Growth of complete ammonia oxidizers on guanidine.

Guanidine is a chemically stable nitrogen compound that is excreted in human urine and is widely used in manufacturing of plastics, as a flame retardant and as a component of propellants, and is well known as a protein denaturant in biochemistry1-3. Guanidine occurs widely in nature and is used by several microorganisms as a nitrogen source, but microorganisms growing on guanidine as the only substrate have not yet been identified. Here we show that the complete ammonia oxidizer (comammox) Nitrospira inopinata and probably most other comammox microorganisms can grow on guanidine as the sole source of energy, reductant and nitrogen. Proteomics, enzyme kinetics and the crystal structure of a N. inopinata guanidinase homologue demonstrated that it is a bona fide guanidinase. Incubation experiments with comammox-containing agricultural soil and wastewater treatment plant microbiomes suggested that guanidine serves as substrate for nitrification in the environment. The identification of guanidine as a growth substrate for comammox shows an unexpected niche of these globally important nitrifiers and offers opportunities for their isolation.

Long-term effect of mavacamten in obstructive hypertrophic cardiomyopathy.

BACKGROUND AND AIMS: Long-term safety and efficacy of mavacamten in patients with obstructive hypertrophic cardiomyopathy (HCM) are unknown. MAVA-LTE (NCT03723655) is an ongoing, 5-year, open-label extension study designed to evaluate the long-term effects of mavacamten. METHODS: Participants from EXPLORER-HCM (NCT03470545) could enrol in MAVA-LTE upon study completion. RESULTS: At the latest data cut-off, 211 (91.3%) of 231 patients originally enrolled in MAVA-LTE still received mavacamten. Median (range) time on study was 166.1 (6.0-228.1) weeks; 185 (80.1%) and 99 (42.9%) patients had completed the week 156 and 180 visits, respectively. Sustained reductions from baseline to week 180 occurred in left ventricular outflow tract gradients (mean [standard deviation]: resting, -40.3 [32.7] mmHg; Valsalva, -55.3 [33.7] mmHg), NT-proBNP (median [interquartile range]: -562 [-1162.5, -209] ng/L), and EQ-5D-5L score (mean [standard deviation]: 0.09 [0.17]). Mean left ventricular ejection fraction (LVEF) decreased from 73.9% (baseline) to 66.6% (week 24) and 63.9% (week 180). At week 180, 74 (77.9%) of 95 patients improved by at least one New York Heart Association class from baseline. Over 739 patient-years exposure, 20 patients (8.7%; exposure-adjusted incidence: 2.77/100 patient-years) experienced 22 transient reductions in LVEF to <50% resulting in temporary treatment interruption (all recovered LVEF of ≥50%). Five (2.2%) patients died (all considered unrelated to mavacamten). CONCLUSIONS: Long-term mavacamten treatment resulted in sustained improvements in cardiac function and symptoms in patients with obstructive HCM, with no new safety concerns identified. Transient, reversible reductions in LVEF were observed in a small proportion of patients during long-term follow-up.

Soil Analysis by Mobile Multinuclear NMR: Quantification of Phosphorus, Aluminum, and Sodium.

Soil analyses are essential to ensure economically and environmentally sustainable crop production while maintaining the soil fertile for future use. Unfortunately, common soil analyses may be highly demanding in terms of time, chemicals, and costs. This applies, in particular, when total quantities of elements are desired. As an easy and fast alternative without consumption of chemicals, we here present mobile 31P, 27Al, 23Na, and 1H NMR for quantification of phosphorus, aluminum, and sodium contents in soil. This enables accurate on-site analysis and is suitable for direct measurement on fresh, undried soil samples since the water content is quantified as well. For demonstration, 40 various Danish agricultural soil samples were analyzed using a mobile NMR sensor, and the results were compared with external laboratory analyses for P, Al, and Na. The laboratory analyses were conducted with ICP-OES after four-acid digestion, which additionally were compared with aqua regia digestion, showing inadequacies in the performance of the latter. Good agreement between NMR and laboratory analyses (correlation coefficients 0.91 for P, 0.98 for Al, and 0.90 for Na, in the concentration ranges 250-1200 ppm P, 1.4-5% Al, and 0.3-1% Na) were obtained with high accuracy using NMR measuring times of 20 min to 1 h for P, 4-12 min for Al, and 6-20 min for Na. Additionally, the NMR measurements provide information on the amount of P associated with paramagnetic centers (e.g., Fe3+). Good correlations were also observed to other parameters such as the clay content, which is predictable from the intensity of the more fast-relaxing of three 27Al NMR components.

