Effect of collection and preprocessing methods on neutrophil elastase plasma concentrations.

OBJECTIVES: Elevated plasma levels of the elastase alpha 1-proteinase inhibitor complex (E-alpha 1 PI) have been proposed as a marker of bacterial infection and neutrophil activation. Liberation of elastase from neutrophils after collection of blood may cause falsely elevated results. Collection methods have not been validated for critically ill neonates and children. We evaluated the influence of preanalytical methods on E-alpha 1 PI results including the recommended collection into EDTA tubes. DESIGN AND METHODS: First, we compared varying acceleration speeds and centrifugation times. Centrifugation at 1550 g for 3 min resulted in reliable preparation of leukocyte free plasma. Second, we evaluated all collection tubes under consideration for absorption of E-alpha 1 PI. Finally, 12 sets of samples from healthy adults and 42 sets obtained from critically ill neonates and children were distributed into the various sampling tubes. Samples were centrifuged within 15 min of collection and analyzed with a new turbidimetric assay adapted to routine laboratory analyzers. RESULTS: One of the two tubes containing a plasma-cell separation gel absorbed 22.1% of the E-alpha 1 PI content. In the remaining tubes without absorption of E-alpha 1 PI no differences were observed for samples from healthy adult patients. However, in samples from critically ill neonates or children, significantly higher results were obtained for plain Li-heparin tubes (mean = 183 micrograms/L), EDTA tubes (mean = 93 micrograms/L), and citrate tubes (mean = 88.5 micrograms/L) than for the Li-hep tube with cell-plasma separation gel and no absorption of E-alpha 1 PI (mean = 62.4 micrograms/L, p < 0.01). CONCLUSION: Contrary to healthy adults, E-alpha 1 PI results in plasma samples from critically ill neonates and children depend on the type of collection tube.

Document sobre el rebuig dels Testimonis de Jehovà a les transfusions de sang

El document pretén aportar pautes útilsals ciutadans, als centres assistencials i a l'Administració, i que subministra criterisètics i jurídics que ajuden a aclarir el debat sobre si les decisions sanitàries adoptadespels cristians anomenats Testimonis de Jehovà, sobre la base de les seves creencesreligioses, han de ser respectades encara que la seva conseqüència fi nal sigui lapèrdua de la vida.

Generation and function of mouse central memory and effector memory T cells

1. SUMMARY Based on functional and homing properties, two subsets of memory T lymphocytes have been defined both in humans and in mice. Central memory T cells (TCM cells) express the lymph node homing receptors CD62L and CCR7, have poor effector function and proliferate efficiently upon antigenic stimulation. Effector memory T cells (TEM cells) do not express CCR7, are mostly CD62L negative and therefore are excluded from lymph nodes, but are able to migrate to sites of inflammation where they exert immediate effector function by producing inflammatory cytokines and cytotoxic mediators. In the present work we have addressed two questions that emerged since the definition of TCM and TEM cells. Firstly, what are the priming conditions for generation of TCM and TEM and, secondly, what is the migratory capacity of TCM and TEM cells in inflammatory conditions. By using naive TCR-transgenic OT-I CD8+ T cells and OT-II CD4+ T cells and ovalbumin pulsed-mature dendritic cells (DCs) we set up an in vitro system in which the strength of T cell stimulation is controlled by varying the ratio of T cells and DCs and the duration of DC-T cell interaction. Using this system we found that precursors of TCM and TEM cells are generated at different strength of stimulation and that T cells capable of persisting in vivo in the absence of antigen and of mounting recall responses is optimally induced by intermediate stimulatory strength. In addition, we found that lymph nodes draining sites of mature DC or adjuvant inoculation recruit CD8+ CD62L- CCR7- effector and TEM cells. CD8+ T cell recruitment in reactive lymph nodes requires CXCR3 expression on T cells and occurs through high endothelial venules (HEVs) in concert with HEV lurninal expression of the CXCR3 ligand CXCL9. In reactive lymph nodes, recruited T cells establish stable interactions with and kill antigen-bearing DCs, limiting the ability of these DCs to activate CD4+ and CD8+ T cells. Taken togther these data define conditions for the generation of TCM and TEM cells and define an inflammatory pathway of effector T cell migration in lymph nodes. The inducible recruitment of blood-borne effector and TEM CD8+ cells to lymph nodes may represent a mechanism for terminating primary and limiting secondary immune responses.

