Fiber rich food suppressed airway inflammation, GATA3 + Th2 cells, and FcεRIα+ eosinophils in asthma.

Background: Allergic Asthma is a disease presenting various endotypes and no current therapies act curative but alleviate disease symptoms. Dietary interventions are gaining increasing importance in regulating immune responses. Furthermore, short chain fatty acids (SFCA), as the main products of dietary fiber's fermentation by the gut bacteria, ameliorate the pathogenesis and disease burden of different illnesses including asthma. Nevertheless, the connection and crosstalk between the gut and lung is poorly understood. Objective: In this work, the role of high fiber diet on the development of allergic asthma at baseline and after exacerbation of disease induced by respiratory viruses was investigated. Methods: Hereby, SCFA in serum of asthmatic and non-asthmatic pre-school children before and after airway disease symptoms were analyzed. Moreover, the effect of high fiber diet in vivo in a murine model of house dust mite extract (HDM) induced allergic asthma and in the end in isolated lung and spleen cells infected ex vivo with Rhinovirus was analyzed. Results: In this study, a decrease of the SCFA 3-Hydroxybutyric acid in serum of asthmatic children after symptomatic episodes at convalescent visit as compared to asthmatic and control children at baseline visit was observed. In experimental asthma, in mice fed with high fiber diet, a reduced lung GATA3 + Th2 type mediated inflammation, mucus production and collagen deposition and expression of Fc epsilon receptor Ia (FcεRIa) in eosinophils was observed. By contrast, the CD8+ memory effector T cells were induced in the lungs of asthmatic mice fed with high fiber diet. Then, total lung cells from these asthmatic mice fed with either standard food or with fiber rich food were infected with RV ex vivo. Here, RV1b mRNA was found significantly reduced in the lung cells derived from fiber rich food fed mice as compared to those derived from standard food fed asthmatic mice. Looking for the mechanism, an increase in CD8+ T cells in RV infected spleen cells derived from fiber rich fed asthmatic mice, was observed. Conclusion: Convalescent preschool asthmatic children after a symptomatic episode have less serum ß-Hydroxybutyric acid as compared to control and asthmatic children at baseline visit. Fiber rich diet associated with anti-inflammatory effects as well as anti-allergic effects by decreasing Type 2 and IgE mediated immune responses and inducing CD8+ memory effector T cells in a murine model of allergic asthma. Finally, ex vivo infection with Rhinovirus (RV) of total lung cells from asthmatic mice fed with fiber rich food led to a decreased RV load as compared to mice fed with standard food. Moreover, spleen cells derived from asthmatic mice fed with fiber rich food induced CD8+ T cells after ex vivo infection with RV. Clinical implications: Dietary interventions with increased content in natural fibers like pectins would ameliorate asthma exacerbations. Moreover, respiratory infection in asthma downregulated SCFA in the gut contributing to asthma exacerbations.

Water Conservation Overrides Osmotic Diuresis During SGLT2 Inhibition in Patients With Heart Failure.