A Clinical Diagnostic Test for Calcium Release Deficiency Syndrome.

Importance: Sudden death and cardiac arrest frequently occur without explanation, even after a thorough clinical evaluation. Calcium release deficiency syndrome (CRDS), a life-threatening genetic arrhythmia syndrome, is undetectable with standard testing and leads to unexplained cardiac arrest. Objective: To explore the cardiac repolarization response on an electrocardiogram after brief tachycardia and a pause as a clinical diagnostic test for CRDS. Design, Setting, and Participants: An international, multicenter, case-control study including individual cases of CRDS, 3 patient control groups (individuals with suspected supraventricular tachycardia; survivors of unexplained cardiac arrest [UCA]; and individuals with genotype-positive catecholaminergic polymorphic ventricular tachycardia [CPVT]), and genetic mouse models (CRDS, wild type, and CPVT were used to define the cellular mechanism) conducted at 10 centers in 7 countries. Patient tracings were recorded between June 2005 and December 2023, and the analyses were performed from April 2023 to December 2023. Intervention: Brief tachycardia and a subsequent pause (either spontaneous or mediated through cardiac pacing). Main Outcomes and Measures: Change in QT interval and change in T-wave amplitude (defined as the difference between their absolute values on the postpause sinus beat and the last beat prior to tachycardia). Results: Among 10 case patients with CRDS, 45 control patients with suspected supraventricular tachycardia, 10 control patients who experienced UCA, and 3 control patients with genotype-positive CPVT, the median change in T-wave amplitude on the postpause sinus beat (after brief ventricular tachycardia at ≥150 beats/min) was higher in patients with CRDS (P < .001). The smallest change in T-wave amplitude was 0.250 mV for a CRDS case patient compared with the largest change in T-wave amplitude of 0.160 mV for a control patient, indicating 100% discrimination. Although the median change in QT interval was longer in CRDS cases (P = .002), an overlap between the cases and controls was present. The genetic mouse models recapitulated the findings observed in humans and suggested the repolarization response was secondary to a pathologically large systolic release of calcium from the sarcoplasmic reticulum. Conclusions and Relevance: There is a unique repolarization response on an electrocardiogram after provocation with brief tachycardia and a subsequent pause in CRDS cases and mouse models, which is absent from the controls. If these findings are confirmed in larger studies, this easy to perform maneuver may serve as an effective clinical diagnostic test for CRDS and become an important part of the evaluation of cardiac arrest.

Interferon alpha-2 treatment reduces circulating neutrophil extracellular trap levels in myeloproliferative neoplasms.

Neutrophil extracellular traps (NETs) may play a pathogenic role in the thrombosis associated with myeloproliferative neoplasms (MPNs). We measured serum NET levels in 128 pretreatment samples from patients with MPNs and in 85 samples taken after 12 months of treatment with interferon alpha-2 (PEG-IFNα-2) formulations or hydroxyurea (HU). No differences in NET levels were observed across subdiagnoses or phenotypic driver mutations. In PV, a JAK2V617F+ allele burden ≥50% associated with increased NET levels (p = 0.006). Baseline NET levels correlated with neutrophil count (r = 0.29, p = 0.001), neutrophil-to-lymphocyte ratio (r = 0.26, p = 0.004) and JAK2V617F allele burden (r = 0.22, p = 0.03), particularly in patients with PV and with allele burden ≥50% (r = 0.50, p = 0.01, r = 0.56, p = 0.002 and r = 0.45, p = 0.03 respectively). In PV, after 12 months of treatment, NET levels decreased on average by 60% in patients with allele burden ≥50%, compared to only 36% in patients with an allele burden <50%. Overall, treatment with PEG-IFNα-2a or PEG-IFNα-2b reduced NETs levels in 77% and 73% of patients, respectively, versus only 53% of HU-treated patients (average decrease across treatments: 48%). Normalization of blood counts did not per se account for these reductions. In conclusion, baseline NET levels correlated with neutrophil count, NLR and JAK2V617F allele burden, and IFNα was more effective at reducing prothrombotic NET levels than HU.

