Prion infection in cells is abolished by a mutated manganese transporter but shows no relation to zinc

The cellular prion protein has been identified as a metalloprotein that binds copper. There have been some suggestions that prion protein also influences zinc and manganese homeostasis. In this study we used a series of cell lines to study the levels of zinc and manganese under different conditions. We overexpressed either the prion protein or known transporters for zinc and manganese to determine relations between the prion protein and both manganese and zinc homeostasis. Our observations supported neither a link between the prion protein and zinc metabolism nor any effect of altered zinc levels on prion protein expression or cellular infection with prions. In contrast we found that a gain of function mutant of a manganese transporter caused reduction of manganese levels in prion infected cells, loss of observable PrPSc in cells and resistance to prion infection. These studies strengthen the link between manganese and prion disease

CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium

BACKGROUND: During neuroinflammation many chemokines alter the function of the blood-brain barrier (BBB) that regulates the entry of macromolecules and immune cells into the brain. As the milieu of the brain is altered, biochemical and structural changes contribute to the pathogenesis of neuroinflammation and may impact on neurogenesis. The chemokine CCL4, previously known as MIP-1β, is upregulated in a wide variety of central nervous system disorders, including multiple sclerosis, where it is thought to play a key role in the neuroinflammatory process. However, the effect of CCL4 on BBB endothelial cells (ECs) is unknown. MATERIALS AND METHODS: Expression and distribution of CCR5, phosphorylated p38, F-actin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) were analysed in the human BBB EC line hCMEC/D3 by Western blot and/or immunofluorescence in the presence and absence of CCL4. Barrier modulation in response to CCL4 using hCMEC/D3 monolayers was assessed by measuring molecular flux of 70 ​kDa RITC-dextran and transendothelial lymphocyte migration. Permeability changes in response to CCL4 in vivo were measured by an occlusion technique in pial microvessels of Wistar rats and by fluorescein angiography in mouse retinae. RESULTS: CCR5, the receptor for CCL4, was expressed in hCMEC/D3 cells. CCL4 stimulation led to phosphorylation of p38 and the formation of actin stress fibres, both indicative of intracellular chemokine signalling. The distribution of junctional proteins was also altered in response to CCL4: junctional ZO-1 was reduced by circa 60% within 60 ​min. In addition, surface VE-cadherin was redistributed through internalisation. Consistent with these changes, CCL4 induced hyperpermeability in vitro and in vivo and increased transmigration of lymphocytes across monolayers of hCMEC/D3 cells. CONCLUSION: These results show that CCL4 can modify BBB function and may contribute to disease pathogenesis

Prion infection in cells is abolished by a mutated manganese transporter but shows no relation to zinc

The cellular prion protein has been identified as a metalloprotein that binds copper. There have been some suggestions that prion protein also influences zinc and manganese homeostasis. In this study we used a series of cell lines to study the levels of zinc and manganese under different conditions. We overexpressed either the prion protein or known transporters for zinc and manganese to determine relations between the prion protein and both manganese and zinc homeostasis. Our observations supported neither a link between the prion protein and zinc metabolism nor any effect of altered zinc levels on prion protein expression or cellular infection with prions. In contrast we found that a gain of function mutant of a manganese transporter caused reduction of manganese levels in prion infected cells, loss of observable PrPSc in cells and resistance to prion infection. These studies strengthen the link between manganese and prion disease

Multicenter Evaluation of Diagnostic Circulating Biomarkers to Detect Sight-Threatening Diabetic Retinopathy

Importance: It is a global challenge to provide regular retinal screening for all people with diabetes to detect sight-threatening diabetic retinopathy (STDR). Objective: To determine if circulating biomarkers could be used to prioritize people with type 2 diabetes for retinal screening to detect STDR. Design, Setting, and Participants: This cross-sectional study collected data from October 22, 2018, to December 31, 2021. All laboratory staff were masked to the clinical diagnosis, assigned a study cohort, and provided with the database containing the clinical data. This was a multicenter study conducted in parallel in 3 outpatient ophthalmology clinics in the UK and 2 centers in India. Adults 40 years and older were categorized into 4 groups: (1) no history of diabetes, (2) type 2 diabetes of at least 5 years' duration with no evidence of DR, (3) nonproliferative DR with diabetic macular edema (DME), or (4) proliferative DR. STDR comprised groups 3 and 4. Exposures: Thirteen previously verified biomarkers were measured using enzyme-linked immunosorbent assay. Main Outcomes and Measures: Severity of DR and presence of DME were diagnosed using fundus photographs and optical coherence tomography. Weighted logistic regression and receiver operating characteristic curve analysis (ROC) were performed to identify biomarkers that discriminate STDR from no DR beyond the standard clinical parameters of age, disease duration, ethnicity (in the UK) and hemoglobin A1c. Results: A total of 538 participants (mean [SD] age, 60.8 [9.8] years; 319 men [59.3%]) were recruited into the study. A total of 264 participants (49.1%) were from India (group 1, 54 [20.5%]; group 2, 53 [20.1%]; group 3, 52 [19.7%]; group 4, 105 [39.8%]), and 274 participants (50.9%) were from the UK (group 1, 50 [18.2%]; group 2, 70 [25.5%]; group 3, 55 [20.1%]; group 4, 99 [36.1%]). ROC analysis (no DR vs STDR) showed that in addition to age, disease duration, ethnicity (in the UK) and hemoglobin A1c, inclusion of cystatin C had near-acceptable discrimination power in both countries (area under the receiver operating characteristic curve [AUC], 0.779; 95% CI, 0.700-0.857 in 215 patients in the UK with complete data; AUC, 0.696; 95% CI, 0.602-0.791 in 208 patients in India with complete data). Conclusions and Relevance: Results of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Circulating cystatin-C levels may be considered as a test to identify those who require prioritization for retinal screening for STDR

