Federica Alciato

TNF-α, IL-6, and IL-1 expression is inhibited by GAS6 in monocytes/macrophages

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.

Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase

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. Pearsons 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.

The expression pattern of inflammatory mediators in cerebrospinal fluid differentiates Guillain–Barré syndrome from chronic inflammatory demyelinating polyneuropathy

INTRODUCTION 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. PATIENTS AND METHODS 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 Bonferronis correction for repeated measures, non-parametric variance and post hoc test were calculated. RESULTS 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 (p<0.002). 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 (p<0.002). DISCUSSION 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.

Development and Validation of an ELISA Method for Detection of Growth Arrest Specific 6 (GAS6) Protein in Human Plasma

Abstract 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.

Elevation of Gas6 protein concentration in cerebrospinal fluid of patients with chronic inflammatory demyelinating polyneuropathy (CIDP).

INTRODUCTION 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. MATERIALS AND METHODS 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 CVs <10%, recovery 96%). Variance, Tukeys post-hoc test, regression were calculated with a statistical software (Statsoft). RESULTS 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, p<0.005). It was not related to age, CSF total proteins or to CSF/plasma ratio of total proteins (regression, p>0.1). CONCLUSIONS Gas6 is detectable in CSF and may be involved in chronic autoimmune demyelination or myelin repair.

Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome

High-grade epithelial ovarian cancer (HGEOC) is a clinically diverse and molecularly heterogeneous disease comprising subtypes with distinct biological features and outcomes. The receptor tyrosine kinases, expressed by EOC cells, and their ligands, present in the microenvironment, activate signaling pathways, which promote EOC cells dissemination. Herein, we established a molecular link between the presence of Gas6 ligand in the ascites of HGEOCs, the expression and activation of its receptor Axl in ovarian cancer cell lines and biopsies, and the progression of these tumors. We demonstrated that Gas6/Axl signalling converges on the integrin β3 pathway in the presence of the adaptor protein p130Cas, thus inducing tumor cell adhesion to the extracellular matrix and invasion. Accordingly, Axl and p130Cas were significantly co-expressed in HGEOC samples. Clinically, we identified an Axl-associated signature of 62 genes able to portray the HGEOCs with the shortest overall survival. These data biologically characterize a group of HGEOCs and could help guide a more effective therapeutic approach to be taken for these patients.

Gas6 evaluation in patients with acute dyspnea due to suspected pulmonary embolism

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 >or=0.5microg/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 >or=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<or=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.

Growth Arrest Specific 6 Concentration is Increased in the Cerebrospinal Fluid of Patients with Alzheimer's Disease.

Growth arrest specific 6 (Gas6) has neurotrophic and neuroinflammatory functions, and may play a role in Alzheimers disease (AD). In keeping with this hypothesis, we observed that cerebrospinal fluid (CSF) Gas6 is increased in AD patients compared to controls (63 versus 67 subjects; median value 13.3 versus 9.1 ng/ml; p < 0.0001). Thereafter, we assessed whether CSF Gas6 concentration was correlated to the following parameters: disease duration, MMSE score two years after clinical diagnosis, AD CSF biomarkers, and years of formal schooling. We detected an inverse correlation between CSF Gas6 levels at diagnosis and both disease duration (p < 0.0001) and decrease in the MMSE score two years later (p < 0.0001). Conversely, we found no correlation between CSF Gas6 and both AD biomarkers and years of formal schooling. In conclusion, our results suggest that upregulation of CSF Gas6 may be part of a defensive response aimed at counteracting AD progression.