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Laura C Arneson,1,* Kristen J Carroll,1,* Eric M Ruderman2 1Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; 2Department of Medicine, Division of Rheumatology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA*These authors contributed equally to this workCorrespondence: Eric M RudermanDepartment of Medicine, Division of Rheumatology, Northwestern University Feinberg School of Medicine, 675 N. St. Clair, Suite 14-100, Chicago, IL, 60611, USATel +1 312-503-3188Fax +1 312-695-0114Email e-ruderman@northwestern.eduAbstract: Bruton’s tyrosine kinase (BTK) inhibitors are an emerging class of drugs that inhibit B cell receptor activation, FC-γ receptor signaling, and osteoclast proliferation. Following on approval for treatment of hematologic malignancies, BTK inhibitors are now under investigation to treat a number of different autoimmune diseases, including rheumatoid arthritis (RA). While the results of BTK inhibitors in RA animal models have been promising, the ensuing human clinical trial outcomes have been rather equivocal. This review will outline the mechanisms of BTK inhibition and its potential impact on immune mediated disease, the types of BTK inhibitors being studied for RA, the findings from both preclinical and clinical trials of BTK inhibitors in RA, and directions for future research.Keywords: evobrutinib, spebrutinib, acalabrutinib, fenebrutinib, rheumatoid arthritis, Bruton’s tyrosine kinase

We previously demonstrated pharmacokinetic differences among manufacturing batches of a US Food and Drug Administration (FDA)-approved dry powder inhalation product (Advair Diskus 100/50) large enough to establish between-batch bio-inequivalence. Here, we provide independent confirmation of pharmacokinetic bio-inequivalence among Advair Diskus 100/50 batches, and quantify residual and between-batch variance component magnitudes. These variance estimates are used to consider the type I error rate of the FDA's current two-way crossover design recommendation. When between-batch pharmacokinetic variability is substantial, the conventional two-way crossover design cannot accomplish the objectives of FDA's statistical bioequivalence test (i.e., cannot accurately estimate the test/reference ratio and associated confidence interval). The two-way crossover, which ignores between-batch pharmacokinetic variability, yields an artificially narrow confidence interval on the product comparison. The unavoidable consequence is type I error rate inflation, to ∼25%, when between-batch pharmacokinetic variability is nonzero. This risk of a false bioequivalence conclusion is substantially higher than asserted by regulators as acceptable consumer risk (5%).

We previously demonstrated pharmacokinetic differences among manufacturing batches of a US Food and Drug Administration (FDA)-approved dry powder inhalation product (Advair Diskus 100/50) large enough to establish between-batch bio-inequivalence. Here, we provide independent confirmation of pharmacokinetic bio-inequivalence among Advair Diskus 100/50 batches, and quantify residual and between-batch variance component magnitudes. These variance estimates are used to consider the type I error rate of the FDA's current two-way crossover design recommendation. When between-batch pharmacokinetic variability is substantial, the conventional two-way crossover design cannot accomplish the objectives of FDA's statistical bioequivalence test (i.e., cannot accurately estimate the test/reference ratio and associated confidence interval). The two-way crossover, which ignores between-batch pharmacokinetic variability, yields an artificially narrow confidence interval on the product comparison. The unavoidable consequence is type I error rate inflation, to ∼25%, when between-batch pharmacokinetic variability is nonzero. This risk of a false bioequivalence conclusion is substantially higher than asserted by regulators as acceptable consumer risk (5%).

