Aggregates in blood filter chambers used from the plasma donations of anti-D donors: evaluation for monoclonal antibody discovery using phage display.

BACKGROUND: RhD-immunoglobulin (RhIg) prevents anti-D alloimmunisation in D-negative pregnant women when the fetus is D-positive, reducing the incidence of haemolytic disease of the fetus and newborn. Manufacturing RhIg is reliant on the limited supply of plasma donations with anti-D antibodies. Monoclonal antibody (mAb) development platforms such as phage display, require blood samples to be collected from anti-D donors, which may be a complicated process. The blood filter chamber (BFC) discarded after an anti-D donor's donation might provide a source of Ig-encoding RNA. This study aims to evaluate whether used BFCs are a suitable source of Ig-encoding RNA for phage display. MATERIAL AND METHODS: Haemonetics PCS2 BFCs were obtained from 10 anti-D donors for total RNA extraction, cDNA synthesis and amplification of VH and VL IgG sequences for assembly of single-chain variable fragments (scFvs). A scFv-phage display library was constructed and 3 rounds of biopanning were performed using D-positive and D-negative red blood cells (RBCs). Positive phage clones were isolated, Sanger sequenced and, where possible, reformatted into full-length human IgGs to define specificity. The BFC aggregates from 2 anti-D donors underwent a Wright-Giemsa stain and hematological cell count. RESULTS: Of 10 BFCs, a sufficient yield of total RNA for library construction was obtained from BFCs containing cellular aggregates (n=5). Aggregate analysis showed lymphocytes were the cellular source of Ig-encoding RNA. From the 5 samples with aggregates, scFvs were assembled from amplified IgG variable regions. The library constructed from 1 of these samples resulted in the isolation of clones binding to D-positive RBCs with IGHV3 gene usage. Of the 4 reformatted IgG, 3 were anti-D and 1 had undefined specificity. DISCUSSION: BFC aggregates are a new and convenient source of Ig-encoding RNA which can be used to construct Ig gene libraries for mAb isolation and discovery via antibody phage display

Computer-based diabetes self-management interventions for adults with type 2 diabetes mellitus

BACKGROUND: Diabetes is one of the commonest chronic medical conditions, affecting around 347 million adults worldwide. Structured patient education programmes reduce the risk of diabetes-related complications four-fold. Internet-based self-management programmes have been shown to be effective for a number of long-term conditions, but it is unclear what are the essential or effective components of such programmes. If computer-based self-management interventions improve outcomes in type 2 diabetes, they could potentially provide a cost-effective option for reducing the burdens placed on patients and healthcare systems by this long-term condition. OBJECTIVES: To assess the effects on health status and health-related quality of life of computer-based diabetes self-management interventions for adults with type 2 diabetes mellitus. SEARCH METHODS: We searched six electronic bibliographic databases for published articles and conference proceedings and three online databases for theses (all up to November 2011). Reference lists of relevant reports and reviews were also screened. SELECTION CRITERIA: Randomised controlled trials of computer-based self-management interventions for adults with type 2 diabetes, i.e. computer-based software applications that respond to user input and aim to generate tailored content to improve one or more self-management domains through feedback, tailored advice, reinforcement and rewards, patient decision support, goal setting or reminders. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the abstracts and extracted data. A taxonomy for behaviour change techniques was used to describe the active ingredients of the intervention. MAIN RESULTS: We identified 16 randomised controlled trials with 3578 participants that fitted our inclusion criteria. These studies included a wide spectrum of interventions covering clinic-based brief interventions, Internet-based interventions that could be used from home and mobile phone-based interventions. The mean age of participants was between 46 to 67 years old and mean time since diagnosis was 6 to 13 years. The duration of the interventions varied between 1 to 12 months. There were three reported deaths out of 3578 participants.Computer-based diabetes self-management interventions currently have limited effectiveness. They appear to have small benefits on glycaemic control (pooled effect on glycosylated haemoglobin A1c (HbA1c): -2.3 mmol/mol or -0.2% (95% confidence interval (CI) -0.4 to -0.1; P = 0.009; 2637 participants; 11 trials). The effect size on HbA1c was larger in the mobile phone subgroup (subgroup analysis: mean difference in HbA1c -5.5 mmol/mol or -0.5% (95% CI -0.7 to -0.3); P < 0.00001; 280 participants; three trials). Current interventions do not show adequate evidence for improving depression, health-related quality of life or weight. Four (out of 10) interventions showed beneficial effects on lipid profile.One participant withdrew because of anxiety but there were no other documented adverse effects. Two studies provided limited cost-effectiveness data - with one study suggesting costs per patient of less than $140 (in 1997) or 105 EURO and another study showed no change in health behaviour and resource utilisation. AUTHORS' CONCLUSIONS: Computer-based diabetes self-management interventions to manage type 2 diabetes appear to have a small beneficial effect on blood glucose control and the effect was larger in the mobile phone subgroup. There is no evidence to show benefits in other biological outcomes or any cognitive, behavioural or emotional outcomes