Postmenopausal circulating levels of 2- and 16α-hydroxyestrone and risk of endometrial cancer.

Background:It has been suggested that the relative importance of oestrogen-metabolising pathways may affect the risk of oestrogen-dependent tumours including endometrial cancer. One hypothesis is that the 2-hydroxy pathway is protective, whereas the 16α-hydroxy pathway is harmful.Methods:We conducted a case-control study nested within three prospective cohorts to assess whether the circulating 2-hydroxyestrone : 16α-hydroxyestrone (2-OHE1 : 16α-OHE1) ratio is inversely associated with endometrial cancer risk in postmenopausal women. A total of 179 cases and 336 controls, matching cases on cohort, age and date of blood donation, were included. Levels of 2-OHE1 and 16α-OHE1 were measured using a monoclonal antibody-based enzyme assay.Results:Endometrial cancer risk increased with increasing levels of both metabolites, with odds ratios in the top tertiles of 2.4 (95% CI=1.3, 4.6; P(trend)=0.007) for 2-OHE1 and 1.9 (95% CI=1.1, 3.5; P(trend)=0.03) for 16α-OHE1 in analyses adjusting for endometrial cancer risk factors. These associations were attenuated and no longer statistically significant after further adjustment for oestrone or oestradiol levels. No significant association was observed for the 2-OHE1 : 16α-OHE1 ratio.Conclusion:Our results do not support the hypothesis that greater metabolism of oestrogen via the 2-OH pathway, relative to the 16α-OH pathway, protects against endometrial cancer.

Evaluation of arterial compliance-pressure curves. Effect of antihypertensive drugs.

A new high-precision ultrasonic device was developed to determine noninvasively arterial compliance as a function of blood pressure. Because of the nonlinear elastic properties of arterial walls, measurements of compliance can be appropriately compared only if obtained over a range of pressures. This apparatus was used to evaluate in a double-blind, parallel fashion the effect of three different antihypertensive drugs and of a placebo on radial artery compliance. Thirty-two normotensive volunteers were randomly allocated to an 8-day, once-a-day oral treatment with either a placebo, 100 mg atenolol, 20 mg nitrendipine, or 20 mg lisinopril. Blood pressure, heart rate, radial artery diameter, and arterial compliance were measured immediately before as well as 6 hours after dosing on the first and last days of the study. On the eighth day of administration, within 6 hours after dosing, lisinopril induced an acute increase in radial artery diameter, from 2.99 +/- 0.06 to 3.28 +/- 0.09 mm (mean +/- SEM, p less than 0.01). The compliance-pressure curve was shifted upward on day 1 (p less than 0.01) as well as on day 8 (p less than 0.05). None of the other drugs induced any significant modification of these parameters. Arterial compliance has a strong nonlinear dependency on intra-arterial pressure and therefore has to be defined as a function of pressure. Antihypertensive drugs acting by different mechanisms may have different effects on the mechanical properties of large arteries.

Document sobre el rebuig dels Testimonis de Jehovà a les transfusions de sang

El document pretén aportar pautes útilsals ciutadans, als centres assistencials i a l'Administració, i que subministra criterisètics i jurídics que ajuden a aclarir el debat sobre si les decisions sanitàries adoptadespels cristians anomenats Testimonis de Jehovà, sobre la base de les seves creencesreligioses, han de ser respectades encara que la seva conseqüència fi nal sigui lapèrdua de la vida.

Cardiovascular influences of alpha1b-adrenergic receptor defect in mice.