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U

Multicenter Evaluation of Diagnostic Circulating Biomarkers to Detect Sight-Threatening Diabetic Retinopathy

IMPORTANCE: It is a global challenge to provide regular retinal screening for all people with diabetes to detect sight-threatening diabetic retinopathy (STDR). OBJECTIVE: To determine if circulating biomarkers could be used to prioritize people with type 2 diabetes for retinal screening to detect STDR. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study collected data from October 22, 2018, to December 31, 2021. All laboratory staff were masked to the clinical diagnosis, assigned a study cohort, and provided with the database containing the clinical data. This was a multicenter study conducted in parallel in 3 outpatient ophthalmology clinics in the UK and 2 centers in India. Adults 40 years and older were categorized into 4 groups: (1) no history of diabetes, (2) type 2 diabetes of at least 5 years’ duration with no evidence of DR, (3) nonproliferative DR with diabetic macular edema (DME), or (4) proliferative DR. STDR comprised groups 3 and 4. EXPOSURES: Thirteen previously verified biomarkers were measured using enzyme-linked immunosorbent assay. MAIN OUTCOMES AND MEASURES: Severity of DR and presence of DME were diagnosed using fundus photographs and optical coherence tomography. Weighted logistic regression and receiver operating characteristic curve analysis (ROC) were performed to identify biomarkers that discriminate STDR from no DR beyond the standard clinical parameters of age, disease duration, ethnicity (in the UK) and hemoglobin A(1c). RESULTS: A total of 538 participants (mean [SD] age, 60.8 [9.8] years; 319 men [59.3%]) were recruited into the study. A total of 264 participants (49.1%) were from India (group 1, 54 [20.5%]; group 2, 53 [20.1%]; group 3, 52 [19.7%]; group 4, 105 [39.8%]), and 274 participants (50.9%) were from the UK (group 1, 50 [18.2%]; group 2, 70 [25.5%]; group 3, 55 [20.1%]; group 4, 99 [36.1%]). ROC analysis (no DR vs STDR) showed that in addition to age, disease duration, ethnicity (in the UK) and hemoglobin A(1c), inclusion of cystatin C had near-acceptable discrimination power in both countries (area under the receiver operating characteristic curve [AUC], 0.779; 95% CI, 0.700-0.857 in 215 patients in the UK with complete data; AUC, 0.696; 95% CI, 0.602-0.791 in 208 patients in India with complete data). CONCLUSIONS AND RELEVANCE: Results of this cross-sectional study suggest that serum cystatin C had good discrimination power in the UK and India. Circulating cystatin-C levels may be considered as a test to identify those who require prioritization for retinal screening for STDR

Feasibility of reporting results of large randomised controlled trials to participants:experience from the Fluoxetine or Control under supervision (FOCUS) trial

Objectives Informing research participants of the results of studies in which they took part is viewed as an ethical imperative. However, there is little guidance in the literature about how to do this. The Fluoxetine Or Control Under Supervision trial randomised 3127 patients with a recent acute stroke to 6 months of fluoxetine or placebo and was published in the Lancet on 5 December 2018. The trial team decided to inform the participants of the results at exactly the same time as the Lancet publication, and also whether they had been allocated fluoxetine or placebo. In this report, we describe how we informed participants of the results.Design In the 6-month and 12-month follow-up questionnaires, we invited participants to provide an email address if they wished to be informed of the results of the trial. We re-opened our trial telephone helpline between 5 December 2018 and 31 March 2019.Setting UK stroke services.Participants 3127 participants were randomised. 2847 returned 6-month follow-up forms and 2703 returned 12-month follow-up forms; the remaining participants had died (380), withdrawn consent or did not respond.Results Of those returning follow-up questionnaires, a total of 1845 email addresses were provided and a further 50 people requested results to be sent by post. Results were sent to all email and postal addresses provided; 309 emails were returned unrecognised. Seventeen people replied, of whom three called the helpline and the rest responded by email.Conclusion It is feasible to disseminate results of large trials to research participants, though only around 60% of those randomised wanted to receive the results. The system we developed was efficient and required very little resource, and could be replicated by trialists in the future.Trial registration number ISRCTN83290762; Post-results