BACKGROUND: Sodium-glucose cotransporter 2 inhibitors are believed to improve cardiac outcomes due to their osmotic diuretic potential. OBJECTIVES: The goal of this study was to test the hypothesis that vasopressin-driven urine concentration overrides the osmotic diuretic effect of glucosuria induced by dapagliflozin treatment. METHODS: DAPA-Shuttle1 (Hepato-renal Regulation of Water Conservation in Heart Failure Patients With SGLT-2 Inhibitor Treatment) was a single-center, double-blind, randomized, placebo-controlled trial, in which patients with chronic heart failure NYHA functional classes I/II and reduced ejection fraction were randomly assigned to receive dapagliflozin 10 mg daily or placebo (1:1) for 4 weeks. The primary endpoint was change from baseline in urine osmolyte concentration. Secondary endpoints included changes in copeptin levels and solute free water clearance. RESULTS: Thirty-three randomized, sodium-glucose cotransporter 2 inhibitor-naïve participants completed the study, 29 of whom (placebo: n = 14; dapagliflozin: n = 15) provided accurate 24-hour urine collections (mean age 59 ± 14 years; left ventricular ejection fraction 31% ± 9%). Dapagliflozin treatment led to an isolated increase in urine glucose excretion by 3.3 mmol/kg/d (95% CI: 2.51-4.04; P < 0.0001) within 48 hours (early) which persisted after 4 weeks (late; 2.7 mmol/kg/d [95% CI: 1.98-3.51]; P < 0.0001). Dapagliflozin treatment increased serum copeptin early (5.5 pmol/L [95% CI: 0.45-10.5]; P < 0.05) and late (7.8 pmol/L [95% CI: 2.77-12.81]; P < 0.01), leading to proportional reductions in free water clearance (early: -9.1 mL/kg/d [95% CI: -14 to -4.12; P < 0.001]; late: -11.0 mL/kg/d [95% CI: -15.94 to -6.07; P < 0.0001]) and elevated urine concentrations (late: 134 mmol/L [95% CI: 39.28-229.12]; P < 0.01). Therefore, urine volume did not significantly increase with dapagliflozin (mean difference early: 2.8 mL/kg/d [95% CI: -1.97 to 7.48; P = 0.25]; mean difference late: 0.9 mL/kg/d [95% CI: -3.83 to 5.62]; P = 0.70). CONCLUSIONS: Physiological-adaptive water conservation eliminated the expected osmotic diuretic potential of dapagliflozin and thereby prevented a glucose-driven increase in urine volume of approximately 10 mL/kg/d · 75 kg = 750 mL/kg/d. (Hepato-renal Regulation of Water Conservation in Heart Failure Patients With SGLT-2 Inhibitor Treatment [DAPA-Shuttle1]; NCT04080518).

Imatinib treatment and longitudinal growth in pediatric patients with chronic myeloid leukemia: Influence of demographic, pharmacological, and genetic factors in the German CML-PAED cohort.

In children and adolescents, impaired growth due to tyrosine kinase inhibitor therapy remains an insufficiently studied adverse effect. This study examines demographic, pharmacological, and genetic factors associated with impaired longitudinal growth in a uniform pediatric cohort treated with imatinib. We analyzed 94 pediatric patients with chronic myeloid leukemia (CML) diagnosed in the chronic phase and treated with imatinib for >12 months who participated in the Germany-wide CML-PAEDII study between February 2006 and February 2021. During imatinib treatment, significant height reduction occurred, with medians of -0.35 standard deviation score (SDS) at 12 months and -0.76 SDS at 24 months. Cumulative height SDS change (Δheight SDS) showed a more pronounced effect in prepubertal patients during the first year but were similar between prepubertal and pubertal subgroups by the second year (-0.55 vs. -0.50). From months 12 to 18 on imatinib, only 18% patients achieved individually longitudinal growth adequate to the growth standard (Δheight SDS≥0). When patients were divided into two subgroups based on median Δheight SDS (classifier Δheight SDS > or ≤-0.37) after one year on imatinib therapy, cohort 1 (Δheight SDS extending -0.37) showed younger age at diagnosis, a higher proportion of prepubertal children, but also better treatment response and higher imatinib serum levels. Exploring the association of growth parameters with pharmacokinetically relevant single nucleotide polymorphisms, known for affecting imatinib response, showed no correlation. This retrospective study provides new insights into imatinib-related growth impairment. We emphasize the importance of optimizing treatment strategies for pediatric patients to realize their maximum growth potential.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Evaluation of five multisteroid LC‒MS/MS methods used for routine clinical analysis: comparable performance was obtained for nine analytes.

OBJECTIVES: A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step. METHODS: All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone. RESULTS: Good or acceptable overall comparability was found in Bland‒Altman and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max. -18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals. CONCLUSIONS: This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes.

Routine Surveillance of SARS-CoV-2 Serostatus in Pediatrics Allows Monitoring of Humoral Response.

