LDLR-Gene therapy for familial hypercholesterolaemia: Problems, progress, and perspectives

Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency

Molecular Interactions between a Biodegradable Demulsifier and Asphaltenes in an Organic Solvent

A surface forces apparatus (SFA) was used to measure the intermolecular forces between a biodegradable demulsifier (ethyl cellulose, EC) and asphaltenes immobilized individually on molecularly smooth mica surfaces in an organic solvent. A steric repulsion on approach between the immobilized EC layers and asphaltenes was measured despite strong adhesion (Fad/R ≈ −2 mN/m; Wad = 0.42 mJ/m2) during retraction. The measured adhesion was attributed to the interpenetration and tangling of aliphatic branches of swollen asphaltenes and solvated chains of EC macromolecules. Competitive adsorption of EC on/in immobilized asphaltene layers was confirmed by combining SFA force measurements and atomic force microscopy (AFM) imaging. Following the injection of EC-in-toluene solution, an immediate (<5 min) increase in the confined layer thickness of the immobilized asphaltene layers was measured. Irreversibly adsorbed asphaltenes were displaced by EC macromolecules through binding with unoccupied surface sites on mica, followed by the spreading of EC across the mica substrate due to increased surface activity governed by the higher number of hydroxyl groups per EC molecule. AFM imaging confirmed that the increase in confined layer thickness resulted from the formation of larger asphaltene aggregates/clusters protruding from the mica substrate. Molecular level topographical images showed that the asphaltenes were not resolvated in the organic phase but self-associated as the EC macromolecules spread across the hydrophilic mica substrate. The results from this study provide not only fundamental insights into the basic interaction mechanisms of asphaltenes with EC macromolecules as a demulsifier in organic media but also directions toward enhancing demulsification of water-in-oil emulsions

Development of a Microarray system for the Rapid and Simultaneous Detection of Bacterial and Viral Foodborne Pathogens

Foodborne diseases are increasingly recognized as a significant global public health problem despite major advances and improvements in the quality of food, water, sanitation and hygiene. However, detection and characterization of foodborne pathogens during outbreak scenarios remains a laborious and time-consuming task. The aim of this work was to develop an oligonucleotide microarray for rapid detection and characterization of the most important infectious bacterial (Campylobacter, Salmonella and Yersinia) and viral (Noroviruses) pathogens found in swine and associated pork products. A total of 272 target regions and genes were identified that were specific for pathogen identification and characterization of specific antimicrobial resistance and virulence determinants. We designed multiple probes (up to three) per gene to increase the sensitivity and specificity of the microarray. After BLAST analysis, a total of 562 probes were finally selected to be printed on to glass slides. Appropriate control strains that were previously characterized in our laboratories by PCR were selected to test the developed arrays. Preliminary results indicated that the designed probes were highly specific and sensitive for identification of tested pathogens and known res1stance and virulence genes present in the selected control strains

Uncovering the true impacts of remediation

This bulletin investigates the sustainability of remediation through the development and use of a sustainability assessment methodology. It is one of the products of the Sustainable Urban Brownfield: Integrated Management (SUBR:IM) research consortium. The objective of this work was to identify and compare the wider impacts of a range of remediation technologies in use in the UK. A methodology was developed based on multi-criteria and detailed impact analyses, both of which incorporated life-cycle approaches. This was used to compare three options on a particular site, and highlighted the major impacts from each