Background: Chronic lung infection with Pseudomonas aeruginosa is associated with increased exacerbations and mortality in people with bronchiectasis. The PROMIS-I and PROMIS-II trials investigated the efficacy and safety of 12-months of inhaled colistimethate sodium delivered via the I-neb., Methods: Two randomised, double-blind, placebo-controlled trials of twice per day colistimethate sodium versus placebo were conducted in patients with bronchiectasis with P aeruginosa and a history of at least two exacerbations requiring oral antibiotics or one requiring intravenous antibiotics in the previous year in hospitals in Argentina, Australia, Belgium, Canada, France, Germany, Greece, Israel, Italy, Netherlands, New Zealand, Poland, Portugal, Spain, Switzerland, the UK, and the USA. Randomisation was conducted through an interactive web response system and stratified by site and long term use of macrolides. Masking was achieved by providing colistimethate sodium and placebo in identical vials. After random assignment, study visits were scheduled for 1, 3, 6, 9, and 12 months (the end of the treatment period); and telephone calls were scheduled for 7 days after random assignment and 2 weeks after the end of treatment. The primary endpoint was the mean annual exacerbation rate. These trials are registered with EudraCT: number 2015-002743-33 (for PROMIS-I) and 2016-004558-13 (for PROMIS-II), and are now completed., Findings: 377 patients were randomly assigned in PROMIS-I (177 to colistimethate sodium and 200 to placebo; in the modified intention-to-treat population, 176 were in the colistimethate sodium group and 197 were in the placebo group) between June 6, 2017, and April 8, 2020. The annual exacerbation rate was 0·58 in the colistimethate sodium group versus 0·95 in the placebo group (rate ratio 0·61; 95% CI 0·46-0·82; p=0·0010). 287 patients were randomly assigned in PROMIS-II (152 were assigned to colistimethate sodium and 135 were assigned to placebo, in the modified intention-to-treat population), between Feb 12, 2018, and Oct 22, 2021. PROMIS-II was then prematurely terminated due to the effect of the COVID-19 pandemic. No significant difference was observed in the annual exacerbation rate between the colistimethate sodium and placebo groups (0·89 vs 0·89; rate ratio 1·00; 95% CI 0·75-1·35; p=0·98). No major safety issues were identified. The overall frequency of adverse events was 142 (81%) patients in the colistimethate sodium group versus 159 (81%) patients in the placebo group in PROMIS-I, and 123 (81%) patients versus 104 (77%) patients in PROMIS-II. There were no deaths related to study treatment., Interpretation: The data from PROMIS-I suggest a clinically important benefit of colistimethate sodium delivered via the I-neb adaptive aerosol delivery system in patients with bronchiectasis and P aeruginosa infection. These results were not replicated in PROMIS-II, which was affected by the COVID-19 pandemic and prematurely terminated., Funding: Zambon., Competing Interests: Declaration of interests CSH reports consultancy or speakers fees from 30 Technology, AstraZeneca, CSL Behring, Chiesi, Infex, Insmed, Janssen, Lifearc, Meiji, Mylan, Pneumagen, Shionogi, Vertex, and Zambon. MS reports research grants from Glaxosmithkline, Trudell, and Tel Aviv League for Lung Disease; consulting fees or speaker fees for Astrazeneca, Boehringer Ingelheim, Dexcel, Kamada, Rafa, Synchrony Medical, Trumed, Vertex, Zambon, and Sanofi Insmed; and data safety monitoring board participation for Bonus Biotherapeutics and Boehringer Ingelheim. KW reports research grants from Insmed, Paratek, Red Hill Biopharma, AN2, Spero, and Renovion; consulting fees from Insmed, Paratek, Red Hill Biopharma, AN2, Renovion, Spero, Zambon, and Janssen; and data safety monitoring board participation for Janssen and Red Hill Biopharma. AB declares research funding from Zambon; and contributed to UptoDate. FB reports research funding from Astrazeneca, Glaxosmithkline, Chiesi, and Insmed; and consulting fees or speakers fees from Astrazeneca, Chiesi, Glaxosmithkline, Menarini, Grifols, Insmed, Novartis, Pfizer, Sanofi, Vertex, Viatris, and Zambon. KD reports payments for consultancy or lectures from Boehringer Ingelheim, GSK, Menarini, Novartis, Norma Hellas, Chiesi, Astrazeneca, and Zambon. LCM reports a leadership role with Lung Foundation Australia; research grants from Boehringer Ingelheim, Astrazeneca, Zambon, Insmed, and Glaxosmithkline; and consultancy or speaker fees from Boehringer Ingelheim, Astrazeneca, Zambon, Insmed, Sequirius, and Glaxosmithkline. AEO reports research grants from Zambon, Insmed, Boehringer Ingelheim, Armata, and the US COPD foundation; consultancy or speaker fees from Insmed, Boehringer Ingelheim, Zambon, and Electromed; participation in a data safety monitoring board for Paraxel; and fees from Academic CME, Vindico Medical Education, Answers in CME, Peer Review Institute, CE, and RMEI. FCR reports grant from the German Centre for Lung Research, German Centre for Infection Research, EU Innovative Medicines Initiative, Mukoviszidose Institute, Novartis, Insmed, Grifols, Bayer, and InfectoPharm; consulting fees or lecture fees from Parion, Grifols, Zambon, Insmed, Helmholtz-Zentrum fur Infektionsforschung, Astrazeneca, Insmed, and Grifols; participation in data safety monitoring board for Insmed, Grifols, Shionogi, and Boehringer Ingelheim; provided expert testimony to the Social Court Cologne; received support to attend meetings from German Kartagener Syndrome and Primary Ciliary Dyskinesia Patient Advocacy Group and Mukoviszidose; was a coordinator of the ERN-LUNG Bronchiectasis Core Network; was the chair of the German Bronchiectasis Registry PROGNOSIS; was a member of the SteerCo of the European Bronchiectasis Registry EMBARC and SteerCo of the European Nontuberculous Mycobacterial Pulmonary Disease Registry EMBARC-NTM; was a co-speaker of the Medical Advisory Board of the German Kartagener Syndrome and PCD Patient Advocacy Group; was a speaker of the Respiratory Infections and TB group of the German Respiratory Society, the Cystic Fibrosis group of German Respiratory Society (DGP); was a principal investigator of the German Center for Lung Research, Member of the Protocol Review Committee of the PCD-CTN, Member of Physician Association of the German Cystic Fibrosis Patient Advocacy Group; and declares fees to institution from clinical trial work from AstraZeneca, Boehringer Ingelheim, Celtaxsys, Corbus, German Center for Lung Research/KKS Marburg, Insmed, Novartis, Parion, University of Dundee, Vertex, and Zambon. RMT reports research grants from Australian Institute of Infectious Diseases, Herston Infectious Diseases Institute, Metro South Health, and the Asia Pacific Society of Respirology Gallipoli Medical Research Foundation; an eductional grant from Insmed; support for attending a meeting from Beyond Air; and consulting fees from AN2 Therapeutics, Beyond Air, and Insmed. KJC reports consulting fees from Zambon. FP is an employee of Zambon. PC is an employee of Zambon; and reports an international patent application WO 23/012280 in the name of Zambon, designating PC as the inventor. JDC has received research grants from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Grifols, Novartis, Insmed, and Trudell; and received consultancy or speaker fees from Antabio, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Insmed, Janssen, Novartis, Pfizer, Trudell, and Zambon. OS declares no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: In the CALCIPHYX trial, we investigated hexasodium fytate, an inhibitor of vascular calcification, for the treatment of calcific uraemic arteriolopathy (calciphylaxis), a rare condition characterised by painful, non-healing skin lesions., Methods: In this international, phase 3, randomised, double-blind, placebo-controlled trial, adults with an ulcerated calciphylaxis lesion and pain visual analogue scale (VAS) score ≥50/100 were randomised 1:1 to hexasodium fytate 7 mg/kg or placebo intravenously during maintenance haemodialysis. Primary efficacy outcomes were an 8-item modification of the Bates-Jensen Wound Assessment Tool (BWAT-CUA) and Pain VAS in the intention-to-treat population. ClinicalTrials.gov number: NCT04195906., Findings: Overall, 34/37 patients randomised to hexasodium fytate and 26/34 patients randomised to placebo completed the 12-week randomised treatment period. At Week 12, both groups (hexasodium fytate versus placebo) showed similar improvements in BWAT-CUA (mean [standard deviation (SD)], -5.3 [5.2] versus -6.0 [6.2]; least squares mean difference, 0.3 [96% confidence interval (CI): -2.5, 3.0]; p = 0.88) and Pain VAS (mean [SD], -19.5 [26.9] versus -32.2 [38.5]; least squares mean difference, 11.5 [96% CI: -4.8, 27.8]; p = 0.15). One patient randomised to placebo briefly received hexasodium fytate in error. Serious adverse events through Week 12 included: calciphylaxis-related events leading to hospitalisation (2/38 [5%] versus 11/33 [33%]) and death (1/38 [3%] versus 5/33 [15%]). During the subsequent 12 weeks of open-label hexasodium fytate and 4 weeks of follow-up, there were no additional calciphylaxis-related events leading to hospitalisation. Over the course of the entire trial, deaths were 2/38 [5%] for the hexasodium fytate group and 7/33 [21%] for the placebo group., Interpretation: In patients with calciphylaxis, BWAT-CUA and Pain VAS improved similarly in hexasodium fytate- and placebo-treated patients; over the course of the entire trial, there were fewer deaths and calciphylaxis-related events leading to hospitalisation in the hexasodium fytate group., Funding: Funded by Sanifit, a CSL Vifor company., Competing Interests: SS received consulting fees from Sanifit and Inozyme Pharma Inc; honoraria from AstraZeneca, Bayer, Sanofi-Genzyme, Novartis, CSL Vifor, GSK, Menarini, Medscape, and Boehringer-Ingelheim; support for attending meetings and/or travel from Sanifit, AstraZeneca, Novartis, and CSL Vifor. SUN received research funding to institution from Laboratoris Sanifit, CSL/Vifor, Hope Pharma, Inozyme Pharma, Epizon; consulting fees from Laboratoris Sanifit, CSL/Vifor, Inozyme Pharma, and Epizon; medical writing support from Laboratoris Sanifit and CSL/Vifor; royalties from UpToDate; and support for attending meetings and/or travel from Inozyme Pharma. LJG received consulting fees from Sanifit Therapeutics. SMM received consulting fees from Ardelyx, Sanfit, and Inozyme; and holds stock or stock options in Eli Lilly. DKC is an employee of Fresenius Medical Care and declares spousal stock in Amgen. KJC received consulting fees from Sanifit srl and Vifor Pharma. GA is an employee of CSL Vifor. LHK was an employee of and received stock compensation from CSL Vifor during the study. JP received consulting fees and stocks or stock options from Sanifit Therapeutics, which owns or has rights on patents related to this work where JP is a coinventor. AG was an employee of Sanifit Therapeutics and CSL Vifor and received consulting fees from Sanifit Therapeutics and CSL Vifor. GMC received graphics support (no writing support) from Sanifit, Vifor, and CSL Behring; received consulting fees from Sanifit, Akebia, AstraZeneca, CSL Behring, and Vertex; participated on a data safety monitoring board or Advisory Board for Ardelyx, Calico, and Miromatrix; served in a leadership or fiduciary role for Satellite Healthcare; received stock or stock options from CloudCath, Duract, Eliaz Therapeutics Outset, Renibus, and Unicycive. All other authors declare no competing interests., (© 2024 The Authors.)

