N-linked glycan truncation causes enhanced clearance of plasma-derived von Willebrand factor.

Essentials von Willebrands factor (VWF) glycosylation plays a key role in modulating in vivo clearance. VWF glycoforms were used to examine the role of specific glycan moieties in regulating clearance. Reduction in sialylation resulted in enhanced VWF clearance through asialoglycoprotein receptor. Progressive VWF N-linked glycan trimming resulted in increased macrophage-mediated clearance. Click to hear Dr Denis discuss clearance of von Willebrand factor in a free presentation from the ISTH Academy SUMMARY: Background Enhanced von Willebrand factor (VWF) clearance is important in the etiology of both type 1 and type 2 von Willebrand disease (VWD). In addition, previous studies have demonstrated that VWF glycans play a key role in regulating in vivo clearance. However, the molecular mechanisms underlying VWF clearance remain poorly understood. Objective To define the molecular mechanisms through which VWF N-linked glycan structures influence in vivo clearance. Methods By use of a series of exoglycosidases, different plasma-derived VWF (pd-VWF) glycoforms were generated. In vivo clearance of these glycoforms was then assessed in VWF-/- mice in the presence or absence of inhibitors of asialoglycoprotein receptor (ASGPR), or following clodronate-induced macrophage depletion. Results Reduced amounts of N-linked and O-linked sialylation resulted in enhanced pd-VWF clearance modulated via ASGPR. In addition to this role of terminal sialylation, we further observed that progressive N-linked glycan trimming also resulted in markedly enhanced VWF clearance. Furthermore, these additional N-linked glycan effects on clearance were ASGPR-independent, and instead involved enhanced macrophage clearance that was mediated, at least in part, through LDL receptor-related protein 1. Conclusion The carbohydrate determinants expressed on VWF regulate susceptibility to proteolysis by ADAMTS-13. In addition, our findings now further demonstrate that non-sialic acid carbohydrate determinants expressed on VWF also play an unexpectedly important role in modulating in vivo clearance through both hepatic ASGPR-dependent and macrophage-dependent pathways. In addition, these data further support the hypothesis that variation in VWF glycosylation may be important in the pathophysiology underlying type 1C VWD.

A new method to quantify ß-antithrombin glycoform in plasma reveals increased levels during the acute stroke event.

INTRODUCTION: ß-antithrombin, the minor antithrombin glycoform in plasma, is probably the major thrombin inhibitor in vivo because of its high heparin affinity. The levels and variability of this glycoform in general population and its relevance in thromboembolic diseases is unknown since there is no specific method to measure this glycoform in clinical samples. METHODS: Plasma and recombinant α- and ß-antithrombins were purified by heparin affinity chromatography. An anti-FXa chromogenic method in presence of pentassacharide was used with two NaCl concentrations (15mM and 1.1M). This method was applied to plasma samples from 97 healthy subjects and 117 consecutive patients with ischemic cerebrovascular disease during the acute event and one year later. SERPINC1 sequencing was done in cases with antithrombin deficiency. RESULTS: High salt concentrations specifically restricted the pentassacharide-induced activation of antithrombin to the ß glycoform. ß-antithrombin displayed a normal distribution in the general population (89.5%-103.5%), with no significant variations according to age or sex. In patients, whole antithrombin values remained within the normal range. Only five cases had antithrombin deficiency during the thrombotic event, one carrying the L99F mutation in SERPINC1. Interestingly, both ß-antithrombin and the ß/whole antithrombin ratio were significantly higher in patients during the acute event but normalized after one year. CONCLUSIONS: We have developed a rapid, simple, sensitive and specific method to quantify ß-antithrombin activity using 1µL of plasma. ß-antithrombin significantly increases in patients with ischemic cerebrovascular disease during the acute event, probably by its release from the vasculature.

Role of the C-sheet in the maturation of N-glycans on antithrombin: functional relevance of pleiotropic mutations.

BACKGROUND: The characterization of natural mutants identified in patients with antithrombin deficiency has helped to identify functional domains or regions of this key anticoagulant and the mechanisms involved in the deficiency, as well as to define the clinical prognosis. Recently, we described an abnormal glycosylation in a pleiotropic mutant (K241E) that explained the impaired heparin affinity and the mild risk of thrombosis in carriers. OBJECTIVES: To evaluate the effects of different natural pleiotropic mutations on the glycosylation of antithrombin and their functional effects. METHODS: Five pleiotropic mutations identified in patients with antithrombin deficiency and located at each one of the strands of the C-sheet were selected (K241E, M251I, M315K, F402L, and P429L). Recombinant mutants were generated and purified. Glycoform heterogeneity and conformational sensitivity were studied with electrophoresis, proteomic analysis, and glycomic analysis. Heparin affinity was evaluated from intrinsic fluorescence. Reactivity assays with factor Xa, thrombin and neutrophil elastase in the presence or absence of heparin were also performed. RESULTS AND CONCLUSIONS: Pleiotropic mutants, except for that with the M315K mutation, which affects a non-exposed residue, showed two glycoforms. Variant 1, with abnormal glycosylation, had reduced heparin affinity and severely affected reactivity with the target proteases. In contrast, variant 2, with similar electrophoretic mobility and heparin affinity to wild-type antithrombin, had impaired inhibitory activity that was partially compensated for by activation with heparin. Our results suggest the C-sheet of antithrombin as a new region that is relevant for proper maturation of the N-glycans. Therefore, pleiotropic mutations lead to glycosylation defects that are responsible for the reduced heparin affinity.

