Carotid atherosclerosis represents a relevant healthcare problem, since unstable plaques are responsible for approximately 15% of neurologic events, namely transient ischemic attack and stroke. Although statins treatment has proven effective in reducing LDL-cholesterol and the onset of acute clinical events, a residual risk may persist suggesting the need for the detection of reliable molecular markers useful for the identification of patients at higher risk regardless of optimal medical therapy. In this regard, several lines of evidence show a relationship among specific biologically active plasma lipids, atherosclerosis, and acute clinical events. We performed a Selected Reaction Monitoring-based High Performance Liquid Chromatography-tandem Mass Spectrometry (SRM-based HPLC-MS/MS) analysis on plasma HDL, LDL, and VLDL fractions purified, by isopycnic salt gradient ultracentrifugation, from twenty-eight patients undergoing carotid endarterectomy, having either a “hard” or a “soft” plaque, with the aim of characterizing the specific lipidomic patterns associated with features of carotid plaque instability. One hundred and thirty lipid species encompassing different lipid (sub)classes were monitored. Supervised multivariate analysis showed that lipids belonging to phosphatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG) classes mostly contribute to discrimination within each lipoprotein fraction according to the plaque typology. Differential analysis evidenced a significant dysregulation of LDL PE (38:6), SM (32:1), and SM (32:2) between the two groups of patients (adj. p-value threshold = 0.05 and log₂FC ≥ |0.58|). Using this approach, some LDL-associated markers of plaque vulnerability have been identified, in line with the current knowledge of the key roles of these phospholipids in lipoprotein metabolism and cardiovascular disease. This proof-of-concept study reports promising results, showing that lipoprotein lipidomics may present a valuable approach for identifying new biomarkers of potential clinical relevance.

Primary Sjögren’s syndrome (pSS) is a complex autoimmune disorder that particularly affects the salivary and lachrymal glands, generally causing a typical dryness of the eyes and of the mouth. The disease encompasses diverse clinical representations and is characterized by B-cell polyclonal activation and autoantibodies production, including anti-Ro/SSA. Recently, it has been suggested that autoantibody profiling may enable researchers to identify susceptible asymptomatic individuals in a pre-disease state. In this pilot study, we used mass spectrometry to analyze and compare the salivary proteomics of patients with established pSS and patients with pre-clinical SS, identifying a common protein signature in their salivary fluid. We found that several inflammatory, immunity-related, and typical acinar proteins (such as MUC5B, PIP, CST4, and lipocalin 1) were differently expressed in pSS and in pre-clinical SSA+ carriers, compared to healthy controls. This suggests that saliva may closely reflect exocrine gland inflammation from the early phases of the disease. This study confirms the value of salivary proteomics for the identification of reliable biomarkers for SS that could be identified, even in a preclinical phase of the disease.

Background: Atherosclerosis is a chronic inflammatory disease. The balance between pro- and anti-inflammatory factors, acting on the arterial wall, promotes less or more coronary plaque macro-calcification, respectively. We investigated the association between monocyte phenotypic polarization and CTCA-assessed plaque dense-calcium volume (DCV) in patients with stable coronary artery disease (CAD). Methods: In 55 patients, individual DCV component was assessed by quantitative CTCA and normalized to total plaque volume. Flow cytometry expression of CD14, CD16, CD18, CD11b, HLA-DR, CD163, CCR2, CCR5, CX3CR1 and CXCR4 was quantified. Adhesion molecules and cytokines were measured by ELISA. Results: DCV values were significantly associated, by multiple regression analysis, with the expression (RFI) of CCR5 (p = 0.04), CX3CR1 (p = 0.03), CCR2 (p = 0.02), CD163 (p = 0.005) on all monocytes, and with the phenotypic M2-like polarization ratio, RFI CCR5/CD11b (p = 0.01). A positive correlation with the increased expression of chemokines receptors CCR2, CCR5 and CX3CR1 on subsets Mon1 was also present. Among cytokines, the ratio between IL-10 and IL-6 was found to be strongly associated with DCV (p = 0.009). Conclusions: The association between DCV and M2-like phenotypic polarization of circulating monocytes indicates that plaque macro-calcification in stable CAD may be partly modulated by an anti-inflammatory monocyte functional state, as evidenced by cell membrane receptor patterns.

Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-β (TGF-β)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-β. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-β-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

TGF-β is a cytokine implicated in multiple cellular responses, including cell cycle regulation, fibrogenesis, angiogenesis and immune modulation. In response to pro-inflammatory and chemotactic cytokines and growth factors, cholangiocytes prime biliary damage, characteristic of cholangiopathies and pathologies that affect biliary tree. The effects and signaling related to TGF-β in cholangiocyte remains poorly investigated. In this study, the cellular response of human cholangiocytes to TGF-β was examined. Wound-healing assay, proliferation assay and cell cycle analyses were used to monitor the changes in cholangiocyte behavior following 24 and 48 h of TGF-β stimulation. Moreover, proteomic approach was used to identify proteins modulated by TGF-β treatment. Our study highlighted a reduction in cholangiocyte proliferation and a cell cycle arrest in G0/G1 phase following TGF-β treatment. Moreover, proteomic analysis allowed the identification of four downregulated proteins (CaM kinase II subunit delta, caveolin-1, NipSnap1 and calumin) involved in Ca²⁺ homeostasis. Accordingly, Gene Ontology analysis highlighted that the plasma membrane and endoplasmic reticulum are the cellular compartments most affected by TGF-β. These results suggested that the effects of TGF-β in human cholangiocytes could be related to an imbalance of intracellular calcium homeostasis. In addition, for the first time, we correlated calumin and NipSnap1 to TGF-β signaling.