In several therapeutic areas, including chronic kidney disease (CKD) and immunoglobulin A nephropathy (IgAN), there is a growing interest in how best to analyze estimated glomerular filtration rate (eGFR) data over time in randomized clinical trials including how to best accommodate situations where the rate of change is not anticipated to be linear over time, often due to possible short term hemodynamic effects of certain classes of interventions. In such situations, concerns have been expressed by regulatory authorities that the common application of single slope analysis models may induce Type I error inflation. This article aims to offer practical advice and guidance, including SAS codes, on the statistical methodology to be employed in an eGFR rate of change analysis and offers guidance on trial design considerations for eGFR endpoints. A two-slope statistical model for eGFR data over time is proposed allowing for an analysis to simultaneously evaluate short term acute effects and long term chronic effects. A simulation study was conducted under a range of credible null and alternative hypotheses to evaluate the performance of the two-slope model in comparison to commonly used single slope random coefficients models as well as to non-slope based analyses of change from baseline or time normalized area under the curve (TAUC). Importantly, and contrary to preexisting concerns, these simulations demonstrate the absence of alpha inflation associated with the use of single or two-slope random coefficient models, even when such models are misspecified, and highlight that any concern regarding model misspecification relates to power and not to lack of Type I error control., (© 2024 John Wiley & Sons Ltd.)