The occurrence of SARS-CoV-2 infections during the pandemic was mainly based on PCR testing of symptomatic patients. However, with new variants, vaccinations, and the changing of the clinical disease severity, knowledge about general immunity is elusive. For public health systems, timely knowledge of these conditions is essential, but it is particularly scarce for the pediatric population. Therefore, in this study, we wanted to investigate the spike and nucleocapsid seroprevalence in pediatric patients using routine residual blood tests collected during the pandemic. This prospective observational study was conducted over seven one-month periods. Herein, the latest four time periods (November 2021, January 2022, March 2022, and May 2022) are depicted. Each patient of a tertiary-care center in Germany was anonymized after collection of clinical diagnosis (ICD-10) and then routinely tested for the respective spike and nucleocapsid SARS-CoV-2 antibody titer. A total of 3235 blood samples from four time periods were included. Spike seroprevalence rose from 37.6% to 51.9% to 70.5% to 85.1% and nucleocapsid seroprevalence from 11.6% to 17.0% to 36.7% to 58.1% in May 2022. In detail, significant changes in seroprevalence between age groups but not between sex or diagnosis groups were found. Quantitative measures revealed rising spike and constant nucleocapsid antibody levels over the pandemic with a half-life of 102 days for spike and 45 days for nucleocapsid antibodies. Routine laboratory assessment of SARS-CoV-2 in residual blood specimens of pediatric hospitals enables monitoring of the seroprevalence and may allow inferences about general immunity in this cohort.

Vitamin D3 resolved human and experimental asthma via B lymphocyte-induced maturation protein 1 in T cells and innate lymphoid cells.

Background: Vitamin D3 (VitD3) is known to have immunomodulatory functions, and VitD3 deficiency is associated with more severe asthma. Objective: We aimed to assess the immunoregulatory effects of VitD3 food supplementation on asthma manifestation, with particular focus on T cells and type 2 innate lymphoid cells. Methods: Preschool children and adult asthmatic cohorts were analyzed in the context of VitD3 supplementation and serum levels. In a murine model of ovalbumin-induced asthma, effects of diet VitD3 sufficiency and deficiency on T cells and type 2 innate lymphoid cells immune mechanisms were investigated. Results: We found less severe and better-controlled asthma phenotypes along with reduced need for steroid medication in preschool children and asthmatic adults with VitD3 supplementation. VitD3 serum levels correlated with B lymphocyte-induced maturation protein 1 (Blimp-1) expression in blood peripheral mononuclear cells. VitD3-supplement-fed mice showed decreased asthmatic traits, with a decrease in IgE serum levels, reduced airway mucus, and increased IL-10 production by lung cells. Furthermore, we discovered an upregulation of effector T cells and Blimp-1+ lung tissue-resident memory T cells as well as induction of anti-inflammatory Blimp-1+ lung innate lymphoid cells producing IL-10. Conclusion: Supplementing VitD3 resulted in amelioration of clinical asthma manifestations in human studies as well as in experimental allergic asthma, indicating that VitD3 shifts proinflammatory immune responses to anti-inflammatory immune responses via upregulating Blimp-1 in lung innate lymphoid cells and tissue-resident memory cells.

A pipeline for the fully automated estimation of continuous reference intervals using real-world data.

Reference intervals are essential for interpreting laboratory test results. Continuous reference intervals precisely capture physiological age-specific dynamics that occur throughout life, and thus have the potential to improve clinical decision-making. However, established approaches for estimating continuous reference intervals require samples from healthy individuals, and are therefore substantially restricted. Indirect methods operating on routine measurements enable the estimation of one-dimensional reference intervals, however, no automated approach exists that integrates the dependency on a continuous covariate like age. We propose an integrated pipeline for the fully automated estimation of continuous reference intervals expressed as a generalized additive model for location, scale and shape based on discrete model estimates using an indirect method (refineR). The results are free of subjective user-input, enable conversion of test results into z-scores and can be integrated into laboratory information systems. Comparison of our results to established and validated reference intervals from the CALIPER and PEDREF studies and manufacturers' package inserts shows good agreement of reference limits, indicating that the proposed pipeline generates high-quality results. In conclusion, the developed pipeline enables the generation of high-precision percentile charts and continuous reference intervals. It represents the first parameter-less and fully automated solution for the indirect estimation of continuous reference intervals.

Pharmacokinetic-pharmacodynamic modeling of maintenance therapy for childhood acute lymphoblastic leukemia.