Novel homozygous CD46 variant with C-isoform expression affects C3b inactivation in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy that may lead to organ failure. Dysregulation of the complement system can cause aHUS, and various disease-related variants in the complement regulatory protein CD46 are described. We here report a pediatric patient with aHUS carrying a hitherto unreported homozygous variant in CD46 (NM_172359.3:c.602C>T p.(Ser201Leu)). In our functional analyses, this variant caused complement dysregulation through three separate mechanisms. First, CD46 surface expression on the patient's blood cells was significantly reduced. Second, stably expressing CD46(Ser201Leu) cells bound markedly less to patterns of C3b than CD46 WT cells. Third, the patient predominantly expressed the rare isoforms of CD46 (C dominated) instead of the more common isoforms (BC dominated). Using BC1 and C1 expressing cell lines, we found that the C1 isoform bound markedly less C3b than the BC1 isoform. These results highlight the coexistence of multiple mechanisms that may act synergistically to disrupt CD46 function during aHUS development.

Prognosis of acute coronary syndrome stratified by cancer type and status - a nationwide cohort study.

BACKGROUND: To investigated the prognosis of the most prevalent cancers (breast-, gastrointestinal-, and lung cancer), according to cancer status (i.e., active-, non-active-, history of-, and no cancer), following first-time of acute coronary syndrome (ACS). METHODS: Danish nationwide registers were used to identify patients with first-time ACS from 2000-2018. Patients were stratified according to cancer type and status. Hazard ratios (HR) estimated by adjusted Cox regression models for 1year all-cause mortality reported. Further absolute risks of 1year cardiovascular versus non-cardiovascular death and 30-day cumulative incidence of coronary angiograms (CAG) was estimated, using the Aalen-Johansen non-parametric method, with competing risk of death. RESULTS: We identified 150,478 (95.7%) with no cancer, 2,370 (1.5%) with history of cancer, 2,712 (1.7%) with non-active cancer and 1,704 (1.1%) with active cancer. Cancer patients were older with more comorbidities than patients with no cancer. When compared with no cancer, we found HRs (95% confidence intervals) of 1.71 (1.44-2.02), 2.47 (2.23-2.73) and 4.22 (3.87-4.60) correspondingly for active breast-, gastrointestinal-, and lung cancer. Increased HRs were also found for non-active cancers, but not for history of cancer. Cardiovascular disease was the leading cause of death in all patients. Among patients with active breast-, gastrointestinal-, and lung cancer 43%, 43%, and 31% underwent CAG, correspondingly, compared with 77% of patients without cancer. CONCLUSIONS: Active- and non-active cancers were associated with an increased 1-year all-cause mortality compared with patients with history of cancer and no cancer. Cardiovascular disease was the leading cause of death; notably CAG was less frequently performed in cancer patients.

Amplicon-guided isolation and cultivation of previously uncultured microbial species from activated sludge.

IMPORTANCE: Biological wastewater treatment relies on complex microbial communities that assimilate nutrients and break down pollutants in the wastewater. Knowledge about the physiology and metabolism of bacteria in wastewater treatment plants (WWTPs) may therefore be used to improve the efficacy and economy of wastewater treatment. Our current knowledge is largely based on 16S rRNA gene amplicon profiling, fluorescence in situ hybridization studies, and predictions based on metagenome-assembled genomes. Bacterial isolates are often required to validate genome-based predictions as they allow researchers to analyze a specific species without interference from other bacteria and with simple bulk measurements. Unfortunately, there are currently very few pure cultures representing the microbes commonly found in WWTPs. To address this, we introduce an isolation strategy that takes advantage of state-of-the-art microbial profiling techniques to uncover suitable growth conditions for key WWTP microbes. We furthermore demonstrate that this information can be used to isolate key organisms representing global WWTPs.

Long-term stability of thyroid peroxidase antibody (anti-TPO) in serum in the Danish General Suburban Population Study.

