Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.

BACKGROUND: The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects. METHODS: 0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis. RESULTS: Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone>1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone<1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established. CONCLUSION: Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations

Treatment With Recombinant Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Alleviates the Severity of Streptozotocin-Induced Diabetes

OBJECTIVE: To evaluate the potential therapeutic effect of recombinant human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment in a model of type 1 diabetes. RESEARCH DESIGN AND METHODS: Recombinant TRAIL was added in vitro to primary human and mouse peripheral blood mononuclear cells (PBMCs) and isolated human islets to evaluate the expression of the immunoregulatory gene SOCS1. Diabetes was induced by five consecutive daily injections of low-concentration (50 mg/kg) streptozotocin (STZ) in C57 black mice (n = 24). A group of these mice (n = 12) was co-injected with recombinant TRAIL (20 microg/day) for 5 days, and the diabetic status (glycemia and body weight) was followed over time. After 6 weeks, circulating levels of insulin, TNF-alpha, and osteoprotegerin (OPG) were measured, and animals were killed to perform the histological analysis of the pancreas. RESULTS: The in vitro exposure of both PBMCs and human islets to recombinant TRAIL significantly upregulated the expression of SOCS1. With respect to STZ-treated animals, mice co-injected with STZ+TRAIL were characterized by 1) lower levels of hyperglycemia, 2) higher levels of body weight and insulinemia, 3) a partial preservation of pancreatic islets with normal morphology, and 4) a lower expression of both systemic (TNF-alpha and OPG) and pancreatic (vascular cell adhesion molecule [VCAM]-1) inflammatory markers. CONCLUSIONS: Overall, these data demonstrate that the administration of recombinant TRAIL ameliorates the severity of STZ-induced type 1 diabetes, and this effect was accompanied by the upregulation of SOCS1 expressio

Endothelial Cells Obtained from Patients Affected by Chronic Venous Disease Exhibit a Pro-Inflammatory Phenotype

The inflammatory properties of vein endothelium in relation to chronic venous disease (CVD) have been poorly investigated. Therefore, new insights on the characteristics of large vein endothelium would increase our knowledge of large vessel physiopathology. METHODOLOGY/PRINCIPAL FINDINGS: Surgical specimens of veins were obtained from the tertiary venous network (R3) and/or saphenous vein (SF) of patients affected by CVD and from control individuals. Highly purified venous endothelial cell (VEC) cultures obtained from CVD patients were characterized for morphological, phenotypic and functional properties compared to control VEC. An increase of CD31/PECAM-1, CD146 and ICAM-1 surface levels was documented at flow cytometry in pathological VEC with respect to normal controls. Of note, the strongest expression of these pro-inflammatory markers was observed in VEC obtained from patients with more advanced disease. Similarly, spontaneous cell proliferation and resistance to starvation was higher in pathological than in normal VEC, while the migratory response of VEC showed an opposite trend, being significantly lower in VEC obtained from pathological specimens. In addition, in keeping with a higher baseline transcriptional activity of NF-kB, the release of the pro-inflammatory cytokines osteoprotegerin (OPG) and vascular endothelial growth factor (VEGF) was higher in pathological VEC cultures with respect to control VEC. Interestingly, there was a systemic correlation to these in vitro data, as demonstrated by higher serum OPG and VEGF levels in CVD patients with respect to normal healthy controls. CONCLUSION/SIGNIFICANCE: Taken together, these data indicate that large vein endothelial cells obtained from CVD patients exhibit a pro-inflammatory phenotype, which might significantly contribute to systemic inflammation in CVD patients

Special issue of Quantitative Finance on \u2018Interlinkages and Systemic Risk\u2019

This special issue of Quantitative Finance collects eight papers on the relation between interlinkages and systemic risk. The papers cover several types of interlinkages and follow different approaches, from agent-based modelling to empirical investigation of large and sometimes confidential data. The special issue collects some of the contributions presented at the international workshop\u2018Interlinkages and systemic risk \u2019 , which took place in Ancona (Italy) on 4 \u2013 5 July 2013. The workshop, organized within the research project\u2018. New tools in the credit network modeling with agents \u2019 heterogeneity \u2019 funded by the Institute for New Economic Thinking, was attended by a balanced mix of scholars from academia and economists from central banks and regulatory authorities

Reduced expression of cell cycle-associated genes in B lymphocytes purified from the peripheral blood of early-stage B chronic lymphocytic leukaemia patients.

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The oncogene DEK promotes leukemic cell survival and is downregulated by both Nutlin-3 and chlorambucil in B-chronic lymphocytic leukemic cells.

