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We previously published that in patients with infantile hemangioma (IH) at the onset (T0) colony forming unit-fibroblasts (CFU-Fs) are present in in vitro cultures from PB. Herein, we characterize these CFU-Fs and investigate their potential role in IH pathogenesis, before and after propranolol therapy. The CFU-F phenotype (by flow cytometry), their differentiation capacity and ability to support angiogenesis (by in vitro cultures) and their gene expression (by RT-PCR) were evaluated. We found that CFU-Fs are actual circulating MSCs (cMSCs). In patients at T0, cMSCs had reduced adipogenic potential, supported the formation of tube-like structures in vitro and showed either inflammatory (IL1β and ESM1) or angiogenic (F3) gene expression higher than that of cMSCs from CTRLs. In patients receiving one-year propranolol therapy, the cMSC differentiation in adipocytes improved, while their support in in vitro tube-like formation was lost; no difference was found between patient and CTRL cMSC gene expressions. In conclusion, in patients with IH at T0 the cMSC reduced adipogenic potential, their support in angiogenic activity and the inflammatory/angiogenic gene expression may fuel the tumor growth. One-year propranolol therapy modifies this picture, suggesting cMSCs as one of the drug targets.

Surgical site infections are the second major cause of hospital acquired infections, accounting for a large part of overall annual medical costs. Airborne particulate is known to be a potential carrier of pathogenic bacteria. We assessed a mobile air particle filter unit for improvement of air quality in an operating room (OR). A new mobile air decontamination and recirculation unit, equipped with a crystalline ultraviolet C (Illuvia® 500 UV) reactor and a HEPA filter, was tested in an OR. Airborne particulate was monitored in four consecutive phases: I) device OFF and OR at rest; II) device OFF and OR in operation; III) device ON and OR in operation; IV) device OFF and OR in operation. We used a particle counter to measure airborne particles of different sizes: ≥0.3, ≥0.5, ≥1, ≥3, ≥5, >10 µm. Activation of the device (phases III) produced a significant reduction (p < 0.05) in airborne particulate of all sizes. Switching the device OFF (phase IV) led to a statistically significant increase (p < 0.05) in the number of particles of most sizes: ≥0.3, ≥0.5, ≥1, ≥3 µm. The device significantly reduced airborne particulate in the OR, improving air quality and possibly lowering the probability of surgical site infections.

Background Single nucleotide polymorphisms (SNPs) in vascular endothelial growth factor A (VEGFA) are associated with susceptibility to several diseases including cancer. Correlations between VEGFA rs3025020 genotypes with clinical and laboratory features of primary myelofibrosis (PMF) are unstudied. Methods DNA was analyzed by real-time polymerase chain reaction for VEGFA rs3025020 genotypes in a cohort of 844 subjects with PMF and in two cohorts of normal subjects (N = 247 and N = 107). Results Frequency of rs3025020 minor allele (T) was not significantly different in subjects with PMF compared with normals; however, the T-allele was more frequent in PMF subjects with a calreticulin (CALR)-mutated genotype compared with normals (35 vs. 27%; OR = 1.47 [95% CI, 1.09, 1.98] p = 0.011), especially in subjects with a CALR-type 2/type 2-like mutation (43 vs. 27%; OR = 2.01 [1.25, 3.24] p = 0.004). CALR mutants with the rs3025020 TT genotype had higher CXCR4 expression on CD34-positive blood cells, and those who carried CT/TT genotypes had lower platelet concentrations compared with other genotypes at diagnosis. Overall, subjects with the rs3025020 CT/TT genotype had a lower cumulative incidence of deep vein thrombosis in typical sites (1.6 vs. 4.2%; OR = 0.37 [0.15, 0.90] p = 0.029) and longer interval from diagnosis to first thrombosis (HR = 0.37 [0.14, 0.95] p = 0.039). Conclusion Persons with PMF and the VEGFA rs3025020 minor T-allele are more likely to have a CALR mutation compared with other somatic driver mutations and lower cumulative incidence and hazard for deep vein thrombosis in typical sites.

Sensitivity, reproducibility, precision and accuracy are recurring words in this book. Indeed, digital PCR (dPCR) represents a major step forward in the quantification of nucleic acids. In this field, for many years it has been used the quantitative PCR (qPCR), a technology that allows a real-time monitoring of DNA amplification which has proved useful for obtaining a relative measurement, but that only indirectly, through the use of reference standards, provides an absolute quantification....