Psychosocial aspects of closed- and open-loop insulin delivery: closing the loop in adults with Type 1 diabetes in the home setting.

AIMS: To explore the psychosocial experiences of closed-loop technology and to compare ratings of closed- and open-loop technology for adults with Type 1 diabetes taking part in a randomized crossover study. METHODS: Adults (aged > 18 years) on insulin pump therapy were recruited to receive a first phase of either real-time continuous glucose monitoring with overnight closed-loop or real-time continuous glucose monitoring alone (open-loop) followed by a second phase of the alternative treatment in random order, at home for 4 weeks, unsupervised. Participants were invited to share their views in semi-structured interviews. The impact of the closed-loop technology, positive and negative aspects of living with the device overnight, along with the hopes and anxieties of the participants, were explored. RESULTS: The participants in the trial were 24 adults with a mean (sd) age of 43 (12) years, of whom 54% were men. The mean (range) interview duration was 26 (12-46) min. Content and thematic analysis showed the following key positive themes: improved blood glucose control (n = 16); reassurance/reduced worry (n = 16); improved overnight control leading to improved daily functioning and diabetes control (n = 16); and improved sleep (n = 8). The key negative themes were: technical difficulties (n = 24); intrusiveness of alarms (n = 13); and size of equipment (n = 7). Of the 24 participant, 20 would recommend the closed-loop technology. CONCLUSIONS: Closed-loop therapy has positive effects when it works in freeing participants from the demands of self-management. The downside was technical difficulties, particularly concerning the pump and 'connectivity', which it is hoped will improve. Future research should continue to explore the acceptability of the closed-loop system as a realistic therapy option, taking account of user concerns as new systems are designed. Failure to do this may reduce the eventual utility of new systems.Diabetes UKThis is the accepted manuscript. The final version is available at http://dx.doi.org/10.1111/dme.12706

The pain experiences of powered wheelchair users

Copyright © 2012 Informa UK, Ltd. This is the author's accepted manuscript. The final published article is available from the link below.Purpose: To explore the experience of pain and discomfort in users of electric-powered indoor/outdoor wheelchairs (EPIOCs) provided by a National Health Service. Methods: EPIOC users receiving their chair between February and November 2002 (N=74) were invited to participate in a telephone questionnaire/interview and 64 (aged 1081 years) agreed. Both specific and open-ended questions examined the presence of pain/discomfort, its severity, minimizing and aggravating factors, particularly in relation to the EPIOC and its use. Results: Most EPIOC users described experiences of pain with 17% reporting severe pain. Over half felt their pain was influenced by the wheelchair and few (25%) considered their chair eased their symptoms. The most common strategy for pain relief was taking medication. Other self-help strategies included changing position, exercise and complementary therapies. Respondents emphasized the provision of backrests, armrests, footrests and cushions which might alleviate or exacerbate pain, highlighting the importance of appropriate assessment for this high dependency group. Conclusions: Users related pain to their underlying medical condition, their wheelchair or a combination of the two. User feedback is essential to ensure that the EPIOC meets health needs with minimal pain. This becomes more important as the health condition of users changes over time

Unsupervised home use of an overnight closed-loop system over 3-4 weeks: a pooled analysis of randomized controlled studies in adults and adolescents with type 1 diabetes.