PURPOSE: To characterize the role of the oncogene DEK in modulating the response to either Nutlin-3, a small-molecule inhibitor of the MDM2/p53 interaction, orchlorambucil in primary B-chronic lymphocytic leukemia (B-CLL) cells. EXPERIMENTAL DESIGN: DEK mRNA and protein levels were evaluated in primary B-CLL samples (n = 21), p53(wild-type) SKW6.4, p53(mutated) BJAB lymphoblastoid cell lines, and normal CD19(+) B lymphocytes-treated Nutlin-3 or chlorambucil (10 micromol/L, each). Knocking down experiments with either p53 or DEK small interfering RNA (siRNA) were done to investigate the potential role of p53 in controlling the expression of DEK and the role of DEK in leukemic cell survival/apoptosis. RESULTS: Both Nutlin-3 and chlorambucil downregulated DEK in primary B-CLL samples (n = 21) and SKW6.4 but not in BJAB cells. Knocking down p53 attenuated the effect of Nutlin-3 on DEK expression, whereas knocking down DEK significantly increased both spontaneous and Nutlin-3-induced apoptosis. Conversely, counteracting DEK downmodulation by using p53 small interfering RNA reduced Nutlin-3-mediated apoptosis. On the other hand, Nutlin-3 potently induced p53 accumulation, but it did not affect DEK levels in normal CD19(+) B lymphocytes. CONCLUSIONS: These data show that the downregulation of DEK in response to either Nutlin-3 or chlorambucil represents an important molecular determinant in the cytotoxic response of leukemic cells, and suggest that strategies aimed to downregulate DEK might improve the therapeutic potential of these drugs

Aortocaval fistulae. Clinical and diagnostic aspects and results of 9 cases treated

Background. The rupture of an abdominal aortic aneurysm (AAA) in the inferior vena cava is a rare event, with an incidence of between 0.3 and 10%. However, it is extremely severe and often cannot be diagnosed preoperatively. The aim of this paper is to present our experience regarding the characteristics of its presentation, the methods of treatment and the analysis of perioperative results, comparing them with the main series reported in the literature. Methods. The study included all the cases of aortocaval fistula (ACF) treated by our unit over the past 14 years, evaluating the characteristics of their presentation, the methods of preoperative diagnosis, the diameter of the aneurysm, the type of surgery and the results obtained based on perioperative mortality and morbidity (30 days). This group was also compared with a group of patients treated for ruptured aneurysm and contained ruptured aneurysm. Results. A total of 9 patients with AAA associated with the presence of ACF were operated during this period. Eight patients were symptomatic at the time of observation: right cardiac decompensation was apparent in 3 cases (33 %), abdominaI/lumbar pain and shock were present in 5 cases (55%), symptoms of hepatorenal insufficiency in 2 cases (22 %) and 2 cases of isolated renal insufficiency. There were also 2 cases of lower limb ischemia and 2 of venous stasis. Among the signs of rupture, abdominal bruit was noted in 2 cases (22%). A state of anemia with Hb &lt;12 mg/dl was found in 7 cases (77%). Only 1 patient (11%) was completely asymptomatic. The interval between the onset of symptoms and surgery ranged from a few hours (within 12 hours) to 6 days. Diagnosis was intraoperative in 4 cases (44 %). Preoperative angiography was performed in 3 cases for diagnostic purposes. The mean diameter of the aneurysm was 7.3 cm. In 4 cases, ACF was associated with retroperitoneal rupture. Surgery took the form of aneurysmectomy and prosthetic graft and endoaneurysmatic suture of the caval opening; ligation of the vena cava was only required in 1 case. Perioperative mortality was 1 case (11%): 1 of the 4 patients with ACF associated with ruptured AAA, therefore the mortality in this group was 25%. No deaths occurred in the group with isolated ACF. Two cases of deep vein thrombosis (DVT) and 2 of lower limb ischemia occurred during the postoperative period: of the latter, 1 case was resolved by thrombectomy, while the other required demolitive treatment (amputation at the thigh). Functional parameters returned to normal after surgery in patients with renal and hepatorenal insufficiency. The mortality rate in ruptured aneurysms was 16.6% (8/48) and 8.3% in contained ruptured aneurysms (2/24). Conclusions. The clinical symptoms of ACF are very similar to the fissuration crisis of AAA. In our experience, perioperative mortality was relatively low and was limited to cases with ruptured aneurysm

MOLECULAR-BASES OF CRM(+) FACTOR-X DEFICIENCY - A FREQUENT MUTATION (SER334PRO) IN THE CATALYTIC DOMAIN AND A SUBSTITUTION (GLU102LYS) IN THE 2ND EGF-LIKE DOMAIN

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