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Aim: To evaluate serum growth arrest-specific gene 6 (Gas6) concentration as a biomarker of liver fibrosis progression. Materials & methods: One hundred and thirteen consecutive patients affected by chronic liver disease underwent transient elastography, Gas6 measurement and, if clinically indicated, liver biopsy. Results: Gas6 concentration was directly correlated to liver stiffness (r = 0.67; p < 0.0001) and was significantly higher in patients with advanced fibrosis (Ishak 4–5; p < 0.001). A plasma concentration 42 ng/ml identified severe fibrosis with a sensitivity of 64% and a specificity of 95%; the diagnostic accuracy was comparable to that of transient elastography. Conclusion: Gas6 is a novel biomarker of liver fibrosis, with a potential clinical and pathophysiological relevance.

Background. Growth arrest specific gene 6 (Gas6) protein enhances survival of oligodendrocytes and neurons, and it is involved in autoimmunity. Therefore, we aimed to verify whether cerebrospinal-fluid (CSF) Gas6 concentration may represent a biomarker of disease activity in multiple sclerosis.Methods. Sixty-five patients who underwent a spinal tap during relapse of relapsing/remitting multiple sclerosis (RR-MS)(McDonald-criteria) were studied. Forty patients affected by noninflammatory/nonautoimmune neurological diseases served as controls. Relapse was defined according to Schumacher criteria. Symptoms were grouped according to Kurtzke-Functional System (FS). Clinical characteristics of the relapse, duration, Expanded-Disability-Status Scale (EDSS) change, number of FS involved, completeness of recovery, age, steroid therapy, were categorised. Patients were followed at 6-month intervals to assess relapse rate and EDSS progression. Gas6 was measured (CSF, plasma) by in-house-enzyme-linked immunoassay (ELISA).Results. Higher CSF Gas6 concentrations were observed in relapses lasting ≤60 days (8.7 ± 3.9 ng/mL) versus >60 days (6.5 ± 2.6) or controls (6.5 ± 2.4;P=0.05), with ≤2 FS involved (8.5 ± 3.8) versus >2 FS (5.6 ± 2.5) (P<0.05) and EDSS change ≤2.5 points (8.8 ± 3.7) versus >2.5 (6.5 ± 3.5) (P=0.04). Conversely, CSF Gas6 was not predictive of the completeness of recovery. Plasma and CSF concentrations were not related (R2=0.0003), and neither were predictive of relapse rate or EDSS progression after first relapse.Conclusions. CSF concentration of Gas6 is inversely correlated with the severity of relapse in RR-MS patients but does not predict the subsequent course of the disease.

Axl, a member of the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases, displays an increasingly important role in carcinogenesis. Analysis of 58 cutaneous melanoma lines indicated that Axl was expressed in 38% of them, with significant overrepresentation in NRAS- compared with BRAF-mutated tumors. Axl activation could be induced by autocrine production of its ligand, Gas6, in a significant fraction of Axl-positive tumors. Pearson's correlation analysis on expression data from five data sets of melanoma lines identified several transcripts correlating positively or negatively with Axl. By functionally grouping genes, those inversely correlated were involved in melanocyte development and pigmentation, whereas those positively correlated were involved in motility, invasion, and microenvironment interactions. Accordingly, Axl-positive melanomas did not express microphthalmia transcription factor (MITF) and melanocyte differentiation antigens (MDAs) such as MART-1 and gp100 and possessed a greater in vitro invasive potential compared with Axl-negative ones. Motility, invasivity, and ability to heal a wound or to migrate across an endothelial barrier were inhibited in vitro by Axl knockdown. Pharmacological inhibition of Axl using the selective inhibitor R428 had comparable effects in reducing migration and invasion. These results suggest that targeted inhibition of Axl signaling in the subset of melanomas lacking MITF and MDAs may represent a novel therapeutic strategy.

Summary Background Gas6 protein is involved in pulmonary embolism (PE) and acute inflammation in animal models. Methods We enrolled 82 consecutive patients with acute dyspnea and suspected PE (Geneva score with high (HCP) or low/intermediate clinical probability (LICP)+D-dimer ≥0.5μg/mL) and 29 age-matched healthy volunteers. According to clinical and instrumental evaluations the following diagnoses were obtained: heart failure (HF), pulmonary or systemic infection (I), PE, or no illness (N). Twenty-two patients were excluded due to oral anticoagulation (9), lack of CT angiography or pulmonary scintigraphy (6), plasma creatinine ≥3mg/dL (3), and pulmonary cancer (4). Plasma Gas6 was measured with a validated enzyme-linked immunoassay. Non-parametric tests and accuracy measures were calculated. Results Out of 60 patients included, 8 were N, 12 HF, 11 I and 29 PE. Gas6 median value in the N group (20.4ng/mL, interquartile range 17.6–21.6) matched that of healthy volunteers, 19.1 (17.2–21.4). Median Gas6 values in HF, 26.4 (21.6–33.3) and I groups, 34.1 (30.0–38.7), were significantly higher than those in PE 18.2 (16.3–23.3) or N (Kruskal–Wallis test p ≤0.05) groups. Gas6 test improved PE diagnosis with an area under the curve of 0.80 and 0.91 (in all and LICP patients). A 24ng/mL threshold excluded PE in 33% of LICP patients without loosing any diagnosis. Conclusions The data link Gas6 protein to infection/inflammation, but not to PE, in humans. Gas6 assay was useful in PE diagnosis, improving D-dimer accuracy particularly in LICP patients, and limiting false positives.