Specific and non-overlapping functions of testosterone and 11-ketotestosterone in the regulation of professional phagocyte responses in the teleost fish gilthead seabream.

Sex hormones, both estrogens and androgens, have a strong impact on immunity in mammals. In fish, the role of androgens in immunity has received little attention and contradictory conclusions have been obtained. However, it is well known that sex steroids are involved in fish growth, osmoregulation and gonad remodelation. In this study, we examine the in vitro effects of testosterone and 11-ketotestosterone, the two main fish androgens, on the professional phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). Although both testosterone and 11-ketotestosterone failed to modulate the respiratory burst of seabream phagocytes, testosterone but not 11-ketotestosterone was able to increase the phagocytic ability of non-activated phagocytes. Curiously, 11-ketotestosterone was more powerful than testosterone at inducing the expression of its own receptor, namely androgen receptor b (ARb), in acidophilic granulocytes (AGs), but none of them affected the basal ARb expression levels in macrophages (MØ). Furthermore, although physiological concentrations of testosterone exerted a pro-inflammatory effect on both AGs and MØs, 11-ketotestosterone showed an anti-inflammatory effect in AGs and a strong pro-inflammatory effect in MØs. Interestingly, both androgens modulated the expression of toll-like receptors in these two immune cell types, suggesting that androgens might regulate the sensitivity of phagocytes to pathogens and damage signals. Testosterone and 11-ketotestosterone have a competitive effect, at least, on the modulation of the expression of some genes. Therefore, our results show for the first time a non-overlapping role for testosterone and 11-ketotestosterone in the regulation of professional phagocyte functions in fish.

Desigualdades sociales en la salud mental infantil en Cataluña / Socio-economic inequalities in children's mental health in Catalonia

Objetivos: Analizar los factores socioeconómicos y de salud asociados a la salud mental infantil en una muestra representativa de población de 4–14 años de edad de Cataluña. Métodos: Los datos provienen de la submuestra infantil de la Encuesta de Salud de Cataluña 2006 (n=1.821). La madre, el padre o la persona encargada del menor respondió el cuestionario de salud mental infantil Strengths and Difficulties Questionnaire (SDQ). Se analizaron las puntuaciones de cada dimensión del SDQ y las dificultades totales según los factores socioeconómicos y de salud del niño/a. Se ajustaron modelos de regresión lineal múltiple. Resultados: En general, pertenecer a una familia de clase social desfavorecida, monoparental, con nivel de estudios de la madre menor que universitario y declarar problemas de salud fueron los factores asociados a una peor salud mental infantil en la mayoría de las dimensiones del SDQ. Los factores asociados a las dificultades totales fueron ser chica (coeficiente β: −1,6), pertenecer a las clases sociales menos favorecidas (grupo IV-V: 1,6), con menor nivel de estudios materno (nivel de estudios primario o menos: 1,2), pertenecer a una familia monoparental (1,5) y declarar más problemas de salud (3 o más problemas: 3,9). Conclusiones: Los niños y las niñas de familias con nivel socioeconómico menos favorecido y de familias monoparentales se encuentran a riesgo de presentar peor salud mental. El estudio aporta información útil para la monitorización y el abordaje de los problemas de salud mental infantil (AU)

Objectives: To analyse socio-economic and health status factors associated with poor mental health in a representative sample of children 4 to 14 years old in Catalonia. Methods: The data comes from a subsample of the Catalan Health Interview Survey 2006 (CHIS) (n=1821). Proxy-respondents, mainly mothers, answered the Strengths and Difficulties Questionnaire (SDQ). The SDQ dimension scores and the Total difficulties score were analysed by socio-demographics and health status factors. Multivariate linear regression models were fitted to analyse the influence of socio-economic and health status factors on mental health. Results: In general, being in a disadvantaged family, single-parent families, a family whose mother had lower level of education, and those children reporting physical health problems, have been factors associated with worse child mental health in most of SDQ dimension scores. Factors associated with Total difficulties were, being a girl (beta coefficient: −1.6), from disadvantaged social classes (group IV–V: 1.6), at lower level of maternal education (mother's educational primary level or less: 1.2), being in a single-parent family (1.5), and reporting health problems (3 or more health problems: 3.9). Conclusions: Children from disadvantaged families, and single-parent families are at higher risk of worse mental health than their counterparts in the advantaged groups. The study contributes to collecting information for monitoring and approaching mental health in children (AU)

[Socio-economic inequalities in children's mental health in Catalonia]. / Desigualdades sociales en la salud mental infantil en Cataluña.