In the treatment of childhood acute lymphoblastic leukemia (ALL), current protocols combine initial high-dose multiagent chemotherapy with prolonged oral therapy with 6-mercaptopurine (6MP) and low-dose methotrexate (MTX) maintenance therapy. Decades of research on ALL treatment have resulted in survival rates of approximately 90%. However, dose-response relationships vary widely between patients and insight into the influencing factors, that would allow for improved personalized treatment management, is insufficient. We use a detailed data set with measurements of thioguanine nucleotides and MTX in red blood cells and absolute neutrophil count (ANC) to develop pharmacokinetic models for 6MP and MTX, as well as a pharmacokinetic-pharmacodynamic (PKPD) model capable of predicting individual ANC levels and thus contributing to the development of personalized treatment strategies. Here, we show that integrating metabolite measurements in red blood cells into the full PKPD model improves results when less data is available, but that model predictions are comparable to those of a fixed pharmacokinetic model when data availability is not limited, providing further evidence of the quality of existing models. With this comprehensive model development leading to dynamics similar to simpler models, we validate the suitability of this model structure and provide a foundation for further exploration of maintenance therapy strategies through simulation and optimization.

Frequency and impact of enteric hyperoxaluria in pediatric short bowel syndrome: a retrospective single centre study.

Objectives: The survival of pediatric patients with short bowel syndrome has improved in recent years. Enteric hyperoxaluria as a pathophysiological consequence has been hardly addressed so far. It can be associated with nephrolithiasis, nephrocalcinosis or even renal insufficiency. We assessed the prevalence of hyperoxaluria and its pathogenic consequences in a retrospective single centre study over the last 12 years. Methods: We conducted an internal database search for all pediatric patients suffering from short bowel syndrome treated from 2010 to 2022 in the department of pediatric gastroenterology as well as the pediatric nephrology and dialysis unit. Out of 56 patients identified, 26 patients were analysed for etiology of short bowel syndrome, renal excretion of oxalate (24/26), remaining short bowel and large intestinal length as well as further clinical parameters such as eGFR, nephrocalcinosis/urinary stone formation or stool frequency. Results: Hyperoxaluria was detected in 14/26 patients (54%). Nephrocalcinosis was present in four patients. Out of these four patients, hyperoxaluria could be proven (21% of all hyperoxaluric patients) in three cases, one hyperoxaluric patient had nephrolithiasis (7%). In one patient hyperoxaluria lead to end stage renal disease. We found that 80% of patients with volvulus developed enteric hyperoxaluria. None of the investigated factors had an effect on oxalate excretion. Conclusion: Enteric hyperoxaluria is a relevant pathophysiological finding in patients with short bowel syndrome occurring in about 50% of our cohort with multiple pathogenic complications. Regular screening for hyperoxaluria may be implemented in medical care for patients with short bowel syndrome. If necessary, prophylaxis, e.g., dietary advice or metaphylaxis should be initiated.

Development of a rapid and specific MALDI-TOF mass spectrometric assay for SARS-CoV-2 detection.

We have developed a rapid and highly specific assay for detecting and monitoring SARS-CoV-2 infections by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As MALDI-TOF mass spectrometers are available in a clinical setting, our assay has the potential to serve as alternative to the commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sample preparation prior to MALDI-TOF-MS involves the tryptic digestion of SARS-CoV-2 proteins, followed by an enrichment of virus-specific peptides from SARS-CoV-2 nucleoprotein via magnetic antibody beads. Our MALDI-TOF-MS method allows the detection of SARS-CoV-2 nucleoprotein in sample collection medium as low as 8 amol/µl. MALDI-TOF mass spectra are obtained in just a few seconds, which makes our MS-based assay suitable for a high-throughput screening of SARS-CoV-2 in healthcare facilities in addition to PCR. Due to the specific detection of virus peptides, different SARS-CoV-2 variants are readily distinguished from each other. Specifically, we show that our MALDI-TOF-MS assay discriminates SARS-CoV-2 strain B.1.617.2 "delta variant" from all other variants in patients' samples, making our method highly valuable to monitor the emergence of new virus variants.

L-arginine metabolism as pivotal interface of mutual host-microbe interactions in the gut.

L-arginine (L-arg) is a versatile amino acid and a central intestinal metabolite in mammalian and microbial organisms. Thus, L-arg participates as precursor of multiple metabolic pathways in the regulation of cell division and growth. It also serves as a source of carbon, nitrogen, and energy or as a substrate for protein synthesis. Consequently, L-arg can simultaneously modify mammalian immune functions, intraluminal metabolism, intestinal microbiota, and microbial pathogenesis. While dietary intake, protein turnover or de novo synthesis usually supply L-arg in sufficient amounts, the expression of several key enzymes of L-arg metabolism can change rapidly and dramatically following inflammation, sepsis, or injury. Consequently, the availability of L-arg can be restricted due to increased catabolism, transforming L-arg into an essential amino acid. Here, we review the enzymatic pathways of L-arg metabolism in microbial and mammalian cells and their role in immune function, intraluminal metabolism, colonization resistance, and microbial pathogenesis in the gut.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