Gas6 is able to inhibit pro-inflammatory cytokine secretion by macrophages acting on Mer receptor; the pathway involved is mediated by PI3K, Akt, GSK3β, and NFκB. GAS6 protein has been described to be involved in immune modulation in vitro and in vivo. Some of these effects are probably mediated through the involvement of monocytes/macrophages. To understand the role of GAS6 in modulating the immune response, we evaluated the effect on cytokine secretion by monocytes/macrophages and the molecular pathways involved. GAS6 inhibits TNF-α and IL-6 secretion by LPS-stimulated U937 cells and monocytes/machrophages. We evidenced that among GAS6 receptors, only Mer (but not Axl or Tyro3) is expressed on differentiated U937 cells, and its activation is responsible for the reduction of cytokine expression. In immunoblot analysis, Mer was activated after GAS6 stimulation, giving rise to an increased phosphorylation of Akt. We also observed GSK3β phosphorylation and consequent inhibition of NF-κB nuclear translocation. Therefore, GAS6 modulates macrophage cytokine secretion, triggering an “anti-inflammatory pathway” involving PI3K/Akt/GSK3β and NF-κB.

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) share histopathological features but display different disease courses; we measured the concentration of 50 inflammatory mediators in the cerebrospinal fluid (CSF) of patients with either of these diseases.CSF samples were collected during a diagnostic lumbar puncture and stored at -30 degrees C. We analyzed the CSF of nine subjects with GBS; eight with CIDP; eight with diabetic polyneuropathy (DP) and seven with headache (controls). Fifty inflammatory mediators were simultaneously measured with a multiplex bead-based ELISA on a Suspension Array System. After Bonferroni's correction for repeated measures, non-parametric variance and post hoc test were calculated.Thirty-two inflammatory mediators were expressed. The median concentration of IL-6, IL-9, IL-15, IL-18, CCL4, CXCL1, LIF, MIF, PDGFbb, IFN-gamma2, IL-2ra, IL-12(p40), IL-16, SCGF-b, TRAIL, FGF, G-CSF, GM-CSF, and M-CSF was not different among groups (variance: n.s.). The median concentration of CCL2, CCL7, CCL27, CXCL9, CXCL10, CXCL12, ICAM-1, VCAM1 and VEGF was higher in CIDP and GBS compared with controls (p0.002). The median concentration of IL-8 and IL-1ra was higher in GBS than CIDP or DP or controls, whereas stem cell factor (SCF) and hepatocyte growth factor (HGF) were higher in CIDP than GBS or DP or controls (p0.002).Mediators of the recruitment and activation of lymphocytes and monocytes are expressed in the CSF of CIDP and GBS. IL-8 and IL-1ra are characteristic of GBS, whereas growth factors (SCF, HGF) of CIDP are possibly related to chronicity or to the survival/repair processes of neurons.

Gas6 protein is possibly involved in human diseases, but a validated plasma assay is lacking. So, we developed a sandwich enzyme‐linked immunosorbent assay (ELISA) method using commercially available reagents. An appropriate plasma‐based matrix was prepared to optimize the assay. The ELISA method showed inter‐ and intra‐assay coefficients of variation lower than 15%. Recoveries all fell within 15% of expected values. Plasma Gas6 concentration in 61 healthy donors was 20.3±3.8 ng/mL. Our assay meets FDA requirements for precision and accuracy for the validation of bioanalytical methods and it is suitable for research or diagnostic purposes.

Gas6 enhances survival of Schwann cells and neurons in vitro and participates in autoimmunity in animal models. Since its concentration in human cerebrospinal fluid (CSF) is unknown, we measured it in samples from patients with non-inflammatory/non-autoimmune neurological diseases (NINAD) and autoimmune polyneuropathies.Samples collected after informed consent during diagnostic lumbar puncture in the period 1999-2006 were stored at -30 degrees C. We considered subjects with NINAD (stroke, ALS, headache, psychiatric conditions simulating neurological diseases, otologic dizziness) or with Guillain-Barré syndrome (GBS) or CIDP. CSF and plasma total protein and age were obtained from clinical records. Gas6 was measured with an ELISA developed and validated in our laboratory (inter-, intra-assay CVs10%, recovery 96%). Variance, Tukey's post-hoc test, regression were calculated with a statistical software (Statsoft).Mean Gas6 concentration in patients with NINAD was 6.5+/-2.4 ng/ml, 7.2+/-2.6 ng/ml in GBS and significantly higher (11.5+/-1.7 ng/ml) in CIDP than in the other conditions (post-hoc, p0.005). It was not related to age, CSF total proteins or to CSF/plasma ratio of total proteins (regression, p0.1).Gas6 is detectable in CSF and may be involved in chronic autoimmune demyelination or myelin repair.