Economic Evaluation Plan (EEP) for A Very Early Rehabilitation Trial (AVERT): An international trial to compare the costs and cost-effectiveness of commencing out of bed standing and walking training (very early mobilization) within 24 h of stroke onset with usual stroke unit care

Rationale: A key objective of A Very Early Rehabilitation Trial is to determine if the intervention, very early mobilisation following stroke, is cost-effective. Resource use data were collected to enable an economic evaluation to be undertaken and a plan for the main economic analyses was written prior to the completion of follow up data collection. Aim and hypothesis To report methods used to collect resource use data, pre-specify the main economic evaluation analyses and report other intended exploratory analyses of resource use data. Sample size estimates: Recruitment to the trial has been completed. A total of 2,104 participants from 56 stroke units across three geographic regions participated in the trial. Methods and design: Resource use data were collected prospectively alongside the trial using standardised tools. The primary economic evaluation method is a cost-effectiveness analysis to compare resource use over 12 months with health outcomes of the intervention measured against a usual care comparator. A cost-utility analysis is also intended. Study outcome: The primary outcome in the cost-effectiveness analysis will be favourable outcome (modified Rankin Scale score 0-2) at 12 months. Cost-utility analysis will use health-related quality of life, reported as quality-adjusted life years gained over a 12 month period, as measured by the modified Rankin Scale and the Assessment of Quality of Life. Discussion: Outcomes of the economic evaluation analysis will inform the cost-effectiveness of very early mobilisation following stroke when compared to usual care. The exploratory analysis will report patterns of resource use in the first year following stroke

RTKN2 induces NF-KappaB dependent resistance to intrinsic apoptosis in HEK cells and REgulates BCL-2 genes in human CD4+ lymphocytes

The gene for Rhotekin 2 (RTKN2) was originally identified in a promyelocytic cell line resistant to oxysterol-induced apoptosis. It is differentially expressed in freshly isolated CD4+ T-cells compared with other hematopoietic cells and is down-regulated following activation of the T-cell receptor. However, very little is known about the function of RTKN2 other than its homology to Rho-GTPase effector, rhotekin, and the possibility that they may have similar roles. Here we show that stable expression of RTKN2 in HEK cells enhanced survival in response to intrinsic apoptotic agents; 25-hydroxy cholesterol and camptothecin, but not the extrinsic agent, TNF&alpha;. Inhibitors of NF-KappaB, but not MAPK, reversed the resistance and mitochondrial pro-apoptotic genes, Bax and Bim, were down regulated. In these cells, there was no evidence of RTKN2 binding to the GTPases, RhoA or Rac2. Consistent with the role of RTKN2 in HEK over-expressing cells, suppression of RTKN2 in primary human CD4+ T-cells reduced viability and increased sensitivity to 25-OHC. The expression of the pro-apoptotic genes, Bax and Bim were increased while BCL-2 was decreased. In both cell models RTKN2 played a role in the process of intrinsic apoptosis and this was dependent on either NF-KappaB signaling or expression of downstream BCL-2 genes. As RTKN2 is a highly expressed in CD4+ T-cells it may play a role as a key signaling switch for regulation of genes involved in T-cell survival.<br /

Enhanced immunogenicity of a functional enzyme by T cell epitope modification

BACKGROUND: T helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines. RESULTS: Hartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone. CONCLUSIONS: With a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzyme's original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics

AVERT2(a very early rehabilitation trial, a very effective reproductive trigger): retrospective observational analysis of the number of babies born to trial staff

Objective: To report the number of participants needed to recruit per baby born to trial staff during AVERT, a large international trial on acute stroke, and to describe trial management consequences. Design: Retrospective observational analysis. Setting: 56 acute stroke hospitals in eight countries. Participants: 1074 trial physiotherapists, nurses, and other clinicians. Outcome measures: Number of babies born during trial recruitment per trial participant recruited. Results: With 198 site recruitment years and 2104 patients recruited during AVERT, 120 babies were born to trial staff. Births led to an estimated 10% loss in time to achieve recruitment. Parental leave was linked to six trial site closures. The number of participants needed to recruit per baby born was 17.5 (95% confidence interval 14.7 to 21.0); additional trial costs associated with each birth were estimated at 5736 Australian dollars on average. Conclusion: The staff absences registered in AVERT owing to parental leave led to delayed trial recruitment and increased costs, and should be considered by trial investigators when planning research and estimating budgets. However, the celebration of new life became a highlight of the annual AVERT collaborators’ meetings and helped maintain a cohesive collaborative group

Metagenomics detection and characterisation of viruses in faecal samples from Australian wild birds

We present an optimised metagenomics method for detection and characterisation of all virus types including single and double stranded DNA/RNA and enveloped and non-enveloped viruses. Initial evaluation included both spiked and non-spiked bird faecal samples as well as non-spiked human faecal samples. From the non-spiked bird samples (Australian Muscovy duck and Pacific black ducks) we detected 21 viruses, and we also present a summary of a few viruses detected in human faecal samples. We then present a detailed analysis of selected virus sequences in the avian samples that were somewhat similar to known viruses, and had good quality (Q20 or higher) and quantity of next-generation sequencing reads, and was of interest from a virological point of view, for example, avian coronavirus and avian paramyxovirus 6. Some of these viruses were closely related to known viruses while others were more distantly related with 70% or less identity to currently known/sequenced viruses. Besides detecting viruses, the technique also allowed the characterisation of host mitochondrial DNA present and thus identifying host species, while ribosomal RNA sequences provided insight into the &quot;ribosomal activity microbiome&quot;; of gut parasites; and of food eaten such as plants or insects, which we correlated to non-avian host associated viruses

