Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice.

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgamma null ) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1ß, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

When is a randomised controlled trial health equity relevant? Development and validation of a conceptual framework.

BACKGROUND: Randomised controlled trials can provide evidence relevant to assessing the equity impact of an intervention, but such information is often poorly reported. We describe a conceptual framework to identify health equity-relevant randomised trials with the aim of improving the design and reporting of such trials. METHODS: An interdisciplinary and international research team engaged in an iterative consensus building process to develop and refine the conceptual framework via face-to-face meetings, teleconferences and email correspondence, including findings from a validation exercise whereby two independent reviewers used the emerging framework to classify a sample of randomised trials. RESULTS: A randomised trial can usefully be classified as 'health equity relevant' if it assesses the effects of an intervention on the health or its determinants of either individuals or a population who experience ill health due to disadvantage defined across one or more social determinants of health. Health equity-relevant randomised trials can either exclusively focus on a single population or collect data potentially useful for assessing differential effects of the intervention across multiple populations experiencing different levels or types of social disadvantage. Trials that are not classified as 'health equity relevant' may nevertheless provide information that is indirectly relevant to assessing equity impact, including information about individual level variation unrelated to social disadvantage and potentially useful in secondary modelling studies. CONCLUSION: The conceptual framework may be used to design and report randomised trials. The framework could also be used for other study designs to contribute to the evidence base for improved health equity.

Cyclin-dependent kinase 7 is a therapeutic target in high-grade glioma.

High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase-quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.

Reversal of warfarin anticoagulation using prothrombin complex concentrate at 25 IU kg-1 : results of the RAPID study.

BACKGROUND: Real-world studies of the emergency reversal of warfarin using 4-factor prothrombin complex concentrate (PCC) report unwarranted delays. The delay to receiving PCC was ≥ 8 h in 46·7% of patients with warfarin-associated bleeding (PWAB) treated with a variable PCC dosing protocol in our retrospective audit. OBJECTIVE: To report the impact of a simplified PCC dosing protocol on the interval to reversal of anticoagulation. METHODS: We developed a PCC dosing protocol standardising the initial PCC dose and simplifying dosing calculations. Study end points were the proportion of PWAB achieving international normalised ratio (INR) ≤1·5 and treated within 8 h of presentation, respectively. RESULTS: Of 17, 15 (88·2%) PWABs achieved a post-treatment INR ≤ 1·5; 14 of 17 (82·4%) PWABs were reversed within 8 h. Median intervals between triage and PCC request and PCC request and start of infusion (administration interval) were 126 min (range 39-520) and 30 min (range 5-100), respectively. Compared with the retrospective cohort, RAPID is associated with an improved administration interval (mean 37·7 vs 76 min, P = 0·031) and the proportion of PWABs treated within 30 min (58·8 vs 6·7%, P = 0·009). CONCLUSION: The RAPID protocol reduces unwarranted delays without compromising efficacy.

Protocol for the development of a CONSORT-equity guideline to improve reporting of health equity in randomized trials.

BACKGROUND: Health equity concerns the absence of avoidable and unfair differences in health. Randomized controlled trials (RCTs) can provide evidence about the impact of an intervention on health equity for specific disadvantaged populations or in general populations; this is important for equity-focused decision-making. Previous work has identified a lack of adequate reporting guidelines for assessing health equity in RCTs. The objective of this study is to develop guidelines to improve the reporting of health equity considerations in RCTs, as an extension of the Consolidated Standards of Reporting Trials (CONSORT). METHODS/DESIGN: A six-phase study using integrated knowledge translation governed by a study executive and advisory board will assemble empirical evidence to inform the CONSORT-equity extension. To create the guideline, the following steps are proposed: (1) develop a conceptual framework for identifying "equity-relevant trials," (2) assess empirical evidence regarding reporting of equity-relevant trials, (3) consult with global methods and content experts on how to improve reporting of health equity in RCTs, (4) collect broad feedback and prioritize items needed to improve reporting of health equity in RCTs, (5) establish consensus on the CONSORT-equity extension: the guideline for equity-relevant trials, and (6) broadly disseminate and implement the CONSORT-equity extension. DISCUSSION: This work will be relevant to a broad range of RCTs addressing questions of effectiveness for strategies to improve practice and policy in the areas of social determinants of health, clinical care, health systems, public health, and international development, where health and/or access to health care is a primary outcome. The outcomes include a reporting guideline (CONSORT-equity extension) for equity-relevant RCTs and a knowledge translation strategy to broadly encourage its uptake and use by journal editors, authors, and funding agencies.

Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

Hydrogen peroxide: a Jekyll and Hyde signalling molecule.

Reactive oxygen species (ROS) are a group of molecules produced in the cell through metabolism of oxygen. Endogenous ROS such as hydrogen peroxide (H2O2) have long been recognised as destructive molecules. The well-established roles they have in the phagosome and genomic instability has led to the characterisation of these molecules as non-specific agents of destruction. Interestingly, there is a growing body of literature suggesting a less sinister role for this Jekyll and Hyde molecule. It is now evident that at lower physiological levels, H2O2 can act as a classical intracellular signalling molecule regulating kinase-driven pathways. The newly discovered biological functions attributed to ROS include proliferation, migration, anoikis, survival and autophagy. Furthermore, recent advances in detection and quantification of ROS-family members have revealed that the diverse functions of ROS can be determined by the subcellular source, location and duration of these molecules within the cell. In light of this confounding paradox, we will examine the factors and circumstances that determine whether H2O2 acts in a pro-survival or deleterious manner.

School-based secondary prevention programmes for preventing violence.

BACKGROUND: Early aggressive behaviour is a risk factor for later violence and criminal behaviour. Despite over 20 years of violence prevention interventions being delivered in the school setting, questions remain regarding the effectiveness of different interventions for children exhibiting aggressive behaviour. OBJECTIVES: To examine the effect of school based violence prevention programmes for children identified as aggressive or at risk of being aggressive. SEARCH STRATEGY: We searched CENTRAL, Cochrane Injuries Group specialised register, MEDLINE, EMBASE, other specialised databases and reference lists of articles. We also contacted authors and organisations to identify any further studies. SELECTION CRITERIA: We included trials meeting the following criteria; 1) participants were randomly assigned to intervention and control groups; 2) outcome data were collected concurrently; 3) participants comprised children in mandatory education identified as exhibiting, or at risk of, aggressive behaviour; 4) interventions designed to reduce aggression, violence, bullying, conflict or anger; 5) school based interventions; 6) outcomes included aggressive behaviour, school and agency responses to acts of aggression, or violent injuries. DATA COLLECTION AND ANALYSIS: Data were collected on design, participants, interventions, outcomes and indicators of study quality. Results of any intervention to no intervention were compared immediately post-intervention and at 12 months using meta-analysis where appropriate. MAIN RESULTS: Of 56 trials identified, none reported data on violent injuries. Aggressive behaviour was significantly reduced in intervention groups compared to no intervention groups immediately post intervention in 34 trials with data, (Standardised Mean Difference (SMD) = -0.41; 95% confidence interval (CI) -0.56 to -0.26). This effect was maintained in the seven studies reporting 12 month follow-up (SMD = -0.40, (95% CI -0.73 to -0.06)). School or agency disciplinary actions in response to aggressive behaviour were reduced in intervention groups for nine trials with data, SMD = -0.48; 95% CI -1.16 to 0.19, although this difference may have been due to chance and was not maintained, based on two studies reporting follow-up to two to four months (SMD = 0.03; 95% CI -0.42 to 0.47). Subgroup analyses suggested that interventions designed to improve relationship or social skills may be more effective than interventions designed to teach skills of non-response to provocative situations, but that benefits were similar when delivered to children in primary versus secondary school, and to groups of mixed sex versus boys alone. AUTHORS' CONCLUSIONS: School-based secondary prevention programmes to reduce aggressive behaviour appear to produce improvements in behaviour greater than would have been expected by chance. Benefits can be achieved in both primary and secondary school age groups and in both mixed sex groups and boys-only groups. Further research is required to establish whether such programmes reduce the incidence of violent injuries or if the benefits identified can be maintained beyond 12 months.