When autistic youth are asked to assess their own social skills, they frequently rate themselves more favorably than their parents rate them. The magnitude of this informant discrepancy has been shown to relate to key clinical outcomes such as treatment response. It has been proposed that this discrepancy arises from difficulties with Theory of Mind. Participants were 167 youth 11 to 17 years old; 72% male, and their parents. Youth completed self-report measures of social skills and social cognitive tasks, while their parents completed questionnaires regarding social skills. A repeated-measures ANOVA indicated both non-autistic and autistic youth rated themselves more favorably than their parents rated them across all measures. Zero-order correlations revealed that raw differences between parent- and participant-report were negatively correlated with scores on parent-reported Theory of Mind measures. However, polynomial analysis did not indicate interaction effects between parent- and participant-report on any of the measures used. Polynomial regression revealed that increases in parent-reported social skill predicted larger increases in parent-report Theory of Mind at low levels of parent-reported social skill compared to high levels of parent-reported social skill. Participant-report social skills predicted performance on a behavioral Theory of Mind test in a curvilinear fashion, such that the relationship was positive at low levels of participant-reported social skills, but negative at high levels. This study replicates the finding that raw difference score analyses may result in illusory effects that are not supported when using more contemporary analysis methods, and that more complex and subtle relationships between social insight and perspective-taking exist within autistic youth., (© 2024. The Author(s).)

Random coefficient (RC) models are commonly used in clinical trials to estimate the rate of change over time in longitudinal data. Trials utilizing a surrogate endpoint for accelerated approval with a confirmatory longitudinal endpoint to show clinical benefit is a strategy implemented across various therapeutic areas, including immunoglobulin A nephropathy. Understanding conditional power (CP) and information fraction calculations of RC models may help in the design of clinical trials as well as provide support for the confirmatory endpoint at the time of accelerated approval. This paper provides calculation methods, with practical examples, for determining CP at an interim analysis for a RC model with longitudinal data, such as estimated glomerular filtration rate (eGFR) assessments to measure rate of change in eGFR slope., (© 2023 John Wiley & Sons Ltd.)

