Non-contact vital-sign monitoring of patients undergoing haemodialysis treatment.

A clinical study was designed to record a wide range of physiological values from patients undergoing haemodialysis treatment in the Renal Unit of the Churchill Hospital in Oxford. Video was recorded for a total of 84 dialysis sessions from 40 patients during the course of 1 year, comprising an overall video recording time of approximately 304.1 h. Reference values were provided by two devices in regular clinical use. The mean absolute error between the heart rate estimates from the camera and the average from two reference pulse oximeters (positioned at the finger and earlobe) was 2.8 beats/min for over 65% of the time the patient was stable. The mean absolute error between the respiratory rate estimates from the camera and the reference values (computed from the Electrocardiogram and a thoracic expansion sensor-chest belt) was 2.1 breaths/min for over 69% of the time for which the reference signals were valid. To increase the robustness of the algorithms, novel methods were devised for cancelling out aliased frequency components caused by the artificial light sources in the hospital, using auto-regressive modelling and pole cancellation. Maps of the spatial distribution of heart rate and respiratory rate information were developed from the coefficients of the auto-regressive models. Most of the periods for which the camera could not produce a reliable heart rate estimate lasted under 3 min, thus opening the possibility to monitor heart rate continuously in a clinical environment.

Combining multiple spatial statistics enhances the description of immune cell localisation within tumours.

Digital pathology enables computational analysis algorithms to be applied at scale to histological images. An example is the identification of immune cells within solid tumours. Image analysis algorithms can extract precise cell locations from immunohistochemistry slides, but the resulting spatial coordinates, or point patterns, can be difficult to interpret. Since localisation of immune cells within tumours may reflect their functional status and correlates with patient prognosis, novel descriptors of their spatial distributions are of biological and clinical interest. A range of spatial statistics have been used to analyse such point patterns but, individually, these approaches only partially describe complex immune cell distributions. In this study, we apply three spatial statistics to locations of CD68+ macrophages within human head and neck tumours, and show that images grouped semi-quantitatively by a pathologist share similar statistics. We generate a synthetic dataset which emulates human samples and use it to demonstrate that combining multiple spatial statistics with a maximum likelihood approach better predicts human classifications than any single statistic. We can also estimate the error associated with our classifications. Importantly, this methodology is adaptable and can be extended to other histological investigations or applied to point patterns outside of histology.

Reduction in intestinal colonization and invasion of internal organs after challenge by homologous and heterologous serovars of Salmonella enterica following vaccination of chickens with a novel trivalent inactivated Salmonella vaccine.

A novel inactivated vaccine, comprising three serovars of Salmonella enterica (Enteritidis, serogroup O:9; Typhimurium, serogroup O:4; Infantis, serogroup O:7) grown under conditions of iron restriction and adjuvanted with aluminium hydroxide, was evaluated for efficacy following challenge by homologous and heterologous serovars. Chickens were vaccinated at 6 and 10 weeks of age by the intramuscular route and challenged 4 to 9 weeks after the second vaccination with serovars belonging to serogroup O:9 (Enteritidis), O:4 (Typhimurium and Heidelberg), O:7 (Infantis and Virchow), and O:8 (Hadar). All vaccinated birds produced a marked systemic antibody response against each of the component vaccine antigens by the time of challenge. Significant reductions in both colonization of the intestinal tract and invasion of internal organs were observed in vaccinated birds compared with non-vaccinated controls, irrespective of the challenge serovar. The findings suggest that broad serovar protection within the constitutive serogroups of an inactivated multi-valent vaccine is possible and could, therefore, play an important role in future Salmonella control programmes. RESEARCH HIGHLIGHTS Novel inactivated trivalent Salmonella chicken vaccine was developed and tested. Vaccine induced marked systemic antibody response against all vaccine antigens. Significant reductions in intestinal tract colonization and internal organ invasion. Vaccine efficacy demonstrated against homologous and heterologous serovars.

Safety and efficacy of a novel inactivated trivalent Salmonella enterica vaccine in chickens.

