Effect of dexamethasone in patients with ARDS and COVID-19 (REMED trial)—study protocol for a prospective, multi-centre, open-label, parallel-group, randomized controlled trial

BACKGROUND: Since December 2019, SARS-CoV-2 virus has infected millions of people worldwide. In patients with COVID-19 pneumonia in need of oxygen therapy or mechanical ventilation, dexamethasone 6 mg per day is currently recommended. However, the dose of 6 mg of dexamethasone is currently being reappraised and may miss important therapeutic potential or may prevent potential deleterious effects of higher doses of corticosteroids. METHODS: REMED is a prospective, open-label, randomised controlled trial testing the superiority of dexamethasone 20 mg (dexamethasone 20 mg on days 1-5, followed by dexamethasone 10 mg on days 6-10) vs 6 mg administered once daily intravenously for 10 days in adult patients with moderate or severe ARDS due to confirmed COVID-19. Three hundred participants will be enrolled and followed up for 360 days after randomization. Patients will be randomised in a 1:1 ratio into one of the two treatment arms. The following stratification factors will be applied: age, Charlson Comorbidity Index, CRP levels and trial centre. The primary endpoint is the number of ventilator-free days (VFDs) at 28 days after randomisation. The secondary endpoints are mortality from any cause at 60 days after randomisation; dynamics of the inflammatory marker, change in WHO Clinical Progression Scale at day 14; and adverse events related to corticosteroids and independence at 90 days after randomisation assessed by the Barthel Index. The long-term outcomes of this study are to assess long-term consequences on mortality and quality of life at 180 and 360 days. The study will be conducted in the intensive care units (ICUs) of ten university hospitals in the Czech Republic. DISCUSSION: We aim to compare two different doses of dexamethasone in patients with moderate to severe ARDS undergoing mechanical ventilation regarding efficacy and safety. TRIAL REGISTRATION: EudraCT No. 2020-005887-70. ClinicalTrials.gov NCT04663555. Registered on December 11, 2020

Postoperative inflammation and insulin resistance in relation to body composition, adiposity and carbohydrate treatment: a randomised controlled study

Background and Aims: The aims of this study were to identify whether differences in distribution of adipose tissue and skeletal muscle in obese and non-obese individuals contribute to the magnitude of the postoperative inflammatory response and insulin resistance, with and without preoperative treatment with carbohydrate drinks. Methods: Thirty-two adults (16 obese/16 non-obese) undergoing elective major open abdominal surgery participated in this 22 factorial, randomised, double-blind, placebo-controlled study. Participants received Nutricia preOp® or placebo (800 ml on the night before surgery/400 ml 2-3 h preoperatively) after stratifying for obesity. Insulin sensitivity was measured using the hyperinsulinaemic-euglycaemic clamp pre- and postoperatively. Vastus lateralis, omental and subcutaneous fat biopsies were taken pre- and postoperatively and analysed after RNA extraction. The primary endpoint was within subject differences in insulin sensitivity. Results: Major abdominal surgery was associated with a 42% reduction in insulin sensitivity from mean(SD) M value of 37.3(11.8) μmol kg-1 fat free mass (FFM) to 21.7(7.4) μmol kg-1 67 FFM, but this was not influenced by obesity or preoperative carbohydrate treatment. Activation of the triggering receptor expressed on myeloid cells (TREM1) pathway was seen in response to surgery in omental fat samples. In postoperative muscle samples, gene expression differences indicated activation of the peroxisome proliferator-activated receptor (PPAR-α)/retinoid X-receptor (RXR-α) pathway in obese but not in non-obese participants. There were no significant changes in gene expression pathways associated with carbohydrate treatment. Conclusion: The reduction in insulin sensitivity associated with major abdominal surgery was confirmed but there were no differences associated with preoperative carbohydrates or obesity

Effects of acute starvation and of type 2 diabetes mellitus upon insulin resistance and substrate utilization in obese subjects