Data directly demonstrating the relationship between urinary oxalate (UOx) excretion and stone events in those with enteric hyperoxaluria (EH) are limited. Therefore, we assessed the relationship between UOx excretion and risk of kidney stone events in a retrospective population-based EH cohort. In all, 297 patients from Olmsted County, Minnesota were identified with EH based upon having a 24-h UOx ≥40 mg/24 h preceded by a diagnosis or procedure associated with malabsorption. Diagnostic codes and urologic procedures consistent with kidney stones during follow-up after baseline UOx were considered a new stone event. Logistic regression and accelerated failure time modeling were performed as a function of UOx excretion to predict the probability of new stone event and the annual rate of stone events, respectively, with adjustment for urine calcium and citrate. Mean ± standard deviation age was 51.4 ± 11.4 years and 68% were female. Median (interquartile range) UOx was 55.4 (46.6-73.0) mg/24 h and 81 patients had one or more stone event during a median follow-up time of 4.9 (2.8-7.8) years. Higher UOx was associated with a higher probability of developing a stone event (P < 0.01) and predicted an increased annual risk of kidney stones (P = 0.001). Estimates derived from these analyses suggest that a 20% decrease in UOx is associated with 25% reduction in the annual odds of a future stone event. Thus, these data demonstrate an association between baseline UOx and stone events in EH patients and highlight the potential benefit of strategies to reduce UOx in this patient group., Background: Data directly demonstrating the relationship between urinary oxalate (UOx) excretion and stone events in those with enteric hyperoxaluria (EH) are limited., Methods: We assessed the relationship between UOx excretion and risk of kidney stone events in a retrospective population-based EH cohort. In all, 297 patients from Olmsted County, Minnesota were identified with EH based upon having a 24-h UOx ≥40 mg/24 h preceded by a diagnosis or procedure associated with malabsorption. Diagnostic codes and urologic procedures consistent with kidney stones during follow-up after baseline UOx were considered a new stone event. Logistic regression and accelerated failure time modeling were performed as a function of UOx excretion to predict the probability of new stone event and the annual rate of stone events, respectively, with adjustment for urine calcium and citrate., Results: Mean ± SD age was 51.4 ± 11.4 years and 68% were female. Median (interquartile range) UOx was 55.4 (46.6-73.0) mg/24 h and 81 patients had ≥1 stone event during a median follow-up time of 4.9 (2.8-7.8) years. Higher UOx was associated with a higher probability of developing a stone event (P < 0.01) and predicted an increased annual risk of kidney stones (P = 0.001). Estimates derived from these analyses suggest that a 20% decrease in UOx is associated with 25% reduction in the annual odds of a future stone event., Conclusions: These data demonstrate an association between baseline UOx and stone events in EH patients and highlight the potential benefit of strategies to reduce UOx in this patient group., (© The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Batch-to-batch pharmacokinetic (PK) variability of orally inhaled drug products has been documented and can render single-batch PK bioequivalence (BE) studies unreliable; results from one batch may not be consistent with a repeated study using a different batch, yet the goal of PK BE is to deliver a product comparison that is interpretable beyond the specific batches used in the study. We characterized four multiple-batch PK BE approaches to improve outcome reliability without increasing the number of clinical study participants. Three approaches include multiple batches directly in the PK BE study with batch identity either excluded from the statistical model ("Superbatch") or included as a fixed or random effect ("Fixed Batch Effect," "Random Batch Effect"). A fourth approach uses a bio-predictive in vitro test to screen candidate batches, bringing the median batch of each product into the PK BE study ("Targeted Batch"). Three of these approaches (Fixed Batch Effect, Superbatch, Targeted Batch) continue the single-batch PK BE convention in which uncertainty in the Test/Reference ratio estimate due to batch sampling is omitted from the Test/Reference confidence interval. All three of these approaches provided higher power to correctly identify true bioequivalence than the standard single-batch approach with no increase in clinical burden. False equivalence (type I) error was inflated above the expected 5% level, but multiple batches controlled type I error better than a single batch. The Random Batch Effect approach restored 5% type I error, but had low power for small (e.g., <8) batch sample sizes using standard [0.8000, 1.2500] bioequivalence limits., (© 2021. The Author(s).)

Pharmacokinetic differences between manufacturing batches, well established for inhaled drug products, preclude control of patient risk in the customary two-way (single batch) pharmacokinetic bioequivalence crossover design if batches are randomly chosen. European regulators have recommended selecting a "typical" in vitro batch to represent each product in pharmacokinetic bioequivalence testing. We explored the feasibility of this approach to control patient risk (the "false equivalence", or Type I, error rate). The probability of achieving a Test/Reference 90% confidence interval within (0.80, 1.25) for a true (non-equivalent) value of 1.25 was simulated for a two-way crossover design using the median in vitro batch across a range of number of in vitro batches, in vitro/in vivo correlation (IVIVC) quality (correlation coefficient, r, of zero to one), and within-subject between-batch pharmacokinetic variability. Even under extremely optimistic conditions, e.g., r=0.95 and >100 batches per product screened in vitro, patient risk for typical between-batch variability levels remained at least threefold higher than the 5% regulatory expectation for the significance level (the false equivalence error rate) of the pharmacokinetic bioequivalence test. This elevated error rate in bioequivalence decision-making occurs because of incomplete confidence that the true product average has been identified, and, importantly, omission of this uncertainty from the bioequivalence confidence interval., (© 2021. American Association of Pharmaceutical Scientists.)