AIMS: To compare overnight closed-loop and sensor-augmented pump therapy in patients with type 1 diabetes by combining data collected during free-living unsupervised randomized crossover home studies. METHODS: A total of 40 participants with type 1 diabetes, of whom 24 were adults [mean ± standard deviation (s.d.) age 43 ± 12 years and glycated haemoglobin (HbA1c) 8.0 ± 0.9%] and 16 were adolescents (mean ± s.d. age 15.6 ± 3.6 years and HbA1c 8.1 ± 0.8%), underwent two periods of sensor-augmented pump therapy in the home setting, in combination with or without an overnight closed-loop insulin delivery system that uses a model predictive control algorithm to direct insulin delivery. The order of the two interventions was random; each period lasted 4 weeks in adults and 3 weeks in adolescents. The primary outcome was time during which sensor glucose readings were in the target range of 3.9-8.0 mmol/l. RESULTS: The proportion of time when sensor glucose was in the target range (3.9-8.0 mmol/l) overnight (between 24:00 and 08:00 hours) was 18.5% greater during closed-loop insulin delivery than during sensor-augmented therapy (p < 0.001). Closed-loop therapy significantly reduced mean overnight glucose levels by 0.9 mmol/l (p < 0.001), with no difference in glycaemic variability, as measured by the standard deviation of sensor glucose. Time spent above the target range was reduced (p = 0.001), as was time spent in hypoglycaemia (<3.9 mmol/l; p = 0.014) during closed-loop therapy. Lower mean overnight glucose levels during closed-loop therapy were brought about by increased overnight insulin delivery (p < 0.001) without changes to the total daily delivery (p = 0.84). CONCLUSION: Overnight closed-loop insulin therapy at home in adults and adolescents with type 1 diabetes is feasible, showing improvements in glucose control and reducing the risk of nocturnal hypoglycaemia.Juvenile Diabetes Research Foundation (#22-2009-802) and Diabetes UK (BDA07/0003549) with additional support for the Artificial Pancreas work by National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK085621), and National Institute for Health Research Cambridge Biomedical Research Centre. Abbott Diabetes Care supplied continuous glucose delivery devices and sensors and modified devices to facilitate real-time connectivity.This if the final version of the article. It was originally published by Wiley in Diabetes, Obesity and Metabolism at http://onlinelibrary.wiley.com/doi/10.1111/dom.12427/abstrac

A high throughput screen for next-generation leads targeting malaria parasite transmission

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2–1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male–female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background: Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker. Methodology/Principal Findings: K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker. Conclusions/Significance: K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo

Adenovirus-Vectored Drug-Vaccine Duo as a Rapid-Response Tool for Conferring Seamless Protection against Influenza

Few other diseases exert such a huge toll of suffering as influenza. We report here that intranasal (i.n.) administration of E1/E3-defective (ΔE1E3) adenovirus serotype 5 (Ad5) particles rapidly induced an anti-influenza state as a means of prophylactic therapy which persisted for several weeks in mice. By encoding an influenza virus (IFV) hemagglutinin (HA) HA1 domain, an Ad5-HA1 vector conferred rapid protection as a prophylactic drug followed by elicitation of sustained protective immunity as a vaccine for inducing seamless protection against influenza as a drug-vaccine duo (DVD) in a single package. Since Ad5 particles induce a complex web of host responses, which could arrest influenza by activating a specific arm of innate immunity to impede IFV growth in the airway, it is conceivable that this multi-pronged influenza DVD may escape the fate of drug resistance that impairs the current influenza drugs

Autobiographical memory and hierarchical search strategies in depressed and non-depressed participants

Background: There is a growing body of literature showing individuals with depression and other trauma-related disorders (e.g., posttraumatic stress disorder) recall more overgeneral and less specific autobiographical memories compared to normal participants. Although the mechanisms underlying overgeneral memory are quite clear, the search strategy operated within the autobiographical knowledge base, at time of recollection, requires further exploration. The current study aimed to examine the hierarchical search sequence used to recall autobiographical memories in depressed and non-depressed participants, with a view to determining whether depressed participants exhibited truncated search strategies. Methods: Thirteen depressed and an equal number of non-depressed participants retrieved 15 memories each, in response to 15 commonly used cue words. Participants reported the first memory that entered in their mind. All memory descriptions were recorded and later transcribed verbatim for content analysis.Results: Depressed participants retrieved autobiographical memories faster, produced shorter memory descriptions and were less likely to recall positive memories than non-depressed participants. Non-depressed participants were more likely to commence retrieval by accessing lifetime period knowledge followed by general event and event specific knowledge, whereas depressed participants showed a tendency to terminate retrieval at the general event level. Conclusions: It is concluded that depressed participants do adhere to the same hierarchical search strategy as non-depressed participants when retrieving specific autobiographical memories, but that they terminate their search early, resulting in overgeneral memories