Pier Paolo Sainaghi

Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor

Axl is a tyrosine kinase receptor and although it is expressed in malignancy such as leukemia, colon cancer, melanoma, endometrial, prostate and thyroid cancers, its role has not been completely elucidated yet and appears to be complex. The ligand of Axl, Gas6, is a 75 KDa multimodular protein with an N‐terminal gamma‐carboxy‐glutamic acid that is essential for binding. Gas6 has a mitogenic effect on several normal cell lines. The receptor Axl is expressed in primary prostate carcinoma and in prostate cancer cell lines as such as PC‐3 and DU 145. We demonstrated a mitogenic activity determined by Gas6/Axl interaction in these undifferentiated metastatic human prostatic cancer cell lines. This effect is proportional to Axl expression, not due to inhibition of apoptosis, and induces AKT and MAPK phosphorylation. However, only MEK phosphorylation seems to be essential for growth signaling. Our results suggest that Axl overexpression and activation by Gas6 could be involved in progression of prostate neoplastic disease.

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.

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.

Comparison between HOMA-IR and ISI-gly in detecting subjects with the metabolic syndrome

To verify whether, as index of insulin resistance, ISI‐gly (insulin sensitivity index) is more efficient than HOMA‐IR (homeostatic model assessment) or QUICKI (quantitative insulin sensitivity check index) in detecting patients with the metabolic syndrome.

Peroxisome proliferator-activated receptor-gamma expression in monocytes/macrophages from rheumatoid arthritis patients: relation to disease activity and therapy efficacy—a pilot study

OBJECTIVES Peroxisome proliferator-activated receptor-gamma (PPARγ) is expressed by different cell types in the joints and plays a relevant anti-inflammatory role in various diseases. This pilot study aimed to evaluate PPARγ expression in monocytes/macrophages isolated from RA patients as compared with healthy subjects, the relationships between PPARγ expression, MMP-9 activity and disease, and the influence of therapy with anti-rheumatic drugs on these parameters. METHODS Thirty RA patients of both sexes (treated with CSs and MTX, mainly) and 15 healthy volunteers were enrolled in this study. Disease severity was evaluated by the 28-joint disease activity score (DAS-28). Monocytes and monocyte-derived macrophages (MDMs) were isolated by standard procedures. PPARγ protein and mRNA expression were assessed by immunoblotting and real-time PCR, respectively; MMP-9 activity was determined by gelatin zymography. Moreover, we checked the ability of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ, a PPARγ agonist), MTX and methylprednisolone (MP) to affect PPARγ expression and lipopolysaccharide (LPS)-induced MMP-9 activity. RESULTS Monocytes/MDMs from RA patients have significantly enhanced PPARγ expression (both protein and mRNA) and MMP-9 activity as compared with healthy donors. Interestingly, cells from patients with less active disease (DAS-28 <3.2) present higher PPARγ protein expression and lower MMP-9 activity than RA patients with DAS-28 >3.2. At therapeutic concentrations, MTX and MP increase in vitro PPARγ protein expression and inhibit LPS-induced MMP-9 activity. CONCLUSION PPARγ expression in human monocytes/MDMs could represent an indicator of disease activity and therapy efficacy in RA because patients with a DAS-28 score <3.2 show the highest expression.

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.

Superiority of a High Loading Dose of Cholecalciferol to Correct Hypovitaminosis D in Patients with Inflammatory/Autoimmune Rheumatic Diseases

Objective. To compare 3 different cholecalciferol supplementation regimens in patients with rheumatic diseases. Methods. One hundred fifty-four patients who completed a 6-month course of cholecalciferol supplementation, of whom 111 had an autoimmune/inflammatory rheumatic disease (ARD) and 43 osteoarthritis (NARD), were retrospectively identified from a database of 872 consecutive adult patients who attended a tertiary level immuno-rheumatology clinic from 2007 to 2010. Patients with renal failure or primary hyperparathyroidism were excluded. Plasma 25-hydroxy vitamin D [25(OH)D] and parathyroid hormone (PTH) concentrations were evaluated at baseline and after completion of treatment with (i) a single oral dose of cholecalciferol 300,000 IU, followed by oral cholecalciferol 800–1000 IU daily for 6 months [high-dose loading treatment (HLT) group; n = 40]; (ii) a single oral dose of cholecalciferol 100,000 IU, followed by daily oral cholecalciferol as above [low-dose loading treatment (LLT) group; n = 30]; or (iii) daily oral cholecalciferol as above but without the loading dose [standard therapy (ST); n = 84]. Results. The rates of serum 25(OH)D and PTH normalization (defined as values > 75 nmol/l and < 72.9 pg/ml, respectively) were as follows: HLT, 52.5% (95% CI 37.5–68.5) and 69.2% (95% CI 54.7–83.3); LLT, 36.7% (95% CI 19.7–54.3) and 53.8% (95% CI 36.2–71.8); ST, 31.0% (95% CI 21.1–40.9) and 35.0% (95% CI 14.1–55.9). All regimes increased 25(OH)D (p < 0.001) but only HLT reduced PTH (p < 0.01) in comparison to baseline. The ARD group had a similar 25(OH)D increase but a smaller PTH reduction than the NARD (p < 0.05). Conclusion. An HLT cholecalciferol regimen is needed to correct hypovitaminosis D of patients with rheumatic diseases, with superior 25(OH)D normalization and PTH suppression rates at 6 months.

Unsuppressed parathyroid hormone in patients with autoimmune/inflammatory rheumatic diseases: implications for vitamin D supplementation

OBJECTIVES To verify if autoimmune/inflammatory rheumatic disease (ARD) patients were more refractory to PTH suppression by 25(OH) vitamin D (VITD). METHODS Data from 105 consecutive ARD patients (including RA, PMR, spondyloarthritis and other CTDs) attending a tertiary-level immuno-rheumatology clinic and 1542 subjects tested at our central laboratory from 2008 to 2010 (controls) were collected. After exclusion of patients with renal failure, primary hyperparathyroidism and hypercalcaemia (n = 522), plasma VITD, PTH, calcium and phosphate concentrations were compared between these two groups. RESULTS Plasma VITD concentrations were <25 nmol/l in 257 patients (severe deficit, 22.8%), ≥25 nmol/l but <75 nmol/l in 661 (mild deficit, 58.8%) and ≥ 75 nmol/l in 207 (normal, 18.4%). Despite similar median age, plasma VITD, calcium and phosphate values (P = 0.96, 0.30, 0.94, respectively), PTH was higher in ARD {73.0 [interquartile range (IQR) 54.2-93.7] pg/ml} than in controls [61.4 (46.9-80.3), P < 0.0002], also in all above-defined VITD categories (P < 0.05). Suppressed PTH was observed in 96.9% (95% CI 95.8%, 98.0%) of controls with VITD ≥ 75 nmol/l. However, PTH was increased more frequently in ARD vs controls. At multiple linear regression analysis, plasma VITD, age and the presence of an ARD (partial correlation coefficients -0.21, 0.15, 0.12, respectively, P < 0.0001) were independent predictors for increased PTH. CONCLUSIONS Patients with ARD had, on average, an increased PTH concentration for any plasma VITD range, suggesting an impaired vitamin D metabolism. Therefore, vitamin D supplementation to ARD patients may be targeted to reach PTH suppression and not simply to obtain VITD concentrations considered optimal in other categories of patients.