Luigi Castello

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.

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.

Additive diagnostic and prognostic value of Bioelectrical Impedance Vector Analysis (BIVA) to brain natriuretic peptide ‘grey-zone’ in patients with acute heart failure in the emergency department

Background: Few data are available on diagnostic and prognostic role of quantitative fluid retention evaluated by bioelectrical impedance vector analysis (BIVA) in acute heart failure (AHF) patients at the moment of emergency department presentation. Methods and results: Point vectors and hydration index (HI) by BIVA were obtained in 381 patients referring to an emergency department. For evaluating cardiovascular events, a 30-day follow-up was performed. Patients were divided into AHF (n=270; 70.8%) and no-AHF groups, (n=111; 29.2%). Compared with the no-AHF cohort, the HI value resulted significantly higher in the AHF group (81.2%±6.7 vs. 72.9±3.6%, p<0.001). HI showed a significant diagnostic power for AHF (cut-off 73.4%, area under curve (AUC) 0.87, sensitivity 90%, specificity 54%) and also showed a significant prognostic value both by univariate (odds ratio 1.03 (1–1.07), p =0.025) and multivariate analysis (odds ratio 1.96 (1.05–3.66) p= 0.034) for cardiac events at 30 days. Although in the overall population BIVA did not increase diagnostic accuracy provided by brain natriuretic peptide (BNP), for AHF patients in BNP ‘grey values’ (100–400 pg/ml) HI showed a significant additive improvement for diagnosis (net reclassification improvement (NRI) 77%) and prognosis (NRI 45%). Conclusions: While in the overall population BIVA did not increase diagnostic accuracy provided by BNP, in AHF patients a quantitative evaluation of fluid congestion obtained by BIVA at the time of emergency department arrival provides significant additive diagnostic and 30-day prognostic value to BNP, particularly in the BNP ‘grey-zone’. This could lead to a better management of these patients with possible improvement in reducing subsequent cardiovascular events.

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.

Suicide attempts and emergency room psychiatric consultation

BackgroundSuicidal behaviours are major public health concerns worldwide. They are associated with risk factors that vary with age and gender, occur in combination, and may change over time. The aim of our study was to investigate how frequently patients visiting a hospital emergency room (ER) require a psychiatric consultation for attempted suicide, and to outline the characteristics of this population.MethodsDeterminants of emergency room visits for psychiatric reasons were studied prospectively from 2008 to 2011 at the “Maggiore” Hospital in Novara.Results280 out of 1888 patients requiring psychiatric consultation were referred to the ER because of suicide attempt. Suicide attempters were more often female. The rate of suicide attempters among Italian people was 14.2%, compared to 19.5% in foreigners. Subjects living with parents or own family and those having a permanent job had a higher frequency of suicide attempt. Suicide attempts were more frequent among patients with a history of psychiatric disorders; nonetheless, suicide attempts were more common among those who had not previously been hospitalized in a psychiatric ward or were not under the care of a psychiatrist. The multivariate analysis found that female gender was a risk factor for suicide attempt, while being in the colder months of the year and, surprisingly, unemployment were protective factors.ConclusionsA better understanding of patients referring to the ER due to attempted suicide may allow the identification of at-risk subjects and the implementation of targeted treatment approaches.

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.

Osteopontin at the Crossroads of Inflammation and Tumor Progression

Complex interactions between tumor and host cells regulate systemic tumor dissemination, a process that begins early at the primary tumor site and goes on until tumor cells detach themselves from the tumor mass and start migrating into the blood or lymphatic vessels. Metastatic cells colonize the target organs and are capable of surviving and growing at distant sites. In this context, osteopontin (OPN) appears to be a key determinant of the crosstalk between cancer cells and the host microenvironment, which in turn modulates immune evasion. OPN is overexpressed in several human carcinomas and has been implicated in inflammation, tumor progression, and metastasis. Thus, it represents one of the most attracting targets for cancer therapy. Within the tumor mass, OPN is secreted in various forms either by the tumor itself or by stroma cells, and it can exert either pro- or antitumorigenic effects according to the cell type and tumor microenvironment. Thus, targeting OPN for therapeutic purposes needs to take into account the heterogeneous functions of the multiple OPN forms with regard to cancer formation and progression. In this review, we will describe the role of systemic, tumor-derived, and stroma-derived OPN, highlighting its pivotal role at the crossroads of inflammation and tumor progression.

Methods for the quantitative assessment of electrolyte disturbances in hyperglycaemia.

AIM While empirical calculations are presently used, exact solutions to compute volume and solute changes of hyperosmolar coma (HC) can be obtained by subdividing the patients according to well defined clinical and laboratory conditions. These are represented by PNa(G), the plasma Na concentration that would be present if there were only glucose addition (GA), that discloses prevalent Na depletion when >PNa(1), prevalent water deficit when <PNa(1) (value measured during HC). Exact solutions are available when Na is lost as NaCl, and when patients are subdivided according to Posm(1) (plasma osmolality during HC) >, = or <Posm(0) (the normal value). When Posm(1)=Posm(0), GA must equal the loss of ions induced by the osmotic diuresis (2 x DeltaNa), and the math solution is exact. We herein report data validating these new computational methods. DATA SYNTHESIS We built a mathematical model describing fluid derangements used to execute computer-simulated experiments of HC. The derangements were generated on the computer by adding, to the extra-cellular volume, different amounts of glucose while subtracting variable combinations of ions and solvent. The model yielded true solute concentrations from which our formulas computed the amounts lost or gained. These were identical to the true changes introduced to simulate the derangements (R(2)=1.00, P<0.0001) when the boundary conditions for PNa(G), exclusive NaCl loss and Posm(1)-Posm(0) were met. In patients with HC in whom these same boundary conditions were satisfied, the computations of glucose and Na changes with our new formulas were not significantly different from those estimated after correction of the derangements, considered true values (R(2)=0.60, P<0.05), and showed a satisfactory agreement with the clinical evaluation. CONCLUSIONS Our new methods are more accurate than the traditional ones, as they reach a better quantitative assessment of the entity of the derangements, avoiding iatrogenic dysnatraemias after correction of HC.