Background: The implementation by diagnostic laboratories in England of polymerase chain reaction (PCR) to screen faecal specimens for Shiga toxin-producing Escherichia coli (STEC) has resulted in a significant increase in notifications mainly due to non-O157 strains. The purpose of this study was to develop an approach to public health risk assessment that prioritizes follow-up to cases caused by haemolytic uraemic syndrome (HUS) associated E. coli (HUSEC) strains and minimizes unnecessary actions., Methods: Epidemiological and microbiological data were prospectively collected from 1 November 2013 to 31 March 2017 and used to compare three risk assessment approaches., Results: A history of HUS/bloody diarrhoea/age under 6 years and faecal specimens positive for stx-predicted HUSEC with a diagnostic accuracy of 84% (95% CI; 81-88%). STEC isolated by Gastrointestinal Bacteria Reference Unit (GBRU) and stx2 and eae positive predicted HUSEC with a diagnostic accuracy of 99% (95% CI; 98-100%). Risk assessment combining these two tests predicts the most efficient use of resources, predicting that 18% (97/552) of cases would be eligible for follow-up at some stage, 16% (86/552) following local stx PCR results, 1% (7/552) following GBRU results of stx2 and eae status and 0.7% (4/552) following whole-genome sequencing. Follow-up could be stopped in 78% (76/97) of these cases, 97% (74/76) following second stage risk assessment., Conclusions: This three-stage risk assessment approach prioritizes follow-up to HUSEC and minimizes unnecessary public health actions. We developed it into the algorithm for public health actions included in the updated PHE Guidance for management of STEC published in August 2018., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Background: Calcific uraemic arteriolopathy (CUA; calciphylaxis) is a rare disease seen predominantly in patients receiving dialysis. Calciphylaxis is characterized by poorly healing or non-healing wounds, and is associated with mortality, substantial morbidity related to infection and typically severe pain. In an open-label Phase 2 clinical trial, SNF472, a selective inhibitor of vascular calcification, was well-tolerated and associated with improvement in wound healing, reduction of wound-related pain and improvement in wound-related quality of life (QoL). Those results informed the design of the CALCIPHYX trial, an ongoing, randomized, placebo-controlled, Phase 3 trial of SNF472 for treatment of calciphylaxis., Methods: In CALCIPHYX, 66 patients receiving haemodialysis who have an ulcerated calciphylaxis lesion will be randomized 1:1 to double-blind SNF472 (7 mg/kg intravenously) or placebo three times weekly for 12 weeks (Part 1), then receive open-label SNF472 for 12 weeks (Part 2). All patients will receive stable background care, which may include pain medications and sodium thiosulphate, in accordance with the clinical practices of each site. A statistically significant difference between the SNF472 and placebo groups for improvement of either primary endpoint at Week 12 will demonstrate efficacy of SNF472: change in Bates-Jensen Wound Assessment Tool-CUA (a quantitative wound assessment tool for evaluating calciphylaxis lesions) or change in pain visual analogue scale score. Additional endpoints will address wound-related QoL, qualitative changes in wounds, wound size, analgesic use and safety., Conclusions: This randomized, placebo-controlled Phase 3 clinical trial will examine the efficacy and safety of SNF472 in patients who have ulcerated calciphylaxis lesions. Patient recruitment is ongoing., (© The Author(s) 2021. Published by Oxford University Press on behalf of ERA-EDTA.)

Background: Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized., Methods: Patients were classified as premenopausal (< 45 years), perimenopausal (45-55 years), or postmenopausal (> 55 years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women., Results: Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5 mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5 mL. In Cohort 2, premenopausal women had a higher incidence of FGFR1 (OR 2.75, p = 0.022) and CCND2 (OR 6.91, p = 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups., Conclusions: Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight FGFR1 inhibitors as potential therapeutics of particular interest among premenopausal women.

Background and Aims: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions., Approach and Results: Exposure of zebrafish embryos to 17β-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b -/- mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development., Conclusions: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy., (© 2020 by the American Association for the Study of Liver Diseases.)

A clinical trial often has primary and secondary endpoints and comparisons of high and low doses of a study drug to a control. Multiplicity is not only caused by the multiple comparisons of study drugs versus the control, but also from the hierarchical structure of the hypotheses. Closed test procedures were proposed as general methods to address multiplicity. Two commonly used tests for intersection hypotheses in closed test procedures are the Simes test and the average method. When the treatment effect of a less efficacious dose is not much smaller than the treatment effect of a more efficacious dose for a specific endpoint, the average method has better power than the Simes test for the comparison of two doses versus control. Accordingly, for inferences for primary and secondary endpoints, the matched parallel gatekeeping procedure based on the Simes test for testing intersection hypotheses is extended here to allow the average method for such testing. This procedure is further extended to clinical trials with more than two endpoints as well as to clinical trials with more than two active doses and a control.

Rationale: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far., Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription., Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d , which when overexpressed, led to hypertrophy and heart failure, and Klf15 , which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15 , we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects., Conclusions: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.

Background: Calciphylaxis in end-stage renal disease is characterized by painful necrotic skin ulcers and high mortality. There are no approved therapies. SNF472, an intravenous formulation of myo-inositol hexaphosphate, inhibits the formation and growth of hydroxyapatite crystals, the final common pathway in the pathogenesis of vascular calcification., Methods: In this open-label, single-arm study, calciphylaxis patients on thrice-weekly hemodialysis and standard care, received intravenous SNF472 3 times per week for 12 weeks. The primary endpoint was wound healing assessed using the quantitative Bates-Jensen Wound Assessment Tool (BWAT). Pain visual analog scale (VAS), quality of life (wound-QoL), and qualitative wound image review were secondary endpoints. Quantitative changes from baseline were analyzed by paired t-tests using multiple imputation to account for missing observations., Results: Fourteen patients received SNF472. Improvements from baseline to week 12 were observed for mean BWAT score (- 8.1; P < 0.001), pain VAS (- 23.6 mm; P = 0.015) and wound-QoL global score (- 0.90; P = 0.003). Of the 9 patients with ulcerated lesions at baseline who completed treatment, wound image review showed improvement for 7. SNF472 was well tolerated with no serious treatment-related adverse events. The most common adverse events were infections which occur frequently in patients on hemodialysis. None of these were considered as treatment-related., Conclusions: SNF472 was well-tolerated and improvements from baseline to week 12 in wound healing, pain, and quality of life were observed. A randomized, double-blind, placebo-controlled trial is planned to evaluate SNF472 in patients with calciphylaxis.