Food poisoning in humans caused by Salmonella enterica remains a significant global public health concern, with the majority of infections associated with the consumption of contaminated eggs or poultry products. The safety and efficacy of a novel inactivated trivalent Salmonella enterica vaccine containing in addition to Salmonella serovars Enteritidis (O:9, serogroup D) and Typhimurium (O:4, serogroup B) also serovar Infantis (O:7, serogroup C1) formulated with an aluminium hydroxide-gel adjuvant was evaluated under field conditions. A total of 10,229 broiler breeder pullets, housed under commercial conditions, were vaccinated at 10 and 17 weeks of age by the intramuscular route in the breast muscle. The vaccine was safe with no local or systemic reactions or adverse effects on bird performance related to the vaccine detected. Vaccination resulted in notable increases in serovar specific antibodies that were maintained until at least 56 weeks of age. Vaccinated birds subjected to homologous challenges around onset of lay showed significantly reduced faecal shedding and organ invasion. Following heterologous challenge with S. Hadar (O:8, serogroup C2) faecal shedding was significantly reduced. These results demonstrate that this novel vaccine could play a significant role in a comprehensive Salmonella control programme intended to reduce both the incidence of food poisoning in humans and the use of antibiotics during poultry production.

Heart Rate Variability as an Indicator of Autonomic Nervous System Disturbance in Tetanus.

Autonomic nervous system dysfunction (ANSD) is a significant cause of mortality in tetanus. Currently, diagnosis relies on nonspecific clinical signs. Heart rate variability (HRV) may indicate underlying autonomic nervous system activity and represents a potentially valuable noninvasive tool for ANSD diagnosis in tetanus. HRV was measured from three 5-minute electrocardiogram recordings during a 24-hour period in a cohort of patients with severe tetanus, all receiving mechanical ventilation. HRV measurements from all subjects-five with ANSD (Ablett Grade 4) and four patients without ANSD (Ablett Grade 3)-showed HRV was lower than reported ranges for healthy individuals. Comparing different severities of tetanus, raw data for both time and frequency measurements of HRV were reduced in those with ANSD compared with those without. Differences were statistically significant in all except root mean square SD, indicating HRV may be a valuable tool in ANSD diagnosis.

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.

The clinical academic workforce of the future: a cross-sectional study of factors influencing career decision-making among clinical PhD students at two research-intensive UK universities.

OBJECTIVES: To examine clinical doctoral students' demographic and training characteristics, career intentions, career preparedness and what influences them as they plan their future careers. DESIGN AND SETTING: Online cross-sectional census surveys at two research-intensive medical schools in England in 2015-2016. PARTICIPANTS: All medically qualified PhD students (N=523) enrolled at the University of Oxford and University College London were invited to participate. We report on data from 320 participants (54% male and 44% female), who were representative by gender of the invited population. MAIN OUTCOME MEASURES: Career intentions. RESULTS: Respondents were mainly in specialty training, including close to training completion (25%, n=80), and 18% (n=57) had completed training. Half (50%, n=159) intended to pursue a clinical academic career (CAC) and 62% (n=198) were at least moderately likely to seek a clinical lectureship (CL). However, 51% (n=163) had little or no knowledge about CL posts. Those wanting a CAC tended to have the most predoctoral medical research experience (χ2 (2, N=305)=22.19, p=0.0005). Key reasons cited for not pursuing a CAC were the small number of senior academic appointments available, the difficulty of obtaining research grants and work-life balance. CONCLUSIONS: Findings suggest that urging predoctoral clinicians to gain varied research experience while ensuring availability of opportunities, and introducing more flexible recruitment criteria for CL appointments, would foster CACs. As CL posts are often only open to those still in training, the many postdoctoral clinicians who have completed training, or nearly done so, do not currently gain the opportunity the post offers to develop as independent researchers. Better opportunities should be accompanied by enhanced career support for clinical doctoral students (eg, to increase knowledge of CLs). Finally, ways to increase the number of senior clinical academic appointments should be explored since their lack seems to significantly influence career decisions.

Prolyl hydroxylase 2 inactivation enhances glycogen storage and promotes excessive neutrophilic responses.

