Oestradiol enhances in vitro the histamine release induced by embryonic histamine-releasing factor (EHRF) from uterine mast cells

The relationship between maternal hormones and factors secreted by the implanting embryo is still controversial. We have analysed the in-vitro effect of oestradiol and human embryo-derived histamine-releasing factor (EHRF) on histamine release from rat uterine mast cells. Rat uterine mast cells which were preincubated with oestradiol and then challenged with human EHRF gave histamine release values two- to threefold higher than those without preincubation. The enhancement observed was time- and temperature-dependent. A similar enhancement was obtained with human sensitized basophils but not with rat peritoneal mast cells. Oestradiol, used as a direct challenge, did not induce any histamine release from either rat uterine or peritoneal mast cells, or from human sensitized basophils. Oestradiol preincubation also enhanced the histamine release induced by anti-IgE but did not enhance the histamine release induced by substance P or compound 48/80, two secretagogues that are not mediated by IgE. Moreover, uterine fragments derived from rats at various oestrus phases, with different amounts of endogenous oestrogen, were challenged in vitro with EHRF. The release of histamine by mast cells was higher at the proestrus and preimplantation phases than at dioestrus. All these findings suggest that the interaction of oestradiol with rat uterine mast cells was capable of enhancing in vitro the histamine releasing effect of EHR

Genetic screening of Fabry patients with EcoTILLING and HRM technology

<p>Abstract</p> <p>Background</p> <p>Anderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the <it>GLA </it>gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.</p> <p>Findings</p> <p>We analyzed 740 samples using EcoTILLING, comparing two mismatch-specific<ul/>endonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.</p> <p>Conclusion</p> <p>On the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe that<ul/>HRM analysis of the <it>GLA </it>gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.</p

Novel α-galactosidase A mutation in patients with severe cardiac manifestations of Fabry disease

Fabry disease (FD) is a hereditary metabolic disorder caused by the partial or total inactivation of alpha-galactosidase A (alpha-gal A), a lysosomal hydrolase. This inactivation is responsible for the accumulation of undegraded glycosphingolipids in the lysosomes with subsequent cellular and microvascular dysfunction. Fabry is considered a rare disease, with an incidence of 1:40,000; however, there are good reasons to believe that it is often seen but rarely diagnosed. To date, more than 600 mutations have been identified in human GLA gene that are responsible for FD. We describe the case of a 54-year-old male patient, who presented with left ventricular hypertrophy, chronic renal failure and acroparaesthesias, which are considered to be specific features of FD. Clinical and instrumental investigations showed several cardiovascular manifestations. The molecular analysis of GLA gene revealed a novel mutation in the fifth exon, called N249K, and the enzymatic analysis showed no alpha-galactosidase A activity. Family screening detected the same mutation in some relatives and also the enzymatic analysis confirmed the diagnosis of FD. In conclusion, these data suggest that the N249K mutation may be associated with cardiac manifestations of FD combined with other classical features of the disease. (C) 2013 Elsevier B.V. All rights reserved

Alteration of proteomic profiles in PBMC isolated from patients with Fabry disease: preliminary findings

Fabry disease (FD) is an X-linked progressive multisystem disease due to mutations in the gene encoding the lysosomal enzyme α-galactosidase A (α-GalA). The deficiency in α-GalA activity leads to an intra-lysosomal accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3), in various organs and systems. Enzyme replacement therapy is available and alternative therapeutic approaches are being explored. No diagnostic test, other than sequencing of the α-galactosidase A gene, is available, no biomarker has been proven useful to screen for and predict the disease, and underlying mechanisms are still elusive. The aim of this study is to identify FD specific biomarkers and to better understand the pathophysiological changes that occur over time in FD. We compared peripheral blood mononuclear cells (PBMC) from FD patients (n = 8) with control PBMC from healthy individuals (n = 6), by two-dimensional electrophoresis (2DE) and the detected differentially expressed proteins were then subjected to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). In FD patients we identified, among the down-regulated proteins, Calnexin, Rho GDP-dissociation inhibitor 2, Rho GDP-dissociation inhibitor 1, Chloride intracellular channel protein 1; on the other hand γ-enolase, 14-3-3 protein theta, 14-3-3 protein zeta/delta, and galectin-1 were identified as up-regulated proteins. Calnexin and Rho GDP-dissociation inhibitor-1,2 are related to protein folding, signal transduction and cell proliferation. This is the first time that γ-enolase and galectin-1 are described to be up-regulated in Fabry patients. Levels of γ-enolase increase dramatically in cardiovascular accidents and cerebral trauma, whereas galectins are regulators of acute and chronic inflammation. These findings may improve our understanding of the molecular mechanisms underlying the pathology and provide new insight and knowledge for future studies in this field

BUD affects <i>in vitro</i> the expression of ICOS on peripheral blood T-lymphocytes.

<p>PBMC from controls (n = 15) and from mild intermittent asthmatics (n = 19) were cultured with/without BUD and were assessed for ICOS expression by flow-cytometry in total lymphocytes (<b>A</b>), in CD4+CD25− cells (<b>B</b>) and in CD4+CD25+ (<b>C</b>) cells from controls (n = 15) and from asthmatics (n = 19). Individual results, median and 25–75 percentiles are shown.*p<0.05 baseline vs BUD.**p<0.05 asthmatics vs controls. Arrows indicate the representative dot plots.</p

Demographic and clinical characteristics of the study population.

*<p>Mann Whitney versus controls.</p

In vivo effects of BUD in ICOS and Foxp3 expression in peripheral blood T-lymphocytes.

<p>PBMC were isolated from mild persistent asthmatics (n = 8) before and after inhaled BUD treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048816#s2" target="_blank">Materials and Methods</a> for details). ICOS (<b>A–B</b>) and Foxp3 (<b>C–D</b>) were assessed by flow-cytometry in CD4+CD25− and in CD4+CD25+ cells. Individual results, median and 25–75 percentiles are shown. *p<0.05. Arrows indicate the representative dot plots.</p

BUD affects <i>in vitro</i> the expression of IL-10 in CD4+CD25+.

<p>PBMC were isolated from controls (n = 15) and from mild intermittent asthmatics (n = 19), were cultured with/without BUD and were assessed for IL-10 expression by flow-cytometry in CD4+CD25− (<b>A</b>) and in CD4+CD25+ cells (<b>B</b>). Individual results, median and 25−75 percentiles are shown.*p<0.05 baseline vs BUD. <b>C.</b> The expression of IL-10 was also assessed in CD4+CD25+CD127 dim T cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048816#s2" target="_blank">materials and methods</a> for details). Representative dot plot of cells from a patient with asthma at baseline and following <i>in vitro</i> BUD exposure is shown.</p

In vivo effects of BUD in IL-10 expression in peripheral blood T-lymphocytes.

<p>PBMC were isolated from mild persistent asthmatics (n = 8) before and after inhaled BUD treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048816#s2" target="_blank">Materials and Methods</a> for details). IL-10 expression were assessed by flow-cytometry in CD4+CD25− (<b>A</b>) and in CD4+CD25+ (<b>B</b>) cells. Individual results, median and 25–75 percentiles are shown. *p<0.05. Arrows indicate the representative dot plots.</p