomography index for interstitial lung disease assessment in systemic sclerosis.

Quantitative high resolution computed tomography (HRCT) may objectively assess systemic sclerosis (SSc)-interstitial lung disease (ILD) extent, using three basic densitometric measures: mean lung attenuation (MLA), skewness, and kurtosis. This prospective study aimed to develop a composite index - computerized integrated index (CII) - that accounted for MLA, skewness, and kurtosis by means of Principal Component Analysis over HRCTs of 83 consecutive SSc subjects, thus eliminating redundancies. Correlations among CII, cardiopulmonary function and immune-inflammatory biomarkers (e.g. sIL-2Rα and CCL18 serum levels) were explored. ILD was detected in 47% of patients at visual HRCT assessment. These patients had worse CII values than patients without ILD. The CII correlated with lung function at both baseline and follow-up, and with sIL-2Rα and CCL18 serum levels. The best discriminating CII value for ILD was 0.1966 (AUC = 0.77; sensitivity = 0.81 [95%CI:0.68-0.92]; specificity = 0.66 [95%CI:0.52-0.80]). Thirty-four percent of patients without visual trace of ILD had a CII lower than 0.1966, and 67% of them had a diffusing lung capacity for CO <80% of predicted. We showed that this new composite CT index for SSc-ILD assessment correlates with both lung function and immune-inflammatory parameters and could be sufficiently sensitive for capturing early lung density changes in visually ILD-free patients.

Low mortality rate in Italian rheumatoid arthritis patients from a tertiary center: putative implication of a low anti-carbamylated protein antibodies prevalence.

BACKGROUND AND OBJECTIVE: Anti-carbamylated protein antibodies (anti-CarP Ab) represent a novel kind of autoantibodies specificity detectable in the sera of patients with rheumatoid arthritis (RA). They have been recently reported to be associated with increased mortality in Spanish patients with RA. The aim of our study was to compare the incidence mortality rates (IMRs) detected in RA patients from a tertiary Italian center with those reported in other European tertiary centers and to evaluate the putative role of anti-CarP Ab in modulating the low IMR detected in our patients. METHODS: Clinical charts of patients consecutively admitted to our center, from January 1, 2008, to December 31, 2014, were retrospectively reviewed. The mortality rate (expressed as the number of deaths in the cohort divided by the number of years of IMR follow-up) and causes of death were assessed at December 31, 2015. Sera of 61 patients, representative of the whole cohort, collected at the time of admission to our center were investigated for the presence and the level of anti-CarP Ab. Demographic and clinical features, mortality rates and prevalence of anti-CarP Ab in our series were compared with those reported in other European cohorts. RESULTS: We observed 608 patients for a median of 3.51 years. All-cause and cause-specific IMRs in our cohort were significantly lower than the Better Anti-rheumatic Farmaco-therapy and the Spanish cohort, while only all-cause and cardiovascular IMRs were significantly lower in our series with respect to the Leiden Early Arthritis Clinic cohort. Anti-CarP Ab prevalence was significantly lower in our series than in any other European cohorts. CONCLUSION: We confirm that the mortality rate is lower in our Italian RA cohort with respect to other European cohorts. Whether the low prevalence of anti-CarP Ab might be responsible for this result awaits to be furtherly investigated.

Resveratrol mimics insulin activity in the adipogenic commitment of human bone marrow mesenchymal stromal cells.

Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent cells capable of differentiating toward osteoblatic and adipocytic phenotypes. BM-MSCs play several key roles including bone remodeling, establishment of hematopoietic niche and immune tolerance induction. Here, we investigated the effect of resveratrol (RSV), a therapeutically promising natural polyphenol, on the commitment of human BM-MSCs primary cultures. Cell differentiation was evaluated by means of morphological analysis, specific staining and expression of osteogenic and adipocytic master genes (Runx-2, PPARγ). To maintain BM-MSC multipotency, all experiments were performed on cells at very early passages. At any concentration RSV, added to standard medium, did not affect the phenotype of confluent BM-MSCs, while, when added to osteogenic or adipogenic medium, 1 µM RSV enhances the differentiation toward osteoblasts or adipocytes, respectively. Conversely, the addition of higher RSV concentration (25 µM) to both differentiation media resulted exclusively in BM-MSCs adipogenesis. Surprisingly, the analysis of RSV molecular effects demonstrated that the compound completely substitutes insulin, a key component of adipogenic medium. We also observed that RSV treatment is associated to enhanced phosphorylation of CREB, a critical effector of insulin adipogenic activity. Finally, our observations contribute to the mechanistic elucidation of the well-known RSV positive effect on insulin sensitivity and type 2 diabetes mellitus.

P27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases.

p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.

Resveratrol: from basic studies to bedside.