Fully activated innate immune cells are required for effective responses to infection, but their prompt deactivation and removal are essential for limiting tissue damage. Here, we have identified a critical role for the prolyl hydroxylase enzyme Phd2 in maintaining the balance between appropriate, predominantly neutrophil-mediated pathogen clearance and resolution of the innate immune response. We demonstrate that myeloid-specific loss of Phd2 resulted in an exaggerated inflammatory response to Streptococcus pneumonia, with increases in neutrophil motility, functional capacity, and survival. These enhanced neutrophil responses were dependent upon increases in glycolytic flux and glycogen stores. Systemic administration of a HIF-prolyl hydroxylase inhibitor replicated the Phd2-deficient phenotype of delayed inflammation resolution. Together, these data identify Phd2 as the dominant HIF-hydroxylase in neutrophils under normoxic conditions and link intrinsic regulation of glycolysis and glycogen stores to the resolution of neutrophil-mediated inflammatory responses. These results demonstrate the therapeutic potential of targeting metabolic pathways in the treatment of inflammatory disease.

The Oxford Renal (OxRen) cross-sectional study of chronic kidney disease in the UK.

INTRODUCTION: Chronic kidney disease (CKD) diagnosed with objective measures of kidney damage and function has been recognised as a major public health burden. Independent of age, sex, ethnicity and comorbidity, strong associations exist between cardiovascular disease, mortality, morbidity and CKD, defined by reduced glomerular filtration rate and increased urinary albumin excretion. Detection of CKD within the population is therefore a priority for health systems. METHODS AND ANALYSIS: 15 000 patients aged 60 years or over meeting the inclusion criteria will be invited to the study. Recruitment will be stratified to represent the distribution of socioeconomic position in the UK general population. Patients will be excluded if terminally ill (expected survival <1 year), or if they have received a solid organ transplant. Patients will attend up to two screening visits, to determine if they have CKD, followed by an assessment visit where demographic and physiological parameters will be recorded alongside questionnaires on exercise, diet, cognitive assessment and quality of life. Blood and urine specimens will be taken for immediate routine assays as well as for freezing pending peptide and genetic studies. Patients will have office and home blood pressure measurements as well as pulse wave velocity assessment. Healthcare costs of screening and subsequent monitoring will be calculated. ETHICS AND DISSEMINATION: The protocol and related documents have been approved by NRES Committee South Central-Oxford B-Reference 13/SC/0020.

Detection of BK virus in urine from renal transplant subjects by mass spectrometry.

BACKGROUND: The diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN. RESULTS: Here we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN. CONCLUSIONS: This is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.

Common genetic variants at the 11q13.3 renal cancer susceptibility locus influence binding of HIF to an enhancer of cyclin D1 expression.

Although genome-wide association studies (GWAS) have identified the existence of numerous population-based cancer susceptibility loci, mechanistic insights remain limited, particularly for intergenic polymorphisms. Here, we show that polymorphism at a remote intergenic region on chromosome 11q13.3, recently identified as a susceptibility locus for renal cell carcinoma, modulates the binding and function of hypoxia-inducible factor (HIF) at a previously unrecognized transcriptional enhancer of CCND1 (encoding cyclin D1) that is specific for renal cancers characterized by inactivation of the von Hippel-Lindau tumor suppressor (pVHL). The protective haplotype impairs binding of HIF-2, resulting in an allelic imbalance in cyclin D1 expression, thus affecting a link between hypoxia pathways and cell cycle control.

Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling.

The Krebs cycle enzyme fumarate hydratase (FH) is a human tumor suppressor whose inactivation is associated with the development of leiomyomata, renal cysts, and tumors. It has been proposed that activation of hypoxia inducible factor (HIF) by fumarate-mediated inhibition of HIF prolyl hydroxylases drives oncogenesis. Using a mouse model, we provide genetic evidence that Fh1-associated cyst formation is Hif independent, as is striking upregulation of antioxidant signaling pathways revealed by gene expression profiling. Mechanistic analysis revealed that fumarate modifies cysteine residues within the Kelch-like ECH-associated protein 1 (KEAP1), abrogating its ability to repress the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response pathway, suggesting a role for Nrf2 dysregulation in FH-associated cysts and tumors.

Neuroprotection by dimethyloxalylglycine following permanent and transient focal cerebral ischemia in rats.

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism.

HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.