Primary Myelofibrosis (PMF) is a myeloproliferative disorder associated with JAK2V617F, Calreticulin (CALR) indels, and MPLW515L/K mutations activating the tyrosine kinase JAK2 and its downstream signaling pathway. The nature of signaling abnormalities in primary cells from PMF patients is poorly understood, since most of the work has been performed in cell lines or animal models. By flow cytometry we measured constitutive and cytokine induced phosphorylation of STAT5, STAT3, and ERK1/2 in circulating CD34+ cells from 57 patients with PMF (20 with prefibrotic-PMF) and 13 healthy controls (CTRLs). Levels of constitutive and TPO induced p-STAT5, and IL6 induced p-STAT3 were higher in patients than in CTRLs. Constitutive p-STAT5 values were lower in CALR than homozygous JAK2V617F mutated CD34+ cells from PMF patients. Moreover, constitutive p-STAT5 and IL6 induced p-STAT3 values correlated directly with circulating CD34+ cell number/L, and inversely with the frequency of circulating CD34+ cells expressing CXCR4. Constitutive p-STAT5 values of CD34+ cells were also inversely correlated with hemoglobin levels. When the patients were divided according with presence/absence of JAK2V617F mutation, all the correlations described characterized the JAK2V617F+ patients with prefibrotic-PMF (P-PMF). In conclusion, increased constitutive p-STAT5 and IL6 induced p-STAT3 values in circulating CD34+ cells characterize patients with PMF. Constitutive p-STAT5 and IL6 induced p-STAT3 values correlate with circulating CD34+ cell number/L, the frequency of circulating CD34+ cells expressing CXCR4 and hemoglobin levels within the prefibrotic JAK2V617F+ patient population. Our data point toward a complex activation of STAT5-dependent pathways in the stem/progenitor cell compartment, that characterize the phenotypic diversity of PMF.

Degree Course and «Community Learning»: Modelling Perspectives The recent use, in economic contexts, of the conceptual opposition between «mark-tomarket » and «mark-to-model» expressions would appear to be relevant also in the organizational analysis of university degree courses. To this end, the «case» of a specific Bachelor’s degree course was analysed within an adult education and lifelong learning perspective. From an epistemological standpoint, the analysis was based on the application of the «community of practice» paradigm by focusing on the following aspects: the historical location of the learning process (situated learning); the simultaneous presence of specific «reified objects» within different «communities» (separated and identified by defined organizational «boundaries»); «brokering» connections, that is, «connections provided by people who can introduce elements of one practice into another» (Wenger, 1998). The aforesaid conditions provide a basis in order to align educational design practices with an action strategy including: multi-membership experiences; the styles and discourses typical of different socio-cultural «constellations»; and the institutional participation of co-operating social actors. In view of all the above, this initial «case study» reveals the typical failings of the Italian university system that is widely geared to uncritical «mark-to-market» strategies.

Objective: Hypereosinophilic syndromes are a heterogeneous group of uncommon disorders characterized by the presence of marked peripheral blood eosinophilia, tissue eosinophilia, or both, resulting in a wide variety of clinical manifestations, often without an identifiable cause. Churg-Strauss syndrome is a systemic vasculitis characterized by prominent peripheral eosinophilia, asthma and systemic involvement. The presence of mild to severe eosinophilia and systemic involvement raise the search of many trigger factor that need to be ruled out. Distinguishing CSS from idiopathic hypereosinophilic syndrome may be particularly challenging, especially in ANCA negative patients. Methods: The aim of the present study was to present a small case series of patients referred to a Rheumatology Unit for mild to severe eosinophilia and signs and symptoms of systemic involvement and to outline the clinical significance of molecular biology in the work-up of hypereosinophilia. Results: Eleven patients with moderate to severe peripheral eosinophylia, were referred to our Unit from 1996 to 2007. Female to male ratio was 7/4, mean age 40.54 (range 22-75). Three out of eleven patients resulted positive for molecular biology. The diagnosis of idiopathic hypereosinophylia was confirmed in one out of three on the basis of the clinical picture and bone marrow biopsy. Conclusions: Molecular biology may be useful in the screening and in the follow-up of a new hypereosinophylic patient.

Objective: This study was aimed at verifying any potential correlation between anti-myeloperoxidase antineutrophil cytoplasmic antibodies (ANCA-MPO) and clinical features and outcome indices in Churg-Strauss Syndrome (CSS). Methods: Thirhty-eight Churg-Strauss syndrome patients were selected from the medical records of all vasculitis patients attending the Rheumatology and Immunology Unit at the Department of Internal Medicine of the University of Pisa in the decades between 1989 and 2008. Data were analysed retrospectively. Statistical analyses of the results were carried out using the Mann-Whitney test to determine the correlations between the clinical and serological parameters. Qualitative variables were compared using contingency table analysis and Fisher’s exact test. Results: ANCA-MPO were detected in15/38 (39%) patients. Positive ANCA status was associated with peripheral neuropathy (p=0.0006), whereas negative ANCA status was associated with lung involvement (p=0.002). Relapses were strongly associated with positive ANCA status (p=0.01) and with an increase in- or a reappearence of ANCA-MPO levels (p=0.006). Finally, ANCA-MPO were significantly associated with neurological damage (p=0.003). Conclusions: The presence or absence of ANCA-MPO identify different clinical subsets in CSS. Overall, ANCA-MPO appears as a useful tool in the monitoring of CSS and in particular a good predictor of CSS relapses