Effects of dexamethasone on renal and systemic acid-base metabolism

Effects of dexamethasone on renal and systemic acid-base metabolism. We carried out long-term balance studies in adrenalectomized (ADX) dogs to evaluate the effects of small amounts of a glucocorticoid steroid with minimal mineralocorticoid potency (dexamethasone; DEX) on renal and systemic acid-base metabolism under conditions of constant mineralocorticoid replacement and both normal and increased systemic acid loads. We investigated the effects of low and high dosages of dexamethasone (0.2 mg/day [normal-DEX] vs. 0.8 mg/day [high-DEX]) before and during hydrochloric acid feeding (5 mmol/kg/day) in paired studies on ADX dogs (N = 7) maintained on constant mineralocorticoid replacement (deoxycorticosterone, corticosterone, aldosterone). Prior to hydrochloric acid feeding, no differences in plasma acid-base composition were observed between the two dosages despite greater endogenous acid production with the higher dosage of DEX, evidenced by greater rates of both net acid excretion (NAE) and the excretion of urinary anions other than chloride, bicarbonate, and phosphate (urine anion gap). During hydrochloric acid feeding, mean plasma bicarbonate (PHCO3) decreased from 21.2 ± 0.4 to 13.7 ± 0.5 (normal-DEX) and from 21.1 ± 0.4 to 15.8 ± 0.4 mEq/liter (high-DEX). The difference in the decrements in PHCO3 between groups was significant (P < 0.05). With continued hydrochloric acid feeding in both groups, increasing the DEX dosage from 0.2 to 0.8 mg/day in the normal-DEX group resulted in a. significant increase in NAE (ΣΔNAE, +161 mEq, P < 0.02) and in PHCO3 (+3.6 ± 0.5 mEq/liter, P < 0.01) to steady-state levels by day 10, which were values not significantly different from those in high-DEX. The DEX dose-related increase in NAE was greater than the corresponding increase in endogenous acid production estimated from the change in urine anion gap, and was due largely to an increase in ammonium excretion, which, because urine pH did not decrease, could not be attributed to increased intraluminal trapping of ammonia as a result of more acidic tubular fluid. These studies indicate that the severity of hydrochloric acid-induced metabolic acidosis in mineralocorticoid-replete ADX dogs can be mitigated by increasing the dosage of exogenous glucocorticoid and suggest that this acidosis mitigating effect is mediated in part by the increased NAE associated with the stimulation of renal ammonia production. These studies further indicate that the rate of production of fixed acids of metabolism increases with an increased dosage of exogenous glucocorticoid, but that this acidosis-producing effect is more than offset by independent stimulation of renal net acid excretion, such that metabolic acidosis is prevented (basal condition) or if present (hydrochloric acid feeding) is significantly ameliorated.Effets de la dexaméthasone sur le métabolisme acido-basique rénal et systémique. Les études de bilan qui sont rapportées ont été réalisées chez des chiens surrénalectomisés (ADX) pour évaluer les effets de faibles quantités d'un stéroïde glucocorticoïde, ayant une activité minéralocorticoïde faible (dexaméthasone; DEX), sur le métabolisme acido-basique rénal et systémique dans des conditions de traitement substitutif permanent de minéralocorticoïdes et de charge acide soit normale soit élevée. Nous avons étudié les effets de doses de dexaméthasone (0,2 mg/jour; normale-DEX) ou 0,8 mg/jour (élevée-DEX) avant et pendant l'administration d'acide chlorhydrique à raison de 5 mmol/kg/jour dans des études appariées chez des chiens ADX (N = 7) recevant un traitement substitutif par les minéralocorticoïdes (deoxycorticosterone acetate, corticosterone, aldosterone). Avant l'administration d'acide chlorhydrique, il n'y avait pas de différence dans la composition acido-basique du plasma selon les doses de DEX malgré l'augmentation de production endogène d'acide sous l'effet de la dose la plus élevée de DEX, augmentation traduite par une élévation de l'état stationnaire d'excrétion nette d'acide (NAE) et de la somme des débits d'excrétion des anions urinaires autres que le chlore, le bicarbonate et le phosphate (trou anionique urinaire). Au cours de l'administration d'acide chlorhydrique la concentration plasmatique moyenne de bicarbonate (PHCO3) a diminué de 21,2 ± 0,4 à 13,7 ± 0,5 (normale-DEX) et de 21,1 ± 0,4 à 15,8 ± 0,4 mEq/litre (élevée-DEX). La différence des diminutions de bicarbonate était significative (P < 0,05). Au cours de l'administration prolongée d'acide chlorhydrique aux deux groupes l'augmentation de la dose de DEX de 0,2 à 0,8 mg/jour dans le groupe normale-DEX a eu pour résultat une augmentation significative de NAE (ΣΔNAE, +161 mEq, P < 0,02) et de PHCO3 (+ 3,6 ± 0,5 mEq/litre, P < 0,01), jusqu'à de nouveaux états stationnaires, le jour 10, non sigmficativement différents de ceux observés dans le groupe élevée-DEX. L'augmentation de NAE dépendant de la dose de DEX a été plus grande que l'augmentation correspondante de la production endogène d'acide estimée à partir de la modification du trou anionique urinaire, elle était principalement due à une augmentation de l'excrétion d'ammonium qui, du fait que le pH de l'urine n'a pas diminué, ne peut pas être attribuée à une augmentation de la captation intraluminale d'ammonia. Ces résultats indiquent que la sévérité de l'acidose métabolique déterminée par acide chlorhydrique chez les chiens ADX recevant des minéralocorticoïdes peut être atténuée par l'augmentation de la dose de glucocorticoïdes exogènes et suggère que cet effet d'atténuation a pour médiateur partiel l'augmentation de NAE associée à la stimulation de la production rénale d'ammonia. Ces résultats indiquent, de plus, que le débit de production des acides fixes augmente en même temps que la dose de glucocorticoïdes exogènes mais que cet effet de production d'acidose est plus que compensé par la stimulation indépendante de l'excrétion rénale nette d'acide, de telle sorte que l'acidose métabolique est empêchée (conditions basales) ou significativement améliorée au cours de l'administration acide chlorhydrique