Very-low calorie diet or even total short-term fasting is widely used in clinical practice in order to improve metabolic compensation of patients with type 2 diabetes mellitus. Although benefits of weight reduction are well proven in T2DM, much less is known about effects of acute starvation, during which the interruption of the afflux of energy substrates is not followed by a major change of body composition. We hypothesize the improvement of insulin effects on glucose metabolism in T2DM as these patients may lack the key metabolic responses which impair insulin sensitivity in lean, non-diabetic subjects. Moreover, we assume according to "thrifty genotype hypothesis" that protein wasting during starvation will be positively related to insulin effects on glucose disposal and negatively related to insulin antilipolytic and antiketogenic effects. In the light of this we designed an observational, prospective, in-hospital study, comparing the effects of 60 hours fast on various aspects of insulin resistance, endocrine regulation and metabolism in 10 patients with T2DM and 10 obese controls without diabetes (OB)

Fecal Microbial Transplantation in Critically Ill Patients—Structured Review and Perspectives

The human gut microbiota consists of bacteria, archaea, fungi, and viruses. It is a dynamic ecosystem shaped by several factors that play an essential role in both healthy and diseased states of humans. A disturbance of the gut microbiota, also termed “dysbiosis”, is associated with increased host susceptibility to a range of diseases. Because of splanchnic ischemia, exposure to antibiotics, and/or the underlying disease, critically ill patients loose 90% of the commensal organisms in their gut within hours after the insult. This is followed by a rapid overgrowth of potentially pathogenic and pro-inflammatory bacteria that alter metabolic, immune, and even neurocognitive functions and that turn the gut into the driver of systemic inflammation and multiorgan failure. Indeed, restoring healthy microbiota by means of fecal microbiota transplantation (FMT) in the critically ill is an attractive and plausible concept in intensive care. Nonetheless, available data from controlled studies are limited to probiotics and FMT for severe C. difficile infection or severe inflammatory bowel disease. Case series and observational trials have generated hypotheses that FMT might be feasible and safe in immunocompromised patients, refractory sepsis, or severe antibiotic-associated diarrhea in ICU. There is a burning need to test these hypotheses in randomized controlled trials powered for the determination of patient-centered outcomes

Lactate production without hypoxia in skeletal muscle during electrical cycling: Crossover study of femoral venous-arterial differences in healthy volunteers.

BACKGROUND:Aim of the study was to compare metabolic response of leg skeletal muscle during functional electrical stimulation-driven unloaded cycling (FES) to that seen during volitional supine cycling. METHODS:Fourteen healthy volunteers were exposed in random order to supine cycling, either volitional (10-25-50 W, 10 min) or FES assisted (unloaded, 10 min) in a crossover design. Whole body and leg muscle metabolism were assessed by indirect calorimetry with concomitant repeated measurements of femoral venous-arterial differences of blood gases, glucose, lactate and amino acids. RESULTS:Unloaded FES cycling, but not volitional exercise, led to a significant increase in across-leg lactate production (from -1.1±2.1 to 5.5±7.4 mmol/min, p<0.001) and mild elevation of arterial lactate (from 1.8±0.7 to 2.5±0.8 mM). This occurred without widening of across-leg veno-arterial (VA) O2 and CO2 gaps. Femoral SvO2 difference was directly proportional to VA difference of lactate (R2 = 0.60, p = 0.002). Across-leg glucose uptake did not change with either type of exercise. Systemic oxygen consumption increased with FES cycling to similarly to 25W volitional exercise (138±29% resp. 124±23% of baseline). There was a net uptake of branched-chain amino acids and net release of Alanine from skeletal muscle, which were unaltered by either type of exercise. CONCLUSIONS:Unloaded FES cycling, but not volitional exercise causes significant lactate production without hypoxia in skeletal muscle. This phenomenon can be significant in vulnerable patients' groups

Kinetic characteristics of propofol-induced inhibition of electron-transfer chain and fatty acid oxidation in human and rodent skeletal and cardiac muscles.