Primary myelofibrosis (PMF) is a Philadelphia-negative (Ph-) myeloproliferative disorder, showing abnormal CD34+ progenitor cell trafficking, splenomegaly, marrow fibrosis leading to extensive extramedullary haematopoiesis, and abnormal neoangiogenesis in either the bone marrow or the spleen. Monocytes expressing the angiopoietin-2 receptor (Tie2) have been shown to support abnormal angiogenic processes in solid tumors through a paracrine action that takes place in proximity to the vessels. In this study we investigated the frequency of Tie2 expressing monocytes in the spleen tissue samples of patients with PMF, and healthy subjects (CTRLs), and evaluated their possible role in favouring spleen angiogenesis. We show by confocal microscopy that in the spleen tissue of patients with PMF, but not of CTRLs, the most of the CD14+ cells are Tie2+ and are close to vessels; by flow cytometry, we found that Tie2 expressing monocytes were Tie2+CD14lowCD16brightCDL62-CCR2- (TEMs) and their frequency was higher (p = 0.008) in spleen tissue-derived mononuclear cells (MNCs) of patients with PMF than in spleen tissue-derived MNCs from CTRLs undergoing splenectomy for abdominal trauma. By in vitro angiogenesis assay we evidenced that conditioned medium of immunomagnetically selected spleen tissue derived CD14+ cells of patients with PMF induced a denser tube like net than that of CTRLs; in addition, CD14+Tie2+ cells sorted from spleen tissue derived single cell suspension of patients with PMF show a higher expression of genes involved in angiogenesis than that found in CTRLs. Our results document the enrichment of Tie2+ monocytes expressing angiogenic genes in the spleen of patients with PMF, suggesting a role for these cells in starting/maintaining the pathological angiogenesis in this organ.

Systemic sclerosis is an inflammatory disease of the connective tissue characterized by vasculopathy and accumulation of collagen and other components of the connective matrix, affecting the skin and internal organs. The appearance of skin ulcers as a result of vascular damage is very common in the history of the disease. Skin ulcers, painful and slow healing due to atrophy and local ischemia, get worse the quality of life of patients. Often, the use of conventional therapies (such as calcium channel blockers and prostanoids) does not cause the complete healing of the lesions. We report the case of a patient in whom therapeutic association between endothelin antagonist (bosentan) and phosphodiesterase-V inhibitor (sildenafil) resulted in complete healing of old ulcers both to upper and lower limbs and allowed the interruption of intravenous therapies.

Primary myelofibrosis (PMF) is an acquired clonal disease of the hematopoietic stem cell compartment, characterized by bone marrow fibrosis, anemia, splenomegaly and extramedullary hematopoiesis. About 60% of patients with PMF harbor a somatic mutation of the JAK2 gene (JAK2-V617F) in their hematopoietic lineage. Recently, a splicing isoform of JAK2, lacking exon 14 (JAK2Δ14) was described in patients affected by myeloproliferative diseases.By using a specific RT-qPCR method, we measured the ratio between the splicing isoform and the JAK2 full-length transcript (JAK2+14) in granulocytes, isolated from peripheral blood, of forty-four patients with PMF and nine healthy donors.We found that JAK2Δ14 was only slightly increased in patients and, at variance with published data, the splicing isoform was also detectable in healthy controls. We also found that, in patients bearing the JAK2-V617F mutation, the percentage of mutated alleles correlated with the observed increase in JAK2Δ14. Homozygosity for the mutation was also associated with a higher level of JAK2+14. Bioinformatic analysis indicates the possibility that the G>T transversion may interfere with the correct splicing of exon 14 by modifying a splicing regulatory sequence.Increased levels of JAK2 full-length transcript and a small but significant increase in JAK2 exon 14 skipping, are associated with the JAK2-V617F allele burden in PMF granulocytes. Our data do not confirm a previous claim that the production of the JAK2Δ14 isoform is related to the pathogenesis of PMF.