OBJECTIVES: We evaluated the long-term stability of thyroid peroxidase antibody (anti-TPO). METHODS: In the Danish General Suburban Population Study (GESUS), serum samples were biobanked at -80 °C during 2010-2013. In a paired design with 70 subjects, we compared anti-TPO (30-198 U/mL) measured on fresh serum on Kryptor Classic in 2010-2011 (anti-TPOfresh) with anti-TPO remeasured on frozen serum (anti-TPOfrozen) on Kryptor Compact Plus in 2022. Both instruments used the same reagents and the anti-TPOn automated immunofluorescent assay, which was calibrated against the international standard NIBSC 66/387, based on the Time Resolved Amplified Cryptate Emission (TRACE) technology from BRAHMS. Values greater than 60 U/mL are regarded as positive in Denmark with this assay. Statistical comparisons included Bland-Altman, Passing-Bablok regression, and Kappa statistic. RESULTS: The mean follow-up time was 11.9 years (SD: 0.43). For anti-TPOfrozen vs. anti-TPOfresh, the line of equality was within the confidence interval of the absolute mean difference [5.71 (-0.32; 11.7) U/mL] and the average percentage deviation [+2.22% (-3.89%; +8.34%)]. The average percentage deviation of 2.22% did not exceed analytical variability. Passing-Bablok regression revealed both a statistically significant systematic and proportional difference: Anti-TPOfrozen=-22.6 + 1.22*(anti-TPOfresh). Frozen samples were correctly classified as positive in 64/70 (91.4%; Kappa=71.8%). CONCLUSIONS: Anti-TPO serum samples in the range 30-198 U/mL were stable after 12-years of storage at -80 °C with an estimated nonsignificant average percentage deviation of +2.22%. This comparison is based on Kryptor Classic and Kryptor Compact Plus, which used identical assays, reagents, and calibrator, but for which the agreement in the range 30-198 U/mL is unclarified.

Labor force participation during COVID-19 and risk of depression: a Danish register study.

BACKGROUND: COVID-19 caused economic insecurity for businesses and their employees. Understanding effects of changes in labor force participation on depression risk during economic recession is fundamental for early diagnosis. The study evaluates if changes in labor force participation are associated with depression risk during COVID-19 in Denmark. METHODS: A register-based longitudinal study of Danes aged 25-67 years without depression 2 years prior to baseline defined as February 2020. An eight-level categorical variable on stable or changing labor force participation was defined from monthly employment percentage gradients in the Danish Register-based Evaluation and Marginalization Database from February 2020. The cohort was followed until 31 December 2020 for depressions overall and mild-, moderate- and severe depression. Sex-stratified cox regression models with hazard ratios (HR) and 95% confidence intervals (95% CI) were performed accounting for important confounders. RESULTS: In total, 1 619 240 (50.3%) men of mean age 45.6 years and 1 598 587 (49.7%) women of mean age 45.9 years were included. Becoming unemployed implied an increased HR of depression in men (HR 2.02; 95% CI 1.94-2.10) and women (2.19; 2.12-2.26) compared to a steady-state full-time employment. Being outside the labor force or employed part-time implied an elevated HR in men (3.02; 2.82-3.23 and 2.41; 2.35-2.48) and women (3.13; 2.30-3.31 and 2.30; 2.26-2.35), respectively, compared to a steady-state full-time employment. CONCLUSIONS: Changes in labor force participation were associated with higher risk of depression relative to a steady-state full-time employment particularly among individuals with low labor force participation during COVID-19.

Attenuation of cGAS-STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1.

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62-deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy-associated vesicles. Thus, DNA sensing induces the cGAS-STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.

Indices of iron homeostasis in asymptomatic subjects with HFE mutations and moderate ferritin elevation during iron removal treatment.

