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Brambilla F.,CNR Institute of Biomedical Technologies | Lavatelli F.,University of Pavia | Di Silvestre D.,CNR Institute of Biomedical Technologies | Valentini V.,University of Pavia | And 7 more authors.
Blood | Year: 2012

Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates. © 2012 by The American Society of Hematology.

Canobbio I.,University of Pavia | Cipolla L.,University of Pavia | Consonni A.,University of Pavia | Momi S.,University of Perugia | And 7 more authors.
Blood | Year: 2013

In the present study, we used a knockout murine model to analyze the contribution of the Ca2+-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2- deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin αIIbβ3 and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A2 production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA2 phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein-coupled receptors in supporting platelet aggregation and thrombus formation. © 2013 by The American Society of Hematology.

Calandra S.,Pediatria II | Gallo M.C.,Pediatria II | Consolaro A.,Pediatria II | Pistorio A.,Pediatria II | And 8 more authors.
Journal of Rheumatology | Year: 2014

Objective. To investigate the risk factors for chronic anterior uveitis in patients with juvenile idiopathic arthritis (JIA). Methods. The clinical charts of patients followed between January 1987 and December 2011 were reviewed to establish whether they had uveitis. Inclusion criteria were a diagnosis of JIA and a disease category of persistent oligoarthritis, extended oligoarthritis, rheumatoid factor-negative polyarthritis, psoriatic arthritis, or undifferentiated arthritis. Risk factors included sex, age at arthritis onset, disease category, and antinuclear antibody (ANA) status. The association of risk factors with occurrence of uveitis was evaluated by survival analysis, with first episode of uveitis as the event of interest, and Cox regression analysis. Results. Of a total of 1189 patients, 278 (23.4%) had uveitis a median of 1.1 years after onset of arthritis. There was no difference in the cumulative probability of developing uveitis between males and females and between patients belonging to different JIA categories, whereas uveitis was strongly associated with age at arthritis onset ≤ 3.5 years and positive ANA. Patients possessing the latter 2 factors in combination had a greater probability of having uveitis than patients who had either of them alone. Conclusion. In our patients, the risk of uveitis was related to younger age at onset of arthritis and presence of ANA, but not to female sex and disease category. This finding suggests that the patients who require the most intensive ophthalmologic screening are those who have early-onset JIA and are ANA-positive, regardless of their sex or disease subtype. Copyright © 2014. All rights reserved.

Milosevic Feenstra J.D.,Austrian Academy of Sciences | Nivarthi H.,Austrian Academy of Sciences | Gisslinger H.,Medical University of Vienna | Leroy E.,Ludwig Institute for Cancer Research | And 22 more authors.
Blood | Year: 2016

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and controlsamples from8 patients.Wefound evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patientswith clonal hematopoiesis analyzed byWES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1germline(R321W)MPLmutationweredetected.All of theidentifiedMPLmutationswere gain-of-functionwhenanalyzedin functionalassays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined.We could demonstrate that JAK2-V625F andJAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative casesofETandPMFdo not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

Canobbio I.,University of Pavia | Guidetti G.F.,University of Pavia | Oliviero B.,Fondazione Istituto di Ricovero e Cura rattere Scientifico Policlinico San Matteo | Manganaro D.,University of Pavia | And 3 more authors.
Biochemical Journal | Year: 2014

Alzheimer's disease is associated with the accumulation of Aβ (amyloid β)-peptides in the brain. Besides their cytotoxic effect on neurons, Aβ-peptides are thought to be responsible for the atherothrombotic complications associated with Alzheimer's disease, which are collectively known as cerebrovascular disease. In the present study, we investigated the effect of Aβ-peptides on human platelet signal transduction and function. We discovered that the 25-35 domain of Aβ-peptides induce an increase in platelet intracellular Ca2+ that stimulates a-granule and dense granule secretion and leads to the release of the secondary agonist ADP. Released ADP acts in an autocrine manner as a stimulant for critical signalling pathways leading to the activation of platelets. This includes the activation of the protein kinases Syk, protein kinase C, Akt and mitogen-activated protein kinases. Ca2+ -dependent release of ADP is also the main component of the activation of the small GTPase Rap1b and the fibrinogen receptor integrin aIIbβ3, which leads to increased platelet aggregation and increased thrombus formation in human whole blood. Our discoveries complement existing understanding of cerebrovascular dementia and suggest that Aβ-peptides can induce vascular complications of Alzheimer's disease by stimulating platelets in an intracellular Ca2+ -dependent manner. Despite a marginal ADP-independent component suggested by low levels of signalling activity in the presence of apyrase or P2Y receptor inhibitors, Ca2+ -dependent release of ADP by Aβ-peptides clearly plays a critical role in platelet activation. Targeting ADP signalling may therefore represent an important strategy to manage the cerebrovascular component of Alzheimer's disease. © 2014 Biochemical Society.

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