Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibbeta prevents the normal maturation and surface exposure of GPIb-IX complexes

This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibalpha and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbalpha, GPIbbeta, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbbeta, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbalpha and GPIX. Transient transfection of the mutant GPIbalpha subunit failed to render surface expression of GPIbalpha and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbbeta may associate with GPIX and GPIbalpha, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5- Cys7 disulfide loop in GPIbbeta is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbbeta mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.     

Does the development of new medicinal products in the European Union address global and regional health concerns?

Background  

Since 1995, approval for many new medicinal products has been obtained through a centralized procedure in the European Union. In recent years, the use of summary measures of population health has become widespread. We investigated whether efforts to develop innovative medicines are focusing on the most relevant conditions from a global public health perspective.     

Burden of disease due to cancer in Spain

Background  

Burden of disease is a joint measure of mortality and morbidity which makes it easier to compare health problems in which these two components enjoy different degrees of relative importance. The objective of this study is ascertaining the burden of disease due to cancer in Spain via the calculation of disability-adjusted life years (DALYs).     

In Vitro Growth in Acute Myeloblastic Leukemia: Clinico-Biological Correlations

In the present review we analyse the current knowledge about the growth properties of AML progenitor cells and their relationship with other clinico-bilogical characteristics of the disease. Leukaemic colony forming unit L-CFU is considered to be the clonogenic cell in AML and more immature than the blast cell population. Our studies have shown that in leukaemic hematopoiesis colony forming cells can exist among both cell fractions CD34+ and CD34-. Optimal “in vitro” proliferation of L-CFU is dependent upon the addition of exogenous growth factors. However, it has been observed that leukaemic progenitor cells frequently display a certain degree of autonomous proliferation. In order to quantify the “in vitro” behaviour of L-CFU, we have explored 3 parameters: 1) plating efficiency (PE); 2) autonomous growth (AG); and 3) autonomous proliferative index (API) which was calculated as AG divided by PE and we have correlated them with other clinico-biological data. According to the FAB classification we could observe that patients with M3 subtype showed an higher PE than other AML subgroups and a significantly lower API. Regarding CD34 expression we observed that AG was enhanced in CD34+ cases and also in those showing a higher rh123 elimination. In order to determine whether PE could condition clinical evolution, we analysed this parameter in a large series of patients but failed to demonstrate any relationship. By contrast, we observed that patients who displayed a higher API showed a shorter survival than patients with lower API (18% vs 48% surviving at 3 years).We have also shown that abnormalities in the CFU-GM growth pattern could be associated with risk the of relapse in AML patients; a switch from normal to abnormal “in vitro” growth should alert us. But for the assessment of the real value of these analyses sequential follow-up studies are mandatory. In summary, cell culture studies contribute not only to a better understanding of leukaemic hematopoiesis but may also contribute to better disease monitoring.     

Intracellular triggering of Fas aggregation and recruitment of apoptotic molecules into Fas-enriched rafts in selective tumor cell apoptosis

We have discovered a new and specific cell-killing mechanism mediated by the selective uptake of the antitumor drug 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3), Edelfosine) into lipid rafts of tumor cells, followed by its coaggregation with Fas death receptor (also known as APO-1 or CD95) and recruitment of apoptotic molecules into Fas-enriched rafts. Drug sensitivity was dependent on drug uptake and Fas expression, regardless of the presence of other major death receptors, such as tumor necrosis factor (TNF) receptor 1 or TNF-related apoptosis-inducing ligand R2/DR5 in the target cell. Drug microinjection experiments in Fas-deficient and Fas-transfected cells unable to incorporate exogenous ET-18-OCH(3) demonstrated that Fas was intracellularly activated. Partial deletion of the Fas intracellular domain prevented apoptosis. Unlike normal lymphocytes, leukemic T cells incorporated ET-18-OCH(3) into rafts coaggregating with Fas and underwent apoptosis. Fas-associated death domain protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid were recruited into rafts, linking Fas and mitochondrial signaling routes. Clustering of rafts was necessary but not sufficient for ET-18-OCH(3)-mediated cell death, with Fas being required as the apoptosis trigger. ET-18-OCH(3)-mediated apoptosis did not require sphingomyelinase activation. Normal cells, including human and rat hepatocytes, did not incorporate ET-18-OCH(3) and were spared. This mechanism represents the first selective activation of Fas in tumor cells. Our data set a framework for the development of more targeted therapies leading to intracellular Fas activation and recruitment of downstream signaling molecules into Fas-enriched rafts.     

Relation Between Genetic Factors and Frailty in Older Adults

ObjectivesFrailty is a geriatric syndrome that identifies individuals at higher risk of disability, institutionalization, and death. We previously reported that frailty is related to oxidative stress and cognitive impairment-related biomarkers. The aim of this study was to determine whether frailty is associated with genetic variants.DesignLongitudinal population-based cohort of 2488 community-dwelling people from Toledo, Spain, aged 65 years or older.Setting and participantsWe obtained blood samples from 78 individuals with frailty and 74 nonfrail individuals who were nonfrail (according to Fried criteria) from the Toledo Study of Healthy Ageing and extracted DNA using the Chemagic DNA blood kit.MeasuresSample genotyping was carried out by means of Axiom Exome 319 Genotyping Array (Thermo Fisher Scientific), which contains 295,988 markers [single-nucleotide polymorphisms (SNPs) and rare variants], and transferred to the GeneTitan Instrument (Affymetrix).ResultsWe found 15 SNPs (P < .001), 18 genes (P < .005), and 4 pathways (P < .05) related to cytokine-cytokine receptor interaction, natural killer cell-mediated cytotoxicity, regulation of autophagy, and renin-angiotensin system as the most strongly associated with frailty.Conclusions/ImplicationsThe specific genetic features related to energy metabolism, biological processes regulation, cognition, and inflammation highlighted by this preliminary analysis offer useful insights for finding biologically meaningful biomarkers of frailty that allow early diagnosis and treatment. Further research is needed to confirm our novel findings in a larger population. Indeed, the EU-funded FRAILOMICS research effort will address this question.