We analysed iron biomarkers and their relationships in 30 subjects with HFE mutations and moderate hyperferritinaemia undergoing iron removal at our blood donation centre. Body mass index (BMI) and liver enzymes were assessed. Serum iron (SI), ferritin, transferrin saturation (TSAT), hepcidin and non-transferrin bound iron (NTBI) were measured serially. Seventeen subjects had p.C282Y/p.C282Y, nine p.C282Y/p.H63D, four p.H63D/p.H63D. Median age (p = 0.582), BMI (p = 0.500) and ferritin (p = 0.089) were comparable. At baseline, 12/17 p.C282Y/p.C282Y and 2/9 p.C282Y/p.H63D had measurable NTBI (p = 0.003). The p.C282Y/p.C282Y had higher TSAT (p < 0.001), lower hepcidin (p = 0.031) and hepcidin/ferritin ratio (p = 0.073). After treatment, iron indices were similar among groups, except TSAT (higher in p.C282Y/p.C282Y; p = 0.06). Strong relationships were observed between ferritin and TSAT (R = 0.71), NTBI and TSAT (R = 0.61), NTBI and SI (R = 0.54) in p.C282Y/p.C282Y. Hepcidin correlated weakly with ferritin in p.C282Y/p.C282Y (R = 0.37) but strongly in p.C282Y/p.H63D (R = 0.66) and p.H63D/p.H63D (R = 0.72), while relationships with TSAT were weak (R = 0.27), moderate (R = 0.55) and strong (R = 0.61), respectively. Low penetrance p.C282Y/p.C282Y phenotype displays hepcidin dysregulation and biochemical risk for iron toxicity.

Clonal haematopoiesis of indeterminate potential and impaired kidney function-A Danish general population study with 11 years follow-up.

The myeloproliferative neoplasms are associated with chronic kidney disease but whether clonal haematopoiesis of indeterminate potential (CHIP) is associated with impaired kidney function is unknown. In the Danish General Suburban Population Study (N = 19 958) from 2010 to 2013, 645 individuals were positive for JAK2V617F (N = 613) or CALR (N = 32) mutations. Mutation-positive individuals without haematological malignancy were defined as having CHIP (N = 629). We used multiple and inverse probability weighted (IPW)-adjusted linear regression analysis to estimate adjusted mean (95% confidence interval) differences in estimated glomerular filtration rate (eGFR; ml/min/1.73 m2 ) by mutation status, variant allele frequency (VAF%), blood cell counts, and neutrophil-to-lymphocyte ratio (NLR). We performed 11-year longitudinal follow-up of eGFR in all individuals. Compared to CHIP-negative individuals, the mean differences in eGFR were -5.6 (-10.3, -0.8, p = .02) for CALR, -11.9 (-21.4, -2.4, p = 0.01) for CALR type 2, and -10.1 (-18.1, -2.2, p = .01) for CALR with VAF ≥ 1%. The IPW-adjusted linear regression analyses showed similar results. NLR was negatively associated with eGFR. Individuals with CALR type 2 had a worse 11-year longitudinal follow-up on eGFR compared to CHIP-negative individuals (p = .004). In conclusion, individuals with CALR mutations, especially CALR type 2, had impaired kidney function compared to CHIP-negative individuals as measured by a lower eGFR at baseline and during 11-year follow-up.

Long-term outcomes in young patients with atrioventricular block of unknown aetiology.

AIMS: Atrioventricular block (AVB) of unknown aetiology is rare in the young, and outcome in these patients is unknown. We aimed to assess long-term morbidity and mortality in young patients with AVB of unknown aetiology. METHODS AND RESULTS: We identified all Danish patients younger than 50 years receiving a first pacemaker due to AVB between January 1996 and December 2015. By reviewing medical records, we included patients with AVB of unknown aetiology. A matched control cohort was established. Follow-up was performed using national registries. The primary outcome was a composite endpoint consisting of death, heart failure hospitalization, ventricular tachyarrhythmia, and cardiac arrest with successful resuscitation. We included 517 patients, and 5170 controls. Median age at first pacemaker implantation was 41.3 years [interquartile range (IQR) 32.7-46.2 years]. After a median follow-up of 9.8 years (IQR 5.7-14.5 years), the primary endpoint had occurred in 14.9% of patients and 3.2% of controls [hazard ratio (HR) 3.8; 95% confidence interval (CI) 2.9-5.1; P < 0.001]. Patients with persistent AVB at time of diagnosis had a higher risk of the primary endpoint (HR 10.6; 95% CI 5.7-20.0; P < 0.001), and risk was highest early in the follow-up period (HR 6.8; 95% CI 4.6-10.0; P < 0.001, during 0-5 years of follow-up). CONCLUSION: Atrioventricular block of unknown aetiology presenting before the age of 50 years and treated with pacemaker implantation was associated with a three- to four-fold higher rate of the composite endpoint of death or hospitalization for heart failure, ventricular tachyarrhythmia, or cardiac arrest with successful resuscitation. Patients with persistent AVB were at higher risk. These findings warrant improved follow-up strategies for young patients with AVB of unknown aetiology.