BACKGROUND: The alpha1-adrenergic receptors (alpha1-ARs) play a key role in cardiovascular homeostasis. However, the functional role of alpha1-AR subtypes in vivo is still unclear. The aim of this study was to evaluate the cardiovascular influences of alpha1b-AR. METHODS AND RESULTS: In transgenic mice lacking alpha1-AR (KO) and their wild-type controls (WT), we evaluated blood pressure profile and cardiovascular remodeling induced by the chronic administration (18 days via osmotic pumps) of norepinephrine, angiotensin II, and subpressor doses of phenylephrine. Our results indicate that norepinephrine induced an increase in blood pressure levels only in WT mice. In contrast, the hypertensive state induced by angiotensin II was comparable between WT and KO mice. Phenylephrine did not modify blood pressure levels in either WT or KO mice. The cardiac hypertrophy and eutrophic vascular remodeling evoked by norepinephrine was observed only in WT mice, and this effect was independent of the hypertensive state because it was similar to that observed during subpressor phenylephrine infusion. Finally, the cardiac hypertrophy induced by thoracic aortic constriction was comparable between WT and KO mice. CONCLUSIONS: Our data demonstrate that the lack of alpha1b-AR protects from the chronic increase of arterial blood pressure induced by norepinephrine and concomitantly prevents cardiovascular remodeling evoked by adrenergic activation independently of blood pressure levels.

FOXC2 and NFATc1 control formation and maturation of collecting lymphatic vessels

The mechanisms of blood vessel maturation into distinct parts of the blood vasculature such as arteries, veins, and capillaries have been the subject of intense investigation over recent years. In contrast, our knowledge of lymphatic vessel maturation is still fragmentary. In this study, we provide a molecular and morphological characterization of the major steps in the maturation of the primary lymphatic capillary plexus into collecting lymphatic vessels during development and show that forkhead transcription factor Foxc2 controls this process. We further identify transcription factor NFATc1 as a novel regulator of lymphatic development and describe a previously unsuspected link between NFATc1 and Foxc2 in the regulation of lymphatic maturation. We also provide a genome-wide map of FOXC2-binding sites in lymphatic endothelial cells, identify a novel consensus FOXC2 sequence, and show that NFATc1 physically interacts with FOXC2-binding enhancers. These data provide novel insights into the molecular program of lymphatic vascular specification and suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Preserved capillary density of dorsal finger skin in treated hypertensive patients with or without type 2 diabetes.

OBJECTIVES: Capillary rarefaction is a hallmark of untreated hypertension. Recent data indicate that rarefaction may be reversed by antihypertensive treatment in nondiabetic hypertensive patients. Despite the frequent association of diabetes with hypertension, nothing is known on the capillary density of treated diabetic patients with hypertension. METHODS: We enrolled 21 normotensive healthy, 25 hypertensive only, and 21 diabetic (type 2) hypertensive subjects. All hypertensive patients were treated with a blocker of the renin-angiotensin system, and a majority had a home blood pressure ≤135/85 mmHg. Capillary density was assessed with videomicroscopy on dorsal finger skin and with laser Doppler imaging on forearm skin (maximal vasodilation elicited by local heating). RESULTS: There was no difference between any of the study groups in either dorsal finger skin capillary density (controls 101 ± 11 capillaries/mm(2) , nondiabetic hypertensive 99 ± 16, diabetic hypertensive 96 ± 18, p > 0.5) or maximal blood flow in forearm skin (controls 666 ± 114 perfusion units, nondiabetic hypertensive 612 ± 126, diabetic hypertensive 620 ± 103, p > 0.5). CONCLUSIONS: Irrespective of the presence or not of type 2 diabetes, capillary density is normal in hypertensive patients with reasonable control of blood pressure achieved with a blocker of the renin-angiotensin system.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Blood donor motivation: what is ethical? What works?