OBJECTIVES: An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. METHODS: The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5 days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. RESULTS: The RMP allowed aldosterone quantification within the range of 20-1,200 pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was ≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between -2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be ≤2.3%; expanded uncertainties were ≤4.6% (k=2) for all levels. CONCLUSIONS: The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

Increase of liver stiffness and altered bile acid metabolism after triple CFTR modulator initiation in children and young adults with cystic fibrosis.

BACKGROUND: Novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies (elexacaftor/tezacaftor/ivacaftor-ETI) promise clinically significant and sustained improvements for patients with cystic fibrosis (CF). In this study, we investigated the impact of ETI therapy on liver stiffness and bile acid metabolism in a cohort of children and young adults with CF. METHODS: A prospective observational study (NCT05576324) was conducted from September 2020 to November 2021 enrolling CF patients naive to ETI. Standard laboratory chemistry, sweat test, lung function, share wave velocity (SWV) derived by acoustic radiation force impulse imaging (ARFI) and serum bile acid profiles were assessed before and 6 months after induction of ETI therapy. RESULTS: A total of 20 patients (10 aged <20 years) completed the study. While lung function and BMI improved after ETI therapy, ARFI SWV increased in CF patients <20 years of age (from 1.27 to 1.43 m/s, p = 0.023). Bile acid (BA) profiles revealed a decrease in unconjugated (5.75 vs 1.46, p = 0.007) and increase in glycine-conjugated derivatives (GCDCA) (4.79 vs 6.64 p = 0.016). There was a positive correlation between ARFI SWV values and GCDCA (r = 0.80, p < 0.0001). Glycine-conjugated BA provided high diagnostic accuracy to predict increased ARFI measurements (AUC 0.90) and clinical (Colombo) CFLD grading (AUC 0.97). CONCLUSIONS: ARFI SWV and bile acid profiles provide evidence for early increase in liver stiffness and altered bile acid metabolism in young CF patients after initiation of ETI and may serve as synergistic measures for detection of hepatic complications during ETI therapy.

Angiotensin pathways under therapy with empagliflozin in patients with chronic heart failure.

AIMS: Large outcome studies demonstrated a reduction of heart failure hospitalization or cardiovascular death in patients with chronic heart failure (CHF). The renin-angiotensin system (RAS) is a key player in fluid and sodium regulation. The classic angiotensin-converting enzyme-angiotensin II-angiotensin-1 receptor axis (Ang I-ACE-Ang II receptor axis) is predominantly angiotensin II (Ang-II) induced and promotes vasoconstriction. In contrast, the angiotensin-converting-enzyme-2-angiotensin-(1-7)-Mas axis (Mas-axis) is mediated by the metabolites angiotensin-1-7 (Ang-(1-7)) and angtiotensin-1-5 (Ang-(1-5)) and exerts cardioprotective effects. METHODS: We previously investigated the effect of empagliflozin on the systemic haemodynamic in patients with stable CHF (NYHA II-III) in a randomized placebo-controlled clinical trial 'Analysing the Effect of Empagliflozin on Reduction of Tissue Sodium Content in Patients With Chronic Heart Failure (ELSI)'. In a post hoc analysis, we now analysed whether empagliflozin has an effect on the RAS by measuring detailed RAS profiles (LC-MS/MS-based approach) in 72 patients from ELSI. We compared RAS parameters after 1-month and 3-months treatment with empagliflozin or placebo to baseline. The secondary goal was to analyse whether the effect of empagliflozin on RAS parameters was dependent on angiotensin-receptor-blocking (ARB) or angiotensin-converting-enzyme-inhibitor (ACEI) co-medication. RESULTS: Empagliflozin medication induced a significant rise in Ang-II [68.5 pmol/L (21.3-324.2) vs. 131.5 pmol/L (34.9-564.0), P = 0.001], angiotensin-I (Ang-I) [78.7 pmol/L (21.5-236.6) vs. 125.9 pmol/L (52.6-512.9), P < 0.001], Ang-(1-7) [3.0 pmol/L (3.0-15.0) vs. 10.1 pmol/L (3.0-31.3), P = 0.006], and Ang-(1-5) [5.4 pmol/L (2.0-22.9) vs. 9.9 pmol/L (2.8-36.4), P = 0.004], which was not observed in the placebo group (baseline to 3-months treatment). A significant rise in Ang-II (206.4 pmol/L (64.2-750.6) vs. 568.2 pmol/L (164.7-1616.4), P = 0.001), Ang-(1-7) (3.0 pmol/L (3.0-14.1) vs. 15.0 pmol/L (3.0-31.3), P = 0.017), and Ang-(1-5) [12.2 pmol/L (3.8-46.6) vs. 36.4 pmol/L (11.1-90.7), P = 0.001] under empagliflozin treatment was only seen in the subgroup of patients with ARB co-medication, whereas no change of Ang-II (16.7 pmol/L (2.0-60.8) vs. 26.4 pmol/L (10.7-63.4), P = 0.469), Ang-(1-7) (6.6 pmol/L (3.0-20.7) vs. 10.5 pmol/L (3.0-50.5), P = 0.221), and Ang-(1-5) (2.7 pmol/L (2.0-8.4) vs. 2.8 pmol/L (2.0-6.9), P = 0.851) was observed in patients with empagliflozin that were on ACEI co-medication (baseline to 3-months treatment). CONCLUSIONS: Our data indicate that empagliflozin might lead to an activation of both the Ang I-ACE-Ang II receptor axis and the Mas-axis pathway. Activation of the Ang I-ACE-Ang II receptor axis and the protective Mas-axis pathway after initiating treatment with empagliflozin was only seen in patients with ARB co-medication, in contrast to co-medication with ACEI.