Purpose: This study describes the epidemiology of Shiga toxin-producing Escherichia coli (STEC) infections in a population in the South East of England., Methods: From 1 November 2013 to 31 March 2017 participating diagnostic laboratories reported Shiga toxin gene (stx) positive real-time PCR results to local public health teams. Stx positive faecal samples/isolates were referred to the Gastrointestinal Bacteria Reference Unit (GBRU) for confirmation by culture and typing by whole genome sequencing (WGS). Key clinical information was collected by public health teams.Results/Key findings. Altogether, 548 faecal specimens (420 were non-travel associated) were stx positive locally, 535 were submitted to the GBRU. STEC were isolated from 42 %, confirmed by stx PCR in 21 % and 37 % were PCR negative. The most common non-travel associated STEC serogroups were O157, O26, O146 and O91. The annualized incidence of confirmed STEC infections (PCR or culture) was 5.8 per 100 000. The ratio of O157 to non-O157 STEC serogroups was 1:7. The annualized incidence of non-O157 haemolytic uraemic syndrome-associated Escherichia coli (HUSEC) strains was 0.4 per 100 000. Bloody diarrhoea was reported by 58 % of cases infected with E. coli O157, 33 % of cases infected with non-O157 HUSEC strains and 12 % of other lower risk non-O157 strains. Overall, 76 % of non-O157 HUSEC isolates possessed the eae virulence gene., Conclusions: HUSEC including serogroup O157 were uncommon and more likely to cause bloody diarrhoea than other STEC. The routine use of stx PCR testing can influence clinical management. Understanding the local epidemiology facilitates a proportionate public health response to STEC, based on clinical and microbiological characteristics including stx subtype(s).

Missing data cause challenging issues, particularly in phase III registration trials, as highlighted by the European Medicines Agency (EMA) and the US National Research Council. We explore, as a case study, how the issues from missing data were tackled in a double-blind phase III trial in subjects with autosomal dominant polycystic kidney disease. A total of 1445 subjects were randomized in a 2:1 ratio to receive active treatment (tolvaptan), or placebo. The primary outcome, the rate of change in total kidney volume, favored tolvaptan (P < .0001). The key secondary efficacy endpoints of clinical progression of disease and rate of decline in kidney function also favored tolvaptan. However, as highlighted by Food and Drug Administration and EMA, the interpretation of results was hampered by a high number of unevenly distributed dropouts, particularly early dropouts. In this paper, we outline the analyses undertaken to address the issue of missing data thoroughly. "Tipping point analyses" were performed to explore how extreme and detrimental outcomes among subjects with missing data must be to overturn the positive treatment effect attained in those subjects who had complete data. Nonparametric rank-based analyses were also performed accounting for missing data. In conclusion, straightforward and transparent analyses directly taking into account missing data convincingly support the robustness of the preplanned analyses on the primary and secondary endpoints. Tolvaptan was confirmed to be effective in slowing total kidney volume growth, which is considered an efficacy endpoint by EMA, and in lessening the decline in renal function in patients with autosomal dominant polycystic kidney disease., (Copyright © 2017 John Wiley & Sons, Ltd.)

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs., (© 2016 The Authors.)

Background: The role of change in proteinuria as a surrogate end point for randomized trials in immunoglobulin A nephropathy (IgAN) has previously not been thoroughly evaluated., Study Design: Individual patient-level meta-analysis., Setting & Population: Individual-patient data for 830 patients from 11 randomized trials evaluating 4 intervention types (renin-angiotensin system [RAS] blockade, fish oil, immunosuppression, and steroids) examining associations between changes in urine protein and clinical end points at the individual and trial levels., Selection Criteria for Studies: Randomized controlled trials of IgAN with measurements of proteinuria at baseline and a median of 9 (range, 5-12) months follow-up, with at least 1 further year of follow-up for the clinical outcome., Predictor: 9-month change in proteinuria., Outcome: Doubling of serum creatinine level, end-stage renal disease, or death., Results: Early decline in proteinuria at 9 months was associated with lower risk for the clinical outcome (HR per 50% reduction in proteinuria, 0.40; 95% CI, 0.32-0.48) and was consistent across studies. Proportions of treatment effect on the clinical outcome explained by early decline in proteinuria were estimated at 11% (95% CI, -19% to 41%) for RAS blockade and 29% (95% CI, 6% to 53%) for steroid therapy. The direction of the pooled treatment effect on early change in proteinuria was in accord with the direction of the treatment effect on the clinical outcome for steroids and RAS blockade. Trial-level analyses estimated that the slope for the regression line for the association of treatment effects on the clinical end points and for the treatment effect on proteinuria was 2.15 (95% Bayesian credible interval, 0.10-4.32)., Limitations: Study population restricted to 11 trials, all having fewer than 200 patients each with a limited number of clinical events., Conclusions: Results of this analysis offer novel evidence supporting the use of an early reduction in proteinuria as a surrogate end point for clinical end points in IgAN in selected settings., (Copyright © 2016 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Electrocatalytic hydrogenation (ECH) is a sustainable pathway for the synthesis of value-added organic compounds, provided affordable catalysts with high activity, selectivity and durability are developed. Here, we synthesize Cu/C, Ni/C, and CuNi/C nanoparticles and compare their performance to Pt/C, Ru/C, PtRu/C for the ECH of hydroxyacetone, a bio-derived feedstock surrogate containing a carbonyl and a hydroxyl functional group. The non-precious metal electrocatalysts show promising conversion-time behavior, product selectivities, and Faradaic efficiencies. Ni/C forms propylene glycol with a selectivity of 89 % (at 80 % conversion), while Cu/C catalyzes ECH (52 % selectivity) and hydrodeoxygenation (HDO, 48 % selectivity, accounting for evaporation). CuNi/C shows increased turnover frequencies but reduced ECH selectivity (80 % at 80 % conversion) as compared to the Ni/C catalyst. Importantly, stability studies show that the non-precious metal catalysts do not leach at operating conditions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.