On-Site Measurement of Fat and Protein Contents in Milk Using Mobile NMR Technology.

Robust and easy-to-use NMR sensor technology is proposed for accurate, on-site determination of fat and protein contents in milk. The two parameters are determined using fast consecutive 1H and 35Cl low-field NMR experiments on milk samples upon the 1:1 addition of a low-cost contrast solution. Reliable and accurate measurements are obtained without tedious calibrations and the need for extensive database information and may readily be conducted by non-experts in production site environments. This enables on-site application at farms or dairies, or use in laboratories harvesting significant reductions in costs and time per analysis as compared to wet-chemistry analysis. The performance is demonstrated for calibration samples, various supermarket milk products, and raw milk samples, of which some were analyzed directly in the milking room. To illustrate the wide application range, the supermarket milk products included both conventionally/organically produced, lactose-free milk, cow's, sheep's and goat's milk, homogenized and unhomogenized milk, and a broad nutrient range (0.1-9% fat, 1-6% protein). Excellent agreement between NMR measurements and reference values, without corrections or changes in calibration for various products and during extensive periods of experiment conduction (4 months) demonstrates the robustness of the procedure and instrumentation. For the raw milk samples, correlations between NMR and IR, NMR and wet-chemistry, as well as IR and wet-chemistry results, show that NMR, in terms of accuracy, compares favorably with the other methods.

Mavacamten for treatment of symptomatic obstructive hypertrophic cardiomyopathy (EXPLORER-HCM): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Cardiac muscle hypercontractility is a key pathophysiological abnormality in hypertrophic cardiomyopathy, and a major determinant of dynamic left ventricular outflow tract (LVOT) obstruction. Available pharmacological options for hypertrophic cardiomyopathy are inadequate or poorly tolerated and are not disease-specific. We aimed to assess the efficacy and safety of mavacamten, a first-in-class cardiac myosin inhibitor, in symptomatic obstructive hypertrophic cardiomyopathy. METHODS: In this phase 3, randomised, double-blind, placebo-controlled trial (EXPLORER-HCM) in 68 clinical cardiovascular centres in 13 countries, patients with hypertrophic cardiomyopathy with an LVOT gradient of 50 mm Hg or greater and New York Heart Association (NYHA) class II-III symptoms were assigned (1:1) to receive mavacamten (starting at 5 mg) or placebo for 30 weeks. Visits for assessment of patient status occurred every 2-4 weeks. Serial evaluations included echocardiogram, electrocardiogram, and blood collection for laboratory tests and mavacamten plasma concentration. The primary endpoint was a 1·5 mL/kg per min or greater increase in peak oxygen consumption (pVO2) and at least one NYHA class reduction or a 3·0 mL/kg per min or greater pVO2 increase without NYHA class worsening. Secondary endpoints assessed changes in post-exercise LVOT gradient, pVO2, NYHA class, Kansas City Cardiomyopathy Questionnaire-Clinical Summary Score (KCCQ-CSS), and Hypertrophic Cardiomyopathy Symptom Questionnaire Shortness-of-Breath subscore (HCMSQ-SoB). This study is registered with ClinicalTrials.gov, NCT03470545. FINDINGS: Between May 30, 2018, and July 12, 2019, 429 adults were assessed for eligibility, of whom 251 (59%) were enrolled and randomly assigned to mavacamten (n=123 [49%]) or placebo (n=128 [51%]). 45 (37%) of 123 patients on mavacamten versus 22 (17%) of 128 on placebo met the primary endpoint (difference +19·4%, 95% CI 8·7 to 30·1; p=0·0005). Patients on mavacamten had greater reductions than those on placebo in post-exercise LVOT gradient (-36 mm Hg, 95% CI -43·2 to -28·1; p<0·0001), greater increase in pVO2 (+1·4 mL/kg per min, 0·6 to 2·1; p=0·0006), and improved symptom scores (KCCQ-CSS +9·1, 5·5 to 12·7; HCMSQ-SoB -1·8, -2·4 to -1·2; p<0·0001). 34% more patients in the mavacamten group improved by at least one NYHA class (80 of 123 patients in the mavacamten group vs 40 of 128 patients in the placebo group; 95% CI 22·2 to 45·4; p<0·0001). Safety and tolerability were similar to placebo. Treatment-emergent adverse events were generally mild. One patient died by sudden death in the placebo group. INTERPRETATION: Treatment with mavacamten improved exercise capacity, LVOT obstruction, NYHA functional class, and health status in patients with obstructive hypertrophic cardiomyopathy. The results of this pivotal trial highlight the benefits of disease-specific treatment for this condition. FUNDING: MyoKardia.