Megakaryocytes release platelets into the bloodstream by elongating proplatelets. In this study, we showed that human megakaryocytes constitutively release Transforming Growth Factor β1 and express its receptors. Importantly, Transforming Growth Factor β1 downstream signaling, through SMAD2/3 phosphorylation, was shown to be active in megakaryocytes extending proplatelets, indicating a type of autocrine stimulation on megakaryocyte development. Furthermore, inactivation of Transforming Growth Factor β1 signaling, by the receptor inhibitors SB431542 and Stemolecule ALK5 inhibitor, determined a significant decrease in proplatelet formation. Recent studies indicated a crucial role of Transforming Growth Factor β1 in the pathogenesis of primary myelofibrosis. We demonstrated that primary myelofibrosis-derived megakaryocytes expressed increased levels of bioactive Transforming Growth Factor β1; however, higher levels of released Transforming Growth Factor β1 did not lead to enhanced activation of downstream pathways. Overall, these data propose Transforming Growth Factor β1 as a new element in the autocrine regulation of proplatelet formation in vitro. Despite the increase in Transforming Growth Factor β1 this mechanism seems to be preserved in primary myelofibrosis.

PurposeThe influence of JAK2 V617F mutation on blast transformation (BT) and overall survival (OS) in primary myelofibrosis (PMF) is controversial. In a large cohort of patients we applied competing risks analysis for studying the influence of JAK2V617F mutation on BT in PMF.Patients and methodsIn 462 PMF-fibrotic type patients (bone marrow [BM] fibrosis grade >0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT.ResultsAt the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade = 0), the BT predictive model including JAK2V617F genotype and older age retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92).ConclusionThe accumulation of mutated alleles in the JAK2V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested.

PurposeIn the WHO diagnostic classification, prefibrotic myelofibrosis (pre-MF) is included in the category of primary myelofibrosis (PMF). However, strong evidence for this position is lacking.Patients and methodsWe investigated whether pre-MF may be aligned along a clinical and biological continuum in 683 consecutive patients who received a WHO diagnosis of PMF.ResultsAs compared with PMF-fibrotic type, pre-MF (132 cases) showed female dominance, younger age, higher hemoglobin, higher platelet count, lower white blood cell count, smaller spleen index and higher incidence of splanchnic vein thrombosis. Female to male ratio and hemoglobin steadily decreased, while age increased from pre-MF to PMF- fibrotic type with early and to advanced bone marrow (BM) fibrosis. Likely, circulating CD34+ cells, LDH levels, and frequency of chromosomal abnormalities increased, while CXCR4 expression on CD34+ cells and serum cholesterol decreased along the continuum of BM fibrosis. Median survival of the entire cohort of PMF cases was 21 years. Ninety-eight, eighty-one and fifty-six percent of patients with pre-MF, PMF-fibrotic type with early and with advanced BM fibrosis, respectively, were alive at 10 years from diagnosis.ConclusionPre-MF is a presentation mode of PMF with a very indolent phenotype. The major consequences of this contention is a new clinical vision of PMF, and the need to improve prognosis prediction of the disease.

According to a Gallese linear definition, a “mirror mechanism” is based on the evidence that neural structures active during sensations and emotions are active also when the same sensations and emotions are to be detected in others. In this remark is argued the thesis that this basic psychic disposition is not sufficient to explain the human orientation to the moral evaluation. This lack is deduced from effective relevance , within a moral complex choice, of mental representations of socio-cultural and political alternatives.

RESUMEN: La historiografía italiana de la infancia, como la de otros muchos países europeos, está hoy embarcada en una tarea de desarrollo y profundización que permitirá recuperar los retrasos que la han caracterizado. En efecto, también en Italia la historia de la infancia y su individuación como «objeto» historiografía) constituyen una adquisición reciente, lograda con la ayuda de diversas influencias. La primera, estrechamente vinculada a la historia de las mentalidades, representada por la obra de Ph. Aries, «El niño y la vida familiar en el Antiguo Régimen», traducido al italiano en 1968. La segunda es la historia social, orientada al estudio de los diversos aspectos de la vida de una sociedad, y más atenta a sus acontecimientos. La tercera la representa el estructuralismo, especialmente el francés, y en concreto M. Foucault. La particular influencia que, también en el contexto italiano, han alcanzado los trabajos de Ph. Aries y L. DeMause, muy importantes por haber abierto una línea de investigación, aunque con las obligadas diferencias, ha conducido a privilegiar una perspectiva exclusiva de historia de las mentalidades que se muestra muy ambigua y claramente interpretativa en exceso.

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca 2+ concentration recruits a multitude of endothelial Ca 2+ -dependent pathways, such as Ca 2+ /calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca 2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca 2+ -sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca 2+ oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca 2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca 2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca 2+ release via inositol-1,4,5-trisphosphate receptors (InsP 3 Rs) and store-operated Ca 2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP 3 R1 and InsP 3 R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca 2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca 2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels. [ABSTRACT FROM AUTHOR]