The retention of previous donors and the recruitment of new donors is a serious challenge for many blood donation services in their effort to prevent blood shortages. More and more services make use of some sort of donation incentives. However, the use of (material) incentives to motivate blood donors is fiercely controversial and there is a longstanding (ethical) debate about whether it should be allowed that donors receive material rewards. Interestingly, this debate is dealt with in almost complete absence of systematic empirical evidence on the effectiveness of material incentives in encouraging people to donate. In this paper, we argue that the discussion on what is ethical in motivating blood donors should be enriched with empirical evidence based on field experiments. We confront the Titmuss controversy with recent results from an experiment administering lottery tickets as a motivation device. Moreover, we take up a neglected phenomenon in the study of blood donors: many non-donors are not principally against donating blood they have just never made up their mind about becoming active blood donors. We propose active decisions as a mechanism to transform latent prosocial preferences into actual prosocial behavior.

Proteomic analysis of Intercept-treated platelets.

In the past decades, transfusion medicine has been driven by the quest for increased safety against transfusion-transmitted infections, mainly by better donor selection and by the development of improved serological and nucleic-acid-based screening assays. Recently, pathogen reduction technologies became available and started to be implemented in several countries, with the primary goal to fight against bacterial contamination of blood products, a rare but dramatic event against which there was no definitive measure. Though pathogen reduction technologies represent a quantum leap in transfusion safety, the biomedical efficacy of platelet concentrates (PCs) treated with various pathogen reduction techniques has been recently questioned by clinical studies. Here, a gel-based proteomic analysis of PCs (n=5), Intercept-treated or untreated, from pooled buffy-coat (10 donors per PC) at Days 1, 2 and 8, shows that the Intercept process that is the most widespread pathogen reduction technique to date, has relatively low impact on the proteome of treated platelets: the process induces modifications of DJ-1 protein, glutaredoxin 5, and G(i)alpha 2 protein. As for the impact of storage, chloride intracellular channel protein 4 (CLIC4) and actin increased independently of Intercept treatment during storage. Whereas alteration of the DJ-1 protein and glutaredoxin 5 points out an oxidative stress-associated lesion, modification of G(i)alpha2 directly connects a possible Intercept-associated lesion to haemostatic properties of Intercept-treated platelets. This article is part of a Special Issue entitled: Integrated omics.

Blood profile during and after hemoglobin substitute administration.

The in vivo effects of Diaspirin Crosslinked Hemoglobin (DCLHb, Baxter Healthcare Corp.) on hematology and biochemistry are unknown. This study includes 6 calves (71.2+/-1.3 kg). In each animal a total of 2 litres of blood was exchanged for the same amount of hydroxylethyl starch (Haes, Fresenius) (n=3) or DCLHb (n=3), which is equivalent to 28cc/kg of blood substitute, over a period of 5 hours. The animals were allowed to survive 7 days. Blood samples were taken hourly during the perfusion protocol, at postoperative day (POD) 1, 2 and 7. ANOVA test was used for repeated measurements. Blood cell profiles were similar in both groups. Peak methemoglobinemia was 4.2% in the DCLHb group. Osmolarity was significantly higher in the DCLHb group with the greatest difference at POD 1 and 2. Postmortem analysis of the major organs did not show any sign of hemoglobin deposit in the DCLHb group. In the given setup DCLHb can be administered in a large quantity with good hematological tolerance and without any deposits in major organs. A prolonged plasma expander effect was observed.

Application of direct-infusion ESI-MS/MS for toxicological screening.

BACKGROUND: Direct-infusion ESI-MS/MS is a powerful approach for the identification of substances in complex mixtures. The aim of this work was to investigate its applicability to the toxicological screening of blood samples. A simple protein precipitation was used, followed by a 15 min infusion of the extract in the mass spectrometer. RESULTS: The application of the procedure to commercial quality controls and authentic post-mortem blood samples demonstrated that the direct-infusion ESI-MS/MS approach enables the simultaneous identification of substances that require different chromatographic conditions. However, poor sensitivity was observed for benzodiazepine, amphetamines and opiate compounds. CONCLUSION: Considering the facile implementation and positive performance of direct-infusion ESI-MS/MS, this approach may to be a valuable complementary technique for systematic toxicological analysis procedures.

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving.

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.