OBJECTIVES: To analyse socio-economic and health status factors associated with poor mental health in a representative sample of children 4 to 14 years old in Catalonia. METHODS: The data comes from a subsample of the Catalan Health Interview Survey 2006 (CHIS) (n=1821). Proxy-respondents, mainly mothers, answered the Strengths and Difficulties Questionnaire (SDQ). The SDQ dimension scores and the Total difficulties score were analysed by socio-demographics and health status factors. Multivariate linear regression models were fitted to analyse the influence of socio-economic and health status factors on mental health. RESULTS: In general, being in a disadvantaged family, single-parent families, a family whose mother had lower level of education, and those children reporting physical health problems, have been factors associated with worse child mental health in most of SDQ dimension scores. Factors associated with Total difficulties were, being a girl (beta coefficient: -1.6), from disadvantaged social classes (group IV-V: 1.6), at lower level of maternal education (mother's educational primary level or less: 1.2), being in a single-parent family (1.5), and reporting health problems (3 or more health problems: 3.9). CONCLUSIONS: Children from disadvantaged families, and single-parent families are at higher risk of worse mental health than their counterparts in the advantaged groups. The study contributes to collecting information for monitoring and approaching mental health in children.

[Mortality risk factors in critical surgical patients]. / Factores de riesgo de mortalidad de los pacientes quirúrgicos graves.

OBJECTIVE: To determine mortality risk factors for critically ill postoperative patients. PATIENTS: Two hundred eleven patients undergoing any type of surgery admitted to the intensive care unit (ICU) over a period of one year were enrolled. METHOD: We performed a cross-sectional study that was retrospective for pre- and intraoperative observations and prospective for ICU observations. A univariate analysis was performed and relative risk (95% CI) was calculated. Multivariate analysis was also performed and the adjusted odds ratio calculated (95% CI). The end point of measurement was death in the ICU. RESULTS: The independent risk factors for mortality were age 60 years (OR: 2.8; 95% CI 1.36-5.09), physical status ASA-V (OR: 9.66; 95% CI 5.62-14.31), intracranial surgery to treat severe head injury (OR: 5.33; 95% CI 3.08-9.47), hemorrhagic shock during surgery (OR: 5.20; 95% CI 4.16-8.29), arterial hypotension during surgery (OR: 4.0; 95% CI 2.78-11.65), APACHE III score upon admission to the ICU (OR: 9.29; 95% CI 3.62-21.38), multiple organ dysfunction syndrome (OR: 7.96; 95% CI 3.73-13.92), and cardiopulmonary arrest in the ICU (OR: 5.85; 95% CI 2.21-10.43). The APACHE III score demonstrated high sensitivity (87%) and positive predictive value (96%). CONCLUSIONS: Surgical patients in critical condition continue to account for a large number of admissions to and death in the ICU. The variables analyzed were sufficient to explain patient outcome, such that risk factors for mortality in surgical patients admitted to the ICU could be determined.

Factores de riesgo de mortalidad de los pacientes quirúrgicos graves / Mortality risk factors in critically ill postoperative patients

Objetivo. Determinar los factores de riesgo de la mortalidad de los pacientes quirúrgicos graves.Material y métodos. Se incluyó a los 211 pacientes operados, independientemente del tipo de intervención quirúrgica practicada, que ingresaron durante el período de tiempo de un año en la unidad de cuidados intensivos (UCI). Se realizó un estudio de corte transversal, ambispectivo (retrospectivo en cuanto a las observaciones pre e intraoperatorias y prospectivo en cuanto a las observaciones en la UCI). Se analizaron variables correspondientes a las etapas pre, intra y postoperatoria. Se realizó un análisis univariado y se calculó el riesgo relativo con un intervalo de confianza (IC) del 95 por ciento. También se realizó un análisis multivariado calculándose la odds ratio (OR) ajustada, así como los intervalos de confianza del 95 por ciento. El punto final de la medición fue la mortalidad en la UCI. Resultados. Las variables que constituyeron factores de riesgo independientes para la mortalidad fueron: edad igual o mayor de 60 años (OR: 2,8; IC del 95 por ciento: 1,36-5,09), estado físico 5, según la clasificación de la American Society of Anesthesiologists (OR: 9,66; IC del 95 por ciento: 5,62-14,31), cirugía intracraneal para tratamiento del trauma craneoencefálico grave (OR: 5,33; IC del 95 por ciento: 3,089,47), shock hemorrágico intraoperatorio (OR: 5,20; IC del 95 por ciento: 4,16-8,29), hipotensión arterial intraoperatoria (OR: 4,0; IC del 95 por ciento: 2,78-11,65), índice APACHE III en el momento del ingreso en UCI (OR: 9,29; IC del 95 por ciento: 3,6221,38), síndrome de disfunción orgánica múltiple (OR: 7,96; IC del 95 por ciento: 3,73-13,92), y paro cardiorrespiratorio en la UCI (OR: 5,85; IC del 95 por ciento: 2,21-10,43). El índice APACHE III evidenció una elevada sensibilidad (87 por ciento) y valor predictivo positivo (96 por ciento).Conclusiones. Los pacientes quirúrgicos graves continúan siendo una causa importante de ingreso y mortalidad en la UCI. Las variables consideradas en el estudio fueron suficientes para explicar el desenlace final de los pacientes, por lo que se pudieron determinar los factores de riesgo para la mortalidad de los pacientes quirúrgicos admitidos en la UCI (AU)