The ontogeny of naïve and regulatory CD4(+) T-cell subsets during the first postnatal year: a cohort study

As there is limited knowledge regarding the longitudinal development and early ontogeny of na&iuml;ve and regulatory CD4(+) T-cell subsets during the first postnatal year, we sought to evaluate the changes in proportion of na&iuml;ve (thymic and central) and regulatory (resting and activated) CD4(+) T-cell populations during the first postnatal year. Blood samples were collected and analyzed at birth, 6 and 12 months of age from a population-derived sample of 130 infants. The proportion of na&iuml;ve and regulatory CD4(+) T-cell populations was determined by flow cytometry, and the thymic and central na&iuml;ve populations were sorted and their phenotype confirmed by relative expression of T cell-receptor excision circle DNA (TREC). At birth, the majority (94%) of CD4(+) T cells were na&iuml;ve (CD45RA(+)), and of these, ~80% had a thymic na&iuml;ve phenotype (CD31(+) and high TREC), with the remainder already central na&iuml;ve cells (CD31(-) and low TREC). During the first year of life, the na&iuml;ve CD4(+) T cells retained an overall thymic phenotype but decreased steadily. From birth to 6 months of age, the proportion of both resting na&iuml;ve T regulatory cells (rTreg; CD4(+)CD45RA(+)FoxP3(+)) and activated Treg (aTreg, CD4(+)CD45RA(-)FoxP3(high)) increased markedly. The ratio of thymic to central na&iuml;ve CD4(+) T cells was lower in males throughout the first postnatal year indicating early sexual dimorphism in immune development. This longitudinal study defines proportions of CD4(+) T-cell populations during the first year of postnatal life that provide a better understanding of normal immune development

Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood

Comparison of serum and urinary biomarker panels with albumin/creatinine ratio in the prediction of renal function decline in type 1 diabetes

Abstract: Aims/hypothesis: We examined whether candidate biomarkers in serum or urine can improve the prediction of renal disease progression in type 1 diabetes beyond prior eGFR, comparing their performance with urinary albumin/creatinine ratio (ACR). Methods: From the population-representative Scottish Diabetes Research Network Type 1 Bioresource (SDRNT1BIO) we sampled 50% and 25% of those with starting eGFR below and above 75 ml min−1 [1.73 m]−2, respectively (N = 1629), and with median 5.1 years of follow-up. Multiplexed ELISAs and single molecule array technology were used to measure nine serum biomarkers and 13 urine biomarkers based on our and others’ prior work using large discovery and candidate studies. Associations with final eGFR and with progression to <30 ml min−1 [1.73] m−2, both adjusted for baseline eGFR, were tested using linear and logistic regression models. Parsimonious biomarker panels were identified using a penalised Bayesian approach, and their performance was evaluated through tenfold cross-validation and compared with using urinary ACR and other clinical record data. Results: Seven serum and seven urine biomarkers were strongly associated with either final eGFR or progression to <30 ml min−1 [1.73 m]−2, adjusting for baseline eGFR and other covariates (all at p<2.3 × 10−3). Of these, associations of four serum biomarkers were independent of ACR for both outcomes. The strongest associations with both final eGFR and progression to <30 ml min−1 [1.73 m]−2 were for serum TNF receptor 1, kidney injury molecule 1, CD27 antigen, α-1-microglobulin and syndecan-1. These serum associations were also significant in normoalbuminuric participants for both outcomes. On top of baseline covariates, the r2 for prediction of final eGFR increased from 0.702 to 0.743 for serum biomarkers, and from 0.702 to 0.721 for ACR alone. The area under the receiver operating characteristic curve for progression to <30 ml min−1 [1.73 m]−2 increased from 0.876 to 0.953 for serum biomarkers, and to 0.911 for ACR alone. Other urinary biomarkers did not outperform ACR. Conclusions/interpretation: A parsimonious panel of serum biomarkers easily measurable along with serum creatinine may outperform ACR for predicting renal disease progression in type 1 diabetes, potentially obviating the need for urine testing

Treatment of Hidradenitis Suppurativa Evaluation Study (THESEUS) : protocol for a prospective cohort study

Background: Hidradenitis suppurativa (HS) is a chronic, painful, inflammatory skin disease with estimates of prevalence in the European population of 1%–2%. Despite being a relatively common condition, the evidence base for management of HS is limited. European and North American management guidelines rely on consensus for many aspects of treatment and within the UK variations in management of HS have been identified. The HS James Lind Alliance Priority Setting Partnership (PSP) published a top 10 list of future HS research priorities including both medical and surgical interventions. The aims of the THESEUS study are to inform the design of future HS randomised controlled trials (RCTs) and to understand how HS treatments are currently used. THESEUS incorporates several HS PSP research priorities, including investigation of oral and surgical treatments. Core outcome domains have been established by the HIdradenitis SuppuraTiva cORe outcomes set International Collaboration (HISTORIC) and THESEUS is designed to validate instruments to measure the domains. Methods and analysis: The THESEUS study is a prospective observational cohort study. Participants, adults with active HS of any severity, will be asked to select one of five HS treatment options that is appropriate for their HS care. Participants will be allocated to their chosen treatment intervention and followed for a period of up to 12 months. Outcomes will be assessed at 3-monthly intervals using HISTORIC core outcome instruments. Video recordings of the surgical and laser operations will provide informational and training videos for future trials. Nested mixed-methods studies will characterise the interventions in clinical practice, understand facilitators and barriers to recruitment into future HS RCTs and examine patients’ and clinicians’ perspectives on HS treatment choices. Trial registration number: ISRCTN69985145