Does diagnostic delay lead invariably to damage in episodic pelvi-ureteric junction obstruction?

OBJECTIVE: To determine the relationship between the length of history and renal function in patients with symptomatic pelvi-ureteric junction (PUJ) obstruction in childhood. PATIENTS AND METHODS: The study included 41 children with symptomatic PUJ obstruction. The duration and details of their symptoms were recorded and the differential renal function estimated by isotopic renography. RESULTS: The median (range) duration of symptoms was 24 (0.25-84) months; there was no statistically significant relationship between the duration of symptoms and differential function at presentation (P=0.148). CONCLUSION: There is no correlation between the duration of symptoms and renal function in children with PUJ obstruction. A delay in diagnosis cannot be directly linked to permanent renal compromise.

Elevated anticardiolipin antibodies in acute liver failure.

Antibodies to cardiolipin (aCLA), a phospholipid primarily localized in inner mitochondrial membranes, were transiently elevated (P<0.01) when mice were exposed to an industrial surfactant and then infected with influenza B virus, a model of acute liver failure (ALF). Children with ALF also had elevated levels of aCLA.

Critical role of the transcription factor AP-1 for the constitutive and interferon-induced expression of IFI 16.

IFI 16 is a member of the HIN-200 family of transcriptional regulators that suppress cell growth, modulate the cell cycle and have been linked to cellular differentiation. We hypothesized that the activity of IFI 16 depends on its level of expression and therefore studied the transcriptional activity of the IFI 16 promoter. A discrete sequence within the 5' untranslated region was required for constitutive activity of the promoter and the functional motif within this region was shown to be a consensus AP-1 site. Interestingly, this AP-1 site was also critical for IFN-induced activation of the promoter and consistent with these observations, treatment of cells with IFNgamma resulted in a rapid and robust induction of AP-1 activity that preceded expression of IFI 16. These experiments define the transcriptional mechanisms of IFI 16 gene regulation and provide evidence suggesting that AP-1 activation may be an important event in IFN signaling.

The use of renal scintigraphy in assessing the potential for recovery in the obstructed renal tract in children.

OBJECTIVE: To assess the value of renal scintigraphy with 99mTc-dimercaptosuccinic acid (DMSA) in predicting functional recovery after the surgical relief of obstructed kidneys in children. Patients and methods Forty-three children underwent surgery to relieve upper urinary tract obstruction; 37 had pelvi-ureteric junction obstruction and six had vesico-ureteric junction obstruction. The indication for surgery was a combination of an obstructed renogram with symptoms of either pain or pyelonephritis. Most children (41) had < 40% function on the affected side before surgery, with just two having hyperfunction (> 55%). In all patients intravenous urography before surgery showed hydronephrosis, and a micturating cystogram was used to exclude coexisting reflux in the presence of an associated megaureter. Diuretic renography (using 99mTc-mercaptoacetyl triglycine or 123I-hippuran) and DMSA scintigraphy were both carried out before surgery and the renography repeated 6 months afterward. RESULTS: The renographic drainage curves improved from obstructed to unobstructed or 'dilated unobstructed' on all postoperative studies. Regression analysis showed that preoperative DMSA scan was an excellent predictor of outcome (P < 0.001) whilst the preoperative renogram was a relatively poor predictor of the functional result. In four patients where the initial renographic function was < 10%, DMSA scintigraphy predicted correctly the capacity for recovery in three and the inability to improve in the fourth. Conclusion Before surgery, DMSA scintigraphy in children with upper urinary tract obstruction is a more useful estimate of probable long-term renal function than value from diuresis renography. If there is doubt about the desirability of reconstructive surgery, a DMSA scan may eliminate the need for more invasive methods of estimating recovery, e.g. a period of nephrostomy drainage.