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

Pulmonary Dysfunction after Pediatric COVID-19.

Background Long COVID occurs at a lower frequency in children and adolescents than in adults. Morphologic and free-breathing phase-resolved functional low-field-strength MRI may help identify persistent pulmonary manifestations after SARS-CoV-2 infection. Purpose To characterize both morphologic and functional changes of lung parenchyma at low-field-strength MRI in children and adolescents with post-COVID-19 condition compared with healthy controls. Materials and Methods Between August and December 2021, a cross-sectional clinical trial using low-field-strength MRI was performed in children and adolescents from a single academic medical center. The primary outcome was the frequency of morphologic changes at MRI. Secondary outcomes included MRI-derived functional proton ventilation and perfusion parameters. Clinical symptoms, the duration from positive reverse transcriptase-polymerase chain reaction test result, and serologic parameters were compared with imaging results. Nonparametric tests for pairwise and corrected tests for groupwise comparisons were applied to assess differences in healthy controls, recovered participants, and those with long COVID. Results A total of 54 participants after COVID-19 infection (mean age, 11 years ± 3 [SD]; 30 boys [56%]) and nine healthy controls (mean age, 10 years ± 3; seven boys [78%]) were included: 29 (54%) in the COVID-19 group had recovered from infection and 25 (46%) were classified as having long COVID on the day of enrollment. Morphologic abnormality was identified in one recovered participant. Both ventilated and perfused lung parenchyma (ventilation-perfusion [V/Q] match) was higher in healthy controls (81% ± 6.1) compared with the recovered group (62% ± 19; P = .006) and the group with long COVID (60% ± 20; P = .003). V/Q match was lower in patients with time from COVID-19 infection to study participation of less than 180 days (63% ± 20; P = .03), 180-360 days (63% ± 18; P = .03), and 360 days (41% ± 12; P < .001) as compared with the never-infected healthy controls (81% ± 6.1). Conclusion Low-field-strength MRI showed persistent pulmonary dysfunction in children and adolescents who recovered from COVID-19 and those with long COVID. Clinical trial registration no. NCT04990531 © RSNA, 2022 Supplemental material is available for this article. See also the editorial by Paltiel in this issue.

RIbench: A Proposed Benchmark for the Standardized Evaluation of Indirect Methods for Reference Interval Estimation.

