Evaluation of Genetic or Cellular Impairments in Type I IFN Immunity in a Cohort of Young Adults with Critical COVID-19.

Several genetic and immunological risk factors for severe COVID-19 have been identified, with monogenic conditions relating to 13 genes of type I interferon (IFN) immunity proposed to explain 4.8% of critical cases. However, previous cohorts have been clinically heterogeneous and were not subjected to thorough genetic and immunological analyses. We therefore aimed to systematically investigate the prevalence of rare genetic variants causing inborn errors of immunity (IEI) and functionally interrogate the type I IFN pathway in young adults that suffered from critical COVID-19 yet lacked comorbidities. We selected and clinically characterized a cohort of 38 previously healthy individuals under 50 years of age who were treated in intensive care units due to critical COVID-19. Blood samples were collected after convalescence. Two patients had IFN-α autoantibodies. Genome sequencing revealed very rare variants in the type I IFN pathway in 31.6% of the patients, which was similar to controls. Analyses of cryopreserved leukocytes did not indicate any defect in plasmacytoid dendritic cell sensing of TLR7 and TLR9 agonists in patients carrying variants in these pathways. However, lymphocyte STAT phosphorylation and protein upregulation upon IFN-α stimulation revealed three possible cases of impaired type I IFN signaling in carriers of rare variants. Together, our results suggest a strategy of functional screening followed by genome analyses and biochemical validation to uncover undiagnosed causes of critical COVID-19.

Hypoxic ventilatory response after rocuronium-induced partial neuromuscular blockade in men with obstructive sleep apnoea.

Obstructive sleep apnoea and residual neuromuscular blockade are, independently, known to be risk factors for respiratory complications after major surgery. Residual effects of neuromuscular blocking agents are known to reduce the hypoxic ventilatory response in healthy volunteers. Patients with obstructive sleep apnoea have impaired control of breathing, but it is not known to what extent neuromuscular blocking agents interfere with the regulation of breathing in such patients. In a physiological study in 10 unsedated men with untreated obstructive sleep apnoea, we wished to examine if partial neuromuscular blockade had an effect on hypoxic ventilatory response (isocapnic hypoxia to oxygen saturation of 80%) and hypercapnic ventilatory response (normoxic inspired carbon dioxide 5%). The hypoxic ventilatory response was reduced by 32% (p = 0.016) during residual neuromuscular block (rocuronium to train-of-four ratio 0.7), but the hypercapnic ventilatory response was unaffected. We conclude that neuromuscular blockade specifically depresses peripheral chemosensitivity, and not respiratory muscle function since the hypercapnic ventilatory response was unaffected.

Prolonged attenuation of acetylcholine-induced phosphorylation of extracellular signal-regulated kinase 1/2 following sevoflurane exposure.

BACKGROUND: Volatile anaesthetics are known to affect cholinergic receptors. Perturbation of cholinergic signalling can cause cognitive deficits. In this study, we wanted to evaluate acetylcholine-induced intracellular signalling following sevoflurane exposure. METHODS: Pheochromocytoma12 PC12 cells were exposed to 4.6% sevoflurane for 2 h. Subsequently, Western blotting was used to measure acetylcholine-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) 1/2 and basal Protein kinase B (AKT) phosphorylation. RESULTS: After exposure, acetylcholine-induced ERK 1/2 phosphorylation was reduced to 58 ± 8% [95% confidence interval (CI): 38-77%, P = 0.003] compared with non-exposed controls. At 30 min after the end of sevoflurane administration [at 0.7% sevoflurane (0.102 mM)], ERK 1/2 phosphorylation remained reduced to 57 ± 7% (95% CI: 39-74%, P = 0.001) and was at 120 min [0.02% (0.003 mM] still reduced to 63 ± 10% (95% CI: 37-88%, P = 0.01), compared with control. At 360 min after exposure, acetylcholine-induced ERK 1/2 phosphorylation had recovered to 98 ± 16% (95% CI: 45-152%, P = 0.98) compared with control. In contrast, immediately after sevoflurane exposure, basal AKT phosphorylation was increased by 228 ± 37% (95% CI: 133-324%, P = 0.02) but had returned to control levels at 30 min after exposure, 172 ± 67% (95% CI: 0-356%, P = 0.34). CONCLUSION: Sevoflurane exposure has differential effects on different intracellular signalling pathways. On one hand, we observed a prolonged attenuation of acetylcholine-induced ERK 1/2 phosphorylation that persisted even when sevoflurane concentrations close to detection level. On the other hand, basal AKT phosphorylation was increased twofold during sevoflurane exposure, with a rapid return to baseline levels after exposure. We speculate that the effects on acetylcholine-induced intracellular signalling observed in our in vitro model could be of relevance also for cholinergic signalling in vivo following sevoflurane exposure.