Predicting protein--protein interactions from primary structure.

MOTIVATION: An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. The expectation is that this will provide a fuller appreciation of cellular processes and networks at the protein level, ultimately leading to a better understanding of disease mechanisms and suggesting new means for intervention. This paper addresses the question: can protein-protein interactions be predicted directly from primary structure and associated data? Using a diverse database of known protein interactions, a Support Vector Machine (SVM) learning system was trained to recognize and predict interactions based solely on primary structure and associated physicochemical properties. RESULTS: Inductive accuracy of the trained system, defined here as the percentage of correct protein interaction predictions for previously unseen test sets, averaged 80% for the ensemble of statistical experiments. Future proteomics studies may benefit from this research by proceeding directly from the automated identification of a cell's gene products to prediction of protein interaction pairs.

Enterocystoplasty.

Despite the problems of augmentation cystoplasty, on balance it has been a much better form of management of the lower urinary tract in patients with bladder neuropathy or high pressure detrusor contractions than the alternatives of rectal diversion, indwelling catheter or external urinary diversion. The metabolic consequences do not seem to interfere with general health in the medium term. The risk of perforation appears to be present with other forms of augmentation cystoplasty or bladder replacement. However, the results are far from perfect and the ideal technique will be one that: removes the need for intraperitoneal surgery and prevents the risk of intestinal adhesions; stops the development of intestinal mucus and stone formation; prevents the metabolic complications and potential bony complications during adolescence; at the same time improves the patient's resistance to UTI; maintains the same degree of long-term, good, low-pressure urine storage and the consequent improvement and stability of the upper urinary tract.

Benchmark data from the literature for evaluation of new glucose sensing technologies.

New glucose sensors based on various technologies are being developed to provide information for improved therapy in diabetes. There is a need to establish rational performance standards for these sensors. Frequently sampled, direct blood glucose recordings representative of blood glucose excursions in diabetes are the "gold standard." An extensive literature search revealed a limited number of diabetic and nondiabetic blood glucose recordings suitable for this purpose. Certain blood glucose recordings reflect the diversity of glycemic dynamics and provide sufficient challenge for evaluation of sensor systems. These recordings were converted into an accessible electronic format. An example is given of the use of these benchmark data to estimate aliasing error, or the error due to insufficient sampling frequency, based on a hypothetical sensor system having some properties of conventional "fingerstick" systems. Discrete sampling systems accumulate substantial aliasing error as the sampling period increases.

Paediatric anatrophic nephrolithotomy; stone clearance - at what price?

OBJECTIVE: To evaluate the functional outcome of anatrophic nephrolithotomy in children. PATIENTS AND METHODS: All children undergoing anatrophic nephrolithotomy for complex branching and multiple renal calculi over an 11-year period were studied prospectively. Demographic data, treatment details and outcome, as assessed by X-ray, ultrasonography and isotope studies, were recorded. Anatrophic nephrolithotomy was carried out with surface cooling of the kidney followed by nephrostomy drainage for 5-7 days. RESULTS: Nine children (median age 4 years, range 7 months to 9 years) underwent anatrophic nephrolithotomy. Predisposing factors included urinary tract infection (by Proteus mirabilis) in all and hyper-calciuria in two children. The median (range) total ischaemic time at operation was 25 (15-40) min and the operative duration 150 (120-200) min. Three children required a blood transfusion. Stone clearance was incomplete in one child. There was no recurrent stone formation after a long-term follow-up (median 32 months, range 14-107) in the other patients. Isotope studies showed impaired split renal function (<40%) in six children before surgery; there was a significant decline (>5%) in divided function in five children (range 6-16%) after surgery. CONCLUSION: Anatrophic nephrolithotomy is an effective means of rendering children with branching calculi stone-free, but this study suggests that it leads to some further parenchymal damage.