The hypothalamic ventromedial nuclei couple activity in the hypothalamo-pituitary-adrenal axis to the morning fed or fasted state.

Function in the adrenocortical system is markedly altered by availability of food. Basal activity is lowest and stress responsivity highest in the morning when nocturnal rats eat approximately 90% of their daily calories during the dark. After an overnight fast, basal corticotrophin and corticosteroid levels are elevated, and responsivity to stressors is decreased. Central neural sites that control these changes are unidentified. The hypothalamic ventromedial nuclei (VMN) appear to signal satiety; lesions result in increased food intake, obesity, and elevated basal insulin and corticosteroids. Thus, the VMN are good candidates for calorically mediated control of adrenocortical system function in satiated rats. We injected colchicine into the VMN to cause reversible inhibition of activity (Avrith and Mogenson, 1978) and tested the effects on basal and stimulated function in the adrenocortical system. Colchicine-injected rats that fed ad libitum exhibited increased basal but reduced corticotrophin and corticosterone responses to restraint in the morning compared with controls. By contrast, after an overnight fast, control rats had increased basal adrenocortical hormones and decreased stress responses that did not differ from colchicine-injected rats. Colchicine was visualized within cells in the VMN for up to 5 d using fluorescein/colchicine, and the treatment did not cause increased gliosis; moreover, the functional effects of the injections were reversed within 15 d. We conclude that (1) the VMN serve to couple activity in the adrenocortical system to energy intake and (2) discrete colchicine injections provide a behaviorally and neuroendocrinologically useful period of inhibition without causing permanent functional damage.

Efecto de inmediato de dos herbicidas sobre larvas de Mansonia y Coquilletidia (Diptera, Culicidae) al ser aplicados a cinco malezas acuáticas / Inmediate effect of herbicides on mansoniam and coquilletidia (Dipteria, Culicidae) Apply to five aquatic weeds

Se realizó un estudio en la quebrada Mojapollo, afluente del río Chagres, con el objeto de obtener estimados poblacionales de larvas de Mansonia y Coquielletidia (Dipetera, Culiciadad), antes y después de la aplicación de 2,4-D y el glisofato. Estos últimos son utilizados en la Unidad de Control de malezas Acuáticas de la Comisión del Canal, para controlar las malezas en esta quebrada. Se colectaron 1,100 unidades muestrales (paquete de raíz) cinco especies de malezas a saber: Pistia stratiotes, Echhornia crassipes, Jusssiaea natans, Cyperus sp. y Luzeola sp; las cuales se encontraban distribuídas en tres estaciones experimentales (testigo, 2,4-D y glisofato). Se observaron cinco especies de de mosquitos: mansonia titillans, Aedeomyai squamipennis, Culex Sp; Anopheles albimanus y Anopheles triannulatus. Las dos primeras especies que constituyeron el 95 por ciento de lo colectado se observaron muy asociados a las malezas Pistia stratiotes y Jussiaea natans, respectivamente. Para la especie M. titillans, la cual se adhiere a la raíz de las malezas, podemos concluir que no se presentó un efectos tóxico directo debido a la aplicación de los herbicidas, sino más bien un efecto indirecto donde se produjo una mayor congregación de larvas en las plantas menos afectadas por los herbicidas. Se realizó un bioensayo con larvas de M. titillans para evaluar el efecto directo de los herbicidas sobre las larvas en condiciones controladas, observándose mortalidad significativa en la prueba con la concentración mayor de glisofato (0.1 por ciento). Este último resultado permitió deducir que las concentraciones utilizadas en el campo, que fueron mucho menores (aproximadamente 0.0000035 por ciento de 2,4-D) y 0.0000043 por ciento de glisofato) no produjeron mortalidad significativa en la población de larvas