Plants produce a remarkable amount of low molecular mass natural products endowed with a large array of pivotal biological activities. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an important modulator of cell phenotype with a complex and pleiotropic mode of action. Extensive literature regarding its activity, mainly employing cellular models, suggests that this polyphenol controls cell proliferation, induces differentiation, and activates apoptosis and autophagy. The compound also modulates angiogenesis and inflammation. Similarly, studies on implanted cancers and chemical-induced tumors confirm the potential chemotherapeutical interest of the compound. Likewise, several reports clearly demonstrated, in animal models, that the compound might positively affect the development and evolution of chronic diseases including type 2 diabetes, obesity, coronary heart disease, metabolic syndrome, and neurogenerative pathologies. Finally, a number of investigations stated that the toxicity of the molecule is scarce. Despite these promising observations, few clinical trials have yet been performed to evaluate the effectiveness of the molecule both in prevention and treatment of human chronic disease. Preliminary findings therefore suggest the need for more extensive clinical investigations.

Resveratrol and cancer treatment: is hormesis a yet unsolved matter?

Plants produce many low molecular mass natural compounds endowed with biological activity. Among them, resveratrol (3,5,4'-trihydroxystilbene) has been demonstrated to be able to affect a plethora of pivotal cellular molecular processes, including transduction pathways and gene expression. These activities result, in turn, in several different cell phenotypes. Particularly, frequent effects of resveratrol treatment appear to be the reduction of growth and the activation of programmed cell death. Accordingly, a number of trials are currently under development to evaluate the possibility of using resveratrol in cancer therapy, both as single agent or in association with other anticancer compounds. However, some reports suggest that, at low concentrations, not only resveratrol does not inhibit the proliferation and/or the survival of cells but, conversely, it induces proliferation and/or protects cells against toxic agents. On the basis of these biphasic effects, it has been proposed that resveratrol belongs to the so-called hormetic compounds. Hormesis is an expression employed by toxicologists to describe a U-shaped (or J-shaped) dose response characterized by a beneficial effect at low doses and a toxic (or inhibitory) activity at high dose. In this review, we will reappraise data that might suggest or disprove that resveratrol is endowed with clear hormetic properties.

Inorganic phosphate as a signaling molecule: a potential strategy in osteosarcoma treatment.

Inorganic phosphate (Pi) is an essential nutrient to living organisms. It plays a key role in diverse biological processes, including osteoblast differentiation and skeletal mineralization. Maintenance of proper Pi homeostasis is a critical event, as any deviation from that state can lead to several acute and chronic disease states and influence the ageing process and lifespan. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, renal tubular reabsorption and depends mainly on the activity of Na/Pi cotransporters. Pi is abundant in the diet and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. Relevantly, Pi is emerging as an important signalling molecule capable of modulating multiple cellular functions by altering signal transduction pathways, gene expression and protein abundance in many cell types. However, little is known about the initial events involving the detection of changes in serum or local Pi concentrations and the subsequent downstream regulation cascade. Previously, we provided evidence that Pi inhibits proliferation and aggressiveness of human osteosarcoma U2OS cells identifying adenylate cyclase, beta3 integrin, Rap1, ERK1/2 as proteins whose expression and function are relevantly affected in response to Pi. More recently, we demonstrated that Pi is capable also of inducing sensitization of osteosarcoma cells to doxorubicin in a p53-dependent manner and through a mechanism involving ERK1/2 down-regulation. This review summarizes the current knowledge regarding inorganic phosphate as a novel specific signaling molecule in bone and other cell types in mammals and discuss how targeting Pi levels at local sites might represent a potential strategy for improving osteosarcoma therapy.

Targeting p27Kip1 protein: its relevance in the therapy of human cancer.

INTRODUCTION: Cell division cycle progression is achieved by a sequential and stringently concerted activation of a family of serine-threonine kinases, namely the cyclin-dependent kinases (CDKs). p27(Kip1) is a pivotal CDK inhibitor and a tight modulator of CDK-dependent phenotypes. Thus, p27(Kip1) plays a fundamental role in key cellular processes such as proliferation, differentiation, apoptosis, substrate adhesion and motility. Intriguingly, when p27(Kip1) is localized in the nucleus, it acts as an antiproliferative protein, while, in the cytosol, p27(Kip1) promotes cytoskeleton remodeling and might positively influence metastatization. Downregulation of p27(Kip1) nuclear level or its cytosolic mislocalization are consistently correlated with poor prognosis of numerous types of human epithelial and non-epithelial cancers. AREAS COVERED: This review illustrates the basic structural features of p27(Kip1) protein, its metabolism and alterations in human malignancies, along with describing anticancer strategies based on targeting p27(Kip1). EXPERT OPINION: Given the role of p27(Kip1) in the control of cell proliferation and its decreased level observed in malignancies with poor outcome, drugs able to handle the protein levels and localization might represent an important goal for novel specific and effective anticancer strategies. Although no convincing proofs have been reported, putative negative consequences of p27(Kip1) targeting might be also conceivable.