Cytokine and Gene Expression Profiling in Patients with HFE-Associated Hereditary Hemochromatosis according to Genetic Profile.

BACKGROUND: Hemochromatosis gene (HFE)-associated hereditary hemochromatosis (HH) is characterized by downregulation of hepcidin synthesis, leading to increased intestinal iron absorption. OBJECTIVES: The objectives were to characterize and elucidate a possible association between gene expression profile, hepcidin levels, disease severity, and markers of inflammation in HFE-associated HH patients. METHODS: Thirty-nine HFE-associated HH patients were recruited and assigned to 2 groups according to genetic profile: C282Y homozygotes in 1 group and patients with H63D, as homozygote or in combination with C282Y, in the other group. Eleven healthy first-time blood donors were recruited as controls. Gene expression was characterized from peripheral blood cells, and inflammatory cytokines and hepcidin-25 isoform were quantified in serum. Biochemical disease characteristics were recorded. RESULTS: Elevated levels of interleukin 8 were observed in a significant higher proportion of patients than controls. In addition, compared to controls, gene expression of ζ-globin was significantly increased among C282Y homozygote patients, while gene expression of matrix metalloproteinase 8, and other neutrophil-secreted proteins, was significantly upregulated in patients with H63D. CONCLUSION: Different disease signatures may characterize HH patients according to their HFE genetic profile. Studies on larger populations, including analyses at protein level, are necessary to confirm these findings.

Smoking, blood cells and myeloproliferative neoplasms: meta-analysis and Mendelian randomization of 2·3 million people.

Meta-analyses and Mendelian randomization (MR) may clarify the associations of smoking, blood cells and myeloproliferative neoplasms (MPN). We investigated the association of smoking with blood cells in the Danish General Suburban Population Study (GESUS, n = 11 083), by meta-analyses (including GESUS) of 92 studies (n = 531 741) and MR of smoking variant CHRNA3 (rs1051730[A]) in UK Biobank, and with MPN in a meta-analysis of six studies (n (total/cases):1 425 529/2187), totalling 2 307 745 participants. In the meta-analysis the random-effects standardized mean difference (SMD) in current smokers versus non-smokers was 0·82 (0·75-0·89, P = 2·0 * 10-108 ) for leukocytes, 0·09 (-0·02 to 0·21, P = 0·12) for erythrocytes, 0·53 (0·42-0·64, P = 8·0 * 10-22 ) for haematocrit, 0·42 (0·34-0·51, P = 7·1 * 10-21 ) for haemoglobin, 0·19 (0·08-0·31, P = 1·2 * 10-3 ) for mean corpuscular haemoglobin (MCH), 0·29 (0·19-0·39, P = 1·6 * 10-8 ) for mean corpuscular volume (MCV), and 0·04 (-0·04 to 0·13, P = 0·34) for platelets with trends for ever/ex-/current smokers, light/heavy smokers and female/male smokers. Analyses presented high heterogeneity but low publication bias. Per allele in CHRNA3, cigarettes per day in current smokers was associated with increased blood cell counts (leukocytes, neutrophils), MCH, red cell distribution width (RDW) and MCV. The pooled fixed-effects odds ratio for MPN was 1·44 [95% confidence interval (CI): 1·33-1·56; P = 1·8 * 10-19 ; I2  = 0%] in current smokers, 1·29 (1·15-1·44; P = 8·0 * 10-6 ; I2  = 0%) in ex-smokers, 1·49 (1·26-1·77; P = 4·4 * 10-6 ; I2  = 0%) in light smokers and 2·04 (1·74-2·39, P = 2·3 * 10-18 ; I2  = 51%) in heavy smokers compared with non-smokers. Smoking is observationally and genetically associated with increased leukocyte counts and red blood cell indices (MCH, MCV, RDW) and observationally with risk of MPN in current and ex-smokers versus non/never-smokers.