A cell-based assay system for activators of the environmental cell stress response

This work was funded by The Scottish Government Rural and Environment Science and Analytical Services Division (RESAS), Scotland, United Kingdom and the Biotechnology and Biological Sciences Research Council (BBSRC; East-Bio Doctoral Training Partnership), United Kingdom. The funders had no role in the design of the study, the analysis and interpretation of the data, or the publication process. The authors wish to thank Prof. John Hayes, University of Dundee, for the NQO1-luciferase construct and Prof. Gary Felsenfeld, NIH, Bethesda for the plasmid pJC13-1.Peer reviewedPostprin

Error reduction therapy in reducing struggle and grope behaviours in apraxia of speech

We report an intervention study focused on the speech production difficulties present in acquired apraxia of speech (AOS). The intervention was a self-administered computer therapy that targeted whole word production and incorporated error reduction strategies. The effectiveness of the therapy was contrasted to that of a visuospatial sham computer program, and performance across treated words, and two sets of matched words, was assessed. Two groups of participants completed the study which employed a two-phase cross-over treatment design. Participants were randomly assigned to a speech first or sham first condition. Treatments were administered for six weeks, with a four week rest between interventions. Participants were assessed five times in total; twice at baseline, once following each of the intervention phases, and once following a lapse of eight weeks after the end of the second phase of intervention. The occurrence of accurate word production and speech characterised by struggle and groping behaviours was recorded on a repetition task. Participants showed significant gains in speech accuracy and fluency, and reductions in articulatory groping and struggle behaviours following the use of the speech program. These gains were largely maintained once the therapy was withdrawn

Characterisation of multiple hindered settling regimes in aggregated mineral suspensions

Aqueous suspensions of magnesium hydroxide are shown to exhibit low ζ-potential behavior and highly complex settling dynamics. Two distinct regimes of hindered settling behavior are observed on either side of a threshold concentration, ϕ*, of 2.38% v/v, which is considerably below the gel point, ϕg, observed at 5.4 ± 1.6% v/v. The low-concentration regime was characterized by a very large Richardson and Zaki exponent of 146, a factor of 10 larger than that of the high-concentration regime. Michaels and Bolger analysis of the low-concentration regime implies settling governed by large, low-density macroaggregates of 138–147 μm diameter and low intraaggregate packing fractions on the order of 0.05, which is in good agreement with in situ particle characterization undertaken using particle vision and measurement (PVM) and focused-beam reflectance measurements (FBRM). The large macroaggregates must undergo some shear densification within the higher-concentration hindered settling regime in order for the suspension to gel at a concentration of 5.4% v/v. Consequently, fluid flow past small, shear-resistant primary agglomerates, observed within the aggregates using scanning electron microscopy and flow particle image analysis, during aggregate densification may represent the limiting step for dewatering within the high-concentration regime