Myocardin-Related Transcription Factors A and B (MRTF-A and MRTF-B) are highly homologous proteins that function as powerful coactivators of serum response factor (SRF), a ubiquitously expressed transcription factor essential for cardiac development. The SRF/MRTF complex binds to CArG boxes found in the control regions of genes that regulate cytoskeletal dynamics and muscle contraction, among other processes. While SRF is required for heart development and function, the role of MRTFs in the developing or adult heart has not been explored. Through cardiac-specific deletion of MRTF alleles in mice, we show that either MRTF-A or MRTF-B is dispensable for cardiac development and function, whereas deletion of both MRTF-A and MRTF-B causes a spectrum of structural and functional cardiac abnormalities. Defects observed in MRTF-A/B null mice ranged from reduced cardiac contractility and adult onset heart failure to neonatal lethality accompanied by sarcomere disarray. RNA-seq analysis on neonatal hearts identified the most altered pathways in MRTF double knockout hearts as being involved in cytoskeletal organization. Together, these findings demonstrate redundant but essential roles of the MRTFs in maintenance of cardiac structure and function and as indispensible links in cardiac cytoskeletal gene regulatory networks., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this work we prepared Li1.2Ni0.2Mn0.6O2 (LNMO) using a hydroxide co-precipitation method and investigated the effect of co-modification with NH4F and Al2O3. After surface co-modification, the first cycle Coulombic efficiency of Li1.2Ni0.2Mn0.6O2 improved from 82.7% to 87.5%, and the reversible discharge capacity improved from 253 to 287 mAh g(-1) at C/20. Moreover, the rate capability also increased significantly. A combination of neutron diffraction (ND), high-resolution transmission electron microscopy (HRTEM), aberration-corrected scanning transmission electron microscopy (a-STEM)/electron energy loss spectroscopy (EELS), and X-ray photoelectron spectroscopy (XPS) revealed the changes of surface structure and chemistry after NH4F and Al2O3 surface co-modification while the bulk properties showed relatively no changes. These complex changes on the material's surface include the formation of an amorphous Al2O3 coating, the transformation of layered material to a spinel-like phase on the surface, the formation of nanoislands of active material, and the partial chemical reduction of surface Mn(4+). Such enhanced discharge capacity of the modified material can be primarily assigned to three aspects: decreased irreversible oxygen loss, the activation of cathode material facilitated with preactivated Mn(3+) on the surface, and stabilization of the Ni-redox pair. These insights will provide guidance for the surface modification in high-voltage-cathode battery materials of the future.

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways., (© 2015 AlphaMed Press.)

Using a controllable wet chemical approach, the polyol process, we developed a cobalt carbide nanomagnet consisting of an assembly of Co2C and Co3C nanoparticles as an alternative to rare earth permanent magnets (PMs). The thermodynamically stable mixed phase cobalt carbide nanoparticles are shown to be acicular in morphology. Their exchange-coupled magnetic interaction possessing high maximum energy product of 20.7 kJ m(-3) and room temperature coercivity (2.9 kOe) has been confirmed through vibrating sample magnetometer (VSM) and first order reversal curves (FORCs). These metastable carbide nanoparticles offer improved magnetic properties compared to their pure bulk form. An understanding of the formation mechanism, using in situ time-resolved X-ray spectroscopy (TR-XAS), and the correlation between phase contributions to the properties are described in detail. Our strategy presents a controllable route to preparing the cobalt carbide nanomagnets, which could be potentially useful in permanent magnet clean energy applications. Additionally, the in situ apparatus offers a promising way to directly explore the effects of reaction variables for high-temperature wet chemical reactions.

We have determined the electrochemical characteristics of the high voltage, high capacity Li-ion battery cathode material Li[Li2/12Ni3/12Mn7/12]O2 prepared using three different synthesis routes: sol-gel, hydroxide coprecipitation, and carbonate coprecipitation. Each route leads to distinct morphologies and surface areas while maintaining the same crystal structures. X-ray photoelectron spectroscopy (XPS) measurements reveal differences in their surface chemistries upon cycling, which correlate with voltage fading. Indeed, we observe the valence state of Mn on the surface to decrease upon lithiation, and this reduction is specifically correlated to discharging below 3.6 V. Furthermore, the data shows a correlation of the formation of Li2CO3 with the Mn oxidation state from the decomposition of electrolyte. These phenomena are related to each material's electrochemistry in order to expand upon the reaction mechanisms taking place-specifically in terms of the particle morphology produced by each synthetic approach.