STING agonists enable antiviral cross-talk between human cells and confer protection against genital herpes in mice.

In recent years, there has been an increasing interest in immunomodulatory therapy as a means to treat various conditions, including infectious diseases. For instance, Toll-like receptor (TLR) agonists have been evaluated for treatment of genital herpes. However, although the TLR7 agonist imiquimod was shown to have antiviral activity in individual patients, no significant effects were observed in clinical trials, and the compound also exhibited significant side effects, including local inflammation. Cytosolic DNA is detected by the enzyme cyclic GMP-AMP (2'3'-cGAMP) synthase (cGAS) to stimulate antiviral pathways, mainly through induction of type I interferon (IFN)s. cGAS is activated upon DNA binding to produce the cyclic dinucleotide (CDN) 2'3'-cGAMP, which in turn binds and activates the adaptor protein Stimulator of interferon genes (STING), thus triggering type I IFN expression. In contrast to TLRs, STING is expressed broadly, including in epithelial cells. Here we report that natural and non-natural STING agonists strongly induce type I IFNs in human cells and in mice in vivo, without stimulating significant inflammatory gene expression. Systemic treatment with 2'3'-cGAMP reduced genital herpes simplex virus (HSV) 2 replication and improved the clinical outcome of infection. More importantly, local application of CDNs at the genital epithelial surface gave rise to local IFN activity, but only limited systemic responses, and this treatment conferred total protection against disease in both immunocompetent and immunocompromised mice. In direct comparison between CDNs and TLR agonists, only CDNs acted directly on epithelial cells, hence allowing a more rapid and IFN-focused immune response in the vaginal epithelium. Thus, specific activation of the STING pathway in the vagina evokes induction of the IFN system but limited inflammatory responses to allow control of HSV2 infections in vivo.

Pregnancies, ventricular arrhythmias, and substrate progression in women with arrhythmogenic right ventricular cardiomyopathy in the Nordic ARVC Registry.

AIMS: Women with arrhythmogenic right ventricular cardiomyopathy (ARVC) are at relatively lower risk of ventricular arrhythmias (VAs) than men, but the physical burden associated with pregnancy on VA risk remains insufficiently studied. We aimed to assess the risk of VA in relation to pregnancies in women with ARVC. METHODS AND RESULTS: We included 199 females with definite ARVC (n = 121) and mutation-positive family members without ascertained ARVC diagnosis (n = 78), of whom 120 had at least one childbirth. Ventricular arrhythmia-free survival after the latest childbirth was compared between women with one (n = 20), two (n = 67), and three or more (n = 37) childbirths. Cumulative probability of VA for each pregnancy (n = 261) was assessed from conception through 2 years after childbirth and compared between those pregnancies that occurred before (n = 191) or after (n = 19) ARVC diagnosis and in mutation-positive family members (n = 51). The nulliparous women had lower median age at ARVC diagnosis (38 vs. 42 years, P < 0.001) and first VA (22 vs. 41 years, P < 0.001). Ventricular arrhythmia-free survival after the latest childbirth was not related to the number of pregnancies. No pregnancy-related VA was reported among the family members. Women who gave birth after ARVC diagnosis had elevated risk of VA postpartum (hazard ratio 13.74, 95% confidence interval 2.9-63, P = 0.001), though only two events occurred during pregnancies. CONCLUSION: In women with ARVC, pregnancy was uneventful for the overwhelming majority and the number of prior completed pregnancies was not associated with VA risk. Pregnancy-related VA was primarily related to the phenotypical severity rather than pregnancy itself.