BACKGROUND: Indirect methods leverage real-world data for the estimation of reference intervals. These constitute an active field of research, and several methods have been developed recently. So far, no standardized tool for evaluation and comparison of indirect methods exists. METHODS: We provide RIbench, a benchmarking suite for quantitative evaluation of any existing or novel indirect method. The benchmark contains simulated test sets for 10 biomarkers mimicking routine measurements of a mixed distribution of non-pathological (reference) values and pathological values. The non-pathological distributions represent 4 common distribution types: normal, skewed, heavily skewed, and skewed-and-shifted. To identify strengths and weaknesses of indirect methods, test sets have varying sample sizes and pathological distributions differ in location, extent of overlap, and fraction. For performance evaluation, we use an overall benchmark score and sub-scores derived from absolute z-score deviations between estimated and true reference limits. We illustrate the application of RIbench by evaluating and comparing the Hoffmann method and 4 modern indirect methods -TML (Truncated-Maximum-Likelihood), kosmic, TMC (Truncated-Minimum-Chi-Square), and refineR- against one another and against a nonparametric direct method (n = 120). RESULTS: For the modern indirect methods, pathological fraction and sample size had a strong influence on the results: With a pathological fraction up to 20% and a minimum sample size of 5000, most methods achieved results comparable or superior to the direct method. CONCLUSIONS: We present RIbench, an open-source R-package, for the systematic evaluation of existing and novel indirect methods. RIbench can serve as a tool for enhancement of indirect methods, improving the estimation of reference intervals.

Elevated n-3/n-6 PUFA ratio in early life diet reverses adverse intrauterine kidney programming in female rats.

Intrauterine growth restriction (IUGR) predisposes to chronic kidney disease via activation of proinflammatory pathways, and omega-3 PUFAs (n-3 PUFAs) have anti-inflammatory properties. In female rats, we investigated 1) how an elevated dietary n-3/n-6 PUFA ratio (1:1) during postnatal kidney development modifies kidney phospholipid (PL) and arachidonic acid (AA) metabolite content and 2) whether the diet counteracts adverse molecular protein signatures expected in IUGR kidneys. IUGR was induced by bilateral uterine vessel ligation or intrauterine stress through sham operation 3.5 days before term. Control (C) offspring were born after uncompromised pregnancy. On postnatal (P) days P2-P39, rats were fed control (n-3/n-6 PUFA ratio 1:20) or n-3 PUFA intervention diet (N3PUFA; ratio 1:1). Plasma parameters (P33), kidney cortex lipidomics and proteomics, as well as histology (P39) were studied. We found that the intervention diet tripled PL-DHA content (PC 40:6; P < 0.01) and lowered both PL-AA content (PC 38:4 and lyso-phosphatidylcholine 20:4; P < 0.05) and AA metabolites (HETEs, dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acids) to 25% in all offspring groups. After ligation, our network analysis of differentially expressed proteins identified an adverse molecular signature indicating inflammation and hypercoagulability. N3PUFA diet reversed 61 protein alterations (P < 0.05), thus mitigating adverse IUGR signatures. In conclusion, an elevated n-3/n-6 PUFA ratio in early diet strongly reduces proinflammatory PLs and mediators while increasing DHA-containing PLs regardless of prior intrauterine conditions. Counteracting a proinflammatory hypercoagulable protein signature in young adult IUGR individuals through early diet intervention may be a feasible strategy to prevent developmentally programmed kidney damage in later life.

Microbiota-Derived Propionate Modulates Megakaryopoiesis and Platelet Function.

Rheumatoid arthritis (RA) is associated with an increased risk for cardiovascular events driven by abnormal platelet clotting effects. Platelets are produced by megakaryocytes, deriving from megakaryocyte erythrocyte progenitors (MEP) in the bone marrow. Increased megakaryocyte expansion across common autoimmune diseases was shown for RA, systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS). In this context, we evaluated the role of the microbial-derived short chain fatty acid (SCFA) propionate on hematopoietic progenitors in the collagen induced inflammatory arthritis model (CIA) as we recently showed attenuating effects of preventive propionate treatment on CIA severity. In vivo, propionate treatment starting 21 days post immunization (dpi) reduced the frequency of MEPs in the bone marrow of CIA and naïve mice. Megakaryocytes numbers were reduced but increased the expression of the maturation marker CD61. Consistent with this, functional analysis of platelets showed an upregulated reactivity state following propionate-treatment. This was confirmed by elevated histone 3 acetylation and propionylation as well as by RNAseq analysis in Meg-01 cells. Taken together, we identified a novel nutritional axis that skews platelet formation and function.