IntroductionPropofol causes a profound inhibition of fatty acid oxidation and reduces spare electron transfer chain capacity in a range of human and rodent cells and tissues-a feature that might be related to the pathogenesis of Propofol Infusion Syndrome. We aimed to explore the mechanism of propofol-induced alteration of bioenergetic pathways by describing its kinetic characteristics.MethodsWe obtained samples of skeletal and cardiac muscle from Wistar rat (n = 3) and human subjects: vastus lateralis from hip surgery patients (n = 11) and myocardium from brain-dead organ donors (n = 10). We assessed mitochondrial functional indices using standard SUIT protocol and high resolution respirometry in fresh tissue homogenates with or without short-term exposure to a range of propofol concentration (2.5-100 μg/ml). After finding concentrations of propofol causing partial inhibition of a particular pathways, we used that concentration to construct kinetic curves by plotting oxygen flux against substrate concentration during its stepwise titration in the presence or absence of propofol. By spectrophotometry we also measured the influence of the same propofol concentrations on the activity of isolated respiratory complexes.ResultsWe found that human muscle and cardiac tissues are more sensitive to propofol-mediated inhibition of bioenergetic pathways than rat's tissue. In human homogenates, palmitoyl carnitine-driven respiration was inhibited at much lower concentrations of propofol than that required for a reduction of electron transfer chain capacity, suggesting FAO inhibition mechanism different from downstream limitation or carnitine-palmitoyl transferase-1 inhibition. Inhibition of Complex I was characterised by more marked reduction of Vmax, in keeping with non-competitive nature of the inhibition and the pattern was similar to the inhibition of Complex II or electron transfer chain capacity. There was neither inhibition of Complex IV nor increased leak through inner mitochondrial membrane with up to 100 μg/ml of propofol. If measured in isolation by spectrophotometry, propofol 10 μg/ml did not affect the activity of any respiratory complexes.ConclusionIn human skeletal and heart muscle homogenates, propofol in concentrations that are achieved in propofol-anaesthetized patients, causes a direct inhibition of fatty acid oxidation, in addition to inhibiting flux of electrons through inner mitochondrial membrane. The inhibition is more marked in human as compared to rodent tissues

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle.

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 μM cytochrome c, 10 mM succinate, 2.5 μM oligomycin, 1.5 μM FCCP, 3.5 μM rotenone, 4 μM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible

Factors associated with success in the oral part of the European Diploma in Intensive Care

Introduction: The oral part of European Diploma in Intensive Care diploma examinations changed in 2013 into an objective structured clinical examination-type exam. This step was undertaken to provide a fair and reproducible clinical exam. All candidates face identical questions with predefined correct answers simultaneously in seven high throughput exam centres on the same day. We describe the factors that are associated with success in part 2 European Diploma in Intensive Care exam. Methods: We prospectively collected self-reported data from all candidates sitting European Diploma in Intensive Care part 2 in 2015, namely demographics, professional background and attendance to a European Diploma in Intensive Care part 2 or generic objective structured clinical examination preparatory courses. After testing association with success (with cutoff at p < 0.10) and co-linearity of these factors as independent variables, we performed a multivariate logistical analysis, with binary exam outcome (pass/fail) as the dependent variable. Structural equation modelling was used to gain further insight into relations among determinants of success in the oral part of the European Diploma in Intensive Care. Results: Out of 427 candidates sitting the exam, completed data from 341 (80%) were available for analysis. The following candidates' factors were associated with increased chance of success: English as native language (odds ratio 4.3 (95% CI 1.7–10.7)), use of Patient-centred Acute Care Training e-learning programme module (odds ratios 2.0 (1.2–3.3)), working in an EU country (odds ratios 2.5 (1.5–4.3)), and better results in the written part of the European Diploma in Intensive Care (for each additional SD of 6.1 points odds ratios 1.9 (1.4–2.4)). Chance of success in the European Diploma in Intensive Care 2 decreased with increased candidates ‘age (for each additional SD of 5.5 years odds ratios 0.67 (0.51–0.87)). Exam centres (7 in total) could be clustered into 3 groups with similar success rates. There were significant differences in exam outcomes among these 3 groups of exam centres even after adjustment to known candidates' factors (G1 vs G2 odds ratios 2.4 (1.4–4.1); G1 vs G3 odds ratios 9.7 (4.0–23.1) and G2 vs G3 odds ratios 3.9 (1.7–9.2)). A short data collection period (only one year) and 20% of missing candidates' data are the main limitations of this study. Conclusions: Younger age, English as native language, better results in written part of the exam, working at a European country and the use of PACT for preparation, were factors associated with success in the oral part of the European Diploma in Intensive Care exam. Despite the limitations of this study, the differences in outcome among the exam centres will need further investigation

Additional file 1 of Effect of noradrenaline on propofol-induced mitochondrial dysfunction in human skeletal muscle cells

Additional file 1: Table S1. Study subject characteristics on biopsy day. Figure S1. Cell viability. Results are expressed as the percentage of cell viability relative to the control (= non-treated cells). a) Individual groups represent viability of cells exposed for 96 h to different concentrations of noradrenaline. Data are presented a s the mean ± SEM (n = 4 subjects). Values for each experimental condition were measured in triplicates in each subject. b) Individual groups represent viability of cells exposed for 96 h to ethanol (= propofol vehicle; 0.1%), 0.1 mM noradrenaline alone and c) different concentrations (μg/mL) of either propofol alone or mixture of propofol and 0.1 mM noradrenaline. Data are presented as the mean ± SEM (n = 7 subjects). Values for each experimental condition were measured in triplicates in each subject. Note: NA = noradrenaline. *** p < 0.001 vs. control group. Figure S2. Kinetic graph on XF24 Analyzer demonstrates changes after propofol at various concentrations. Real-time measurement of OCR at baseline and after sequential injection of oligomycin, FCCP and Antimycin A. Each data-point represents the mean of 7 independent samples (subjects) measured in tri- or tetraplicates normalized to protein content. Error bars indicate standard error of the mean. Different colours represent different groups exposed to propofol (0; 2.5; 10 µg/mL). Figure S3. A) Global mitochondrial parameters. Basal respiration, maximal respiratory capacity, ATP production and non-mitochondrial respiration. N = 7 replicates with 21–28 wells for each condition normalized to protein content. Error bars indicate standard error of the mean. B) Mitochondrial mass calculated as a fraction (%) of a cell surface area in 2D cross-sectional images. Figure S4. Uptake of CO2 production and lactic acid metabolism. A) Uptake of [14C]palmitic acid. B) [14C]lactic acid oxidation. C) [14C]lactic acid uptake. Note: NA = noradrenaline. Error bars in each graph indicate standard error of the mean. Note: NA = noradrenaline. Figure S5. Flow cytometry. Histogram showing MTG intensity of individual cell groups. Data are presented as the mean ± SEM (n = 2–3 experiments per each group). Note: NA = noradrenaline. Figure S6. Mitochondrial membrane potential. A) Myoblasts after staining with MitoTracker™ Green FM (left), TMRE (in the middle) and after staining of both agents (right). Experiments were performed at least at 60 cells per each group from n = 3 independent experiments (cells from 3 individual subjects). B) Determination of ∆ψm was expressed as TMRE/MTG ratio. The mitochondrial uncoupling agent FCCP was used as a positive control. Note: MTG = MitoTracker™ Green FM; TRME = tetramethylrhodamine ethyl ester; FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, NA = noradrenaline. Error bars indicate standard error of the mean. *p < 0.05, ***p < 0.001 vs. control group. Figure S7. Analysis of lipid droplets. A) Quantification of LD mass by cross-sectional area of BODIPY 493/503 normalized to cell area. B) LD size assessed by cross-sectional area of individual LDs. C) LD number normalized to cell area. Experiments were performed at least at 38 cells per each condition from 2 independent measurements (= 2 individual subjects). Error bars indicate standard error of the mean. ***p < 0.001 vs. control group