Current concepts in neuromuscular transmission.

The neuromuscular junction (NMJ) is structured and powered to transduce electrical activity from the distal nerve terminal of a motor neurone via the neuromuscular cleft to the post-junctional muscle membrane to ultimately generate muscle contraction. Our understanding of this complex function has expanded over many years, and the NMJ has served as a prototype for how different synapses operate in the peripheral and central nervous systems. The NMJ has a presynaptic part which is synonymous with the distal nerve ending, being responsible for neurotransmitter synthesis, packaging into vesicles, and subsequent vesicle transportation to active release sites where vesicle docking, fusion, and release of acetylcholine and other co-released transmitters finally take place. The synaptic cleft, filled with large molecular complexes that guarantee ultrastructural NMJ arrangement and signal transduction, allows for rapid diffusion and degradation of the neurotransmitter. The postsynaptic part consists of a folded muscle membrane into which nicotinic acetylcholine receptors (nAChRs) directly opposite the presynaptic active release sites are mounted and fixed by a cytoskeleton. This specialized postsynaptic region is closely associated with the perijunctional zone where a high density of sodium channels promote and amplify the signal in order to guarantee the propagation of the electrical activity to generate muscle contraction. The transduction process is maintained at load (i.e. high stimulus frequency) by a presynaptic mechanism allowing for sustained transmitter release over time at high demand. This positive feedback mechanism relies on neuronal nAChRs present on the distal nerve terminal, whereas the continuation of the transduction process at the postsynaptic part relies on the classical muscle type nAChR. In this review, we will focus on recent findings of potential clinical importance that will advance our understanding of the effects of neuromuscular blocking agents and neuromuscular monitoring and also our management of disorders of the neuromuscular system within anaesthesia and intensive care.

Basic principles of neuromuscular transmission.

Neuromuscular transmission at the skeletal muscle occurs when a quantum of acetylcholine from the nerve ending is released and binds to the nicotinic acetylcholine receptors on the postjunctional muscle membrane. The nicotinic acetylcholine receptors on the endplate respond by opening channels for the influx of sodium ions and subsequent endplate depolarisation leads to muscle contraction. The acetylcholine immediately detaches from the receptor and is hydrolysed by acetylcholinesterase enzyme. Suxamethonium is a cholinergic agonist stimulating the muscle nicotinic acetylcholine receptors prior to causing neuromuscular block. Non-depolarising neuromuscular blocking drugs bind to the nicotinic acetylcholine receptors preventing the binding of acetylcholine. Non-depolarising neuromuscular blocking drugs also inhibit prejunctional alpha3beta2 nicotinic acetylcholine autoreceptors, which can be seen in the clinical setting as train-of-four fade. In some pathological states such as denervation, burns, immobilisation, inflammation and sepsis, there is expression of other subtypes of nicotinic acetylcholine receptors with upregulation of these receptors throughout the muscle membrane. The responses of these receptors to suxamethonium and non-depolarising neuromuscular blocking drugs are different and explain some of the aberrant responses to neuromuscular blocking drugs.

An in vitro model for studying neuromuscular transmission in the mouse pharynx.

The muscles of the pharynx are controlled by networks of neurons under the control of specific regions in the brain stem, which have been fairly well studied. However, the transmission between these neurons and the pharyngeal muscles, at the motor end plates, is less well understood. Therefore, an in vitro model for the study of neuromuscular transmission in the pharyngeal muscle of the mouse was developed. Ring preparations from the inferior constrictor and the cricopharyngeus muscles were isolated and mounted for isometric force recording at physiologic temperature. Preparations from the diaphragm and the soleus muscles were examined in parallel. The muscles were stimulated at supramaximal voltage with short tetani at 100 Hz. Following direct stimulation of the muscle fibers, using a longer pulse duration, the rate of force development of the pharyngeal muscles was similar to that of the diaphragm and faster than that of the soleus muscle. By varying the duration of the stimulation pulses, conditions where the nerve-mediated activation contributed to a major extent of the contractile responses were identified. Gallamine completely inhibited the nerve-mediated responses. In separate experiments the dose dependence of gallamine inhibition was examined, showing similar sensitivity in the inferior pharyngeal constrictor compared to the diaphragm and soleus muscles. We conclude that reproducible contractile responses with an identifiable nerve-induced component can be obtained from the mouse inferior pharyngeal constrictor. The pharyngeal muscles have contractile characteristics similar to those of the faster diaphragm. The sensitivity to the neuromuscular blocking agent gallamine of the inferior pharyngeal constrictor was in the same concentration range as that of the diaphragm and soleus muscles.

Pretreatment with magnesium sulphate is associated with less succinylcholine-induced fasciculation and subsequent tracheal intubation-induced hemodynamic changes than precurarization with vecuronium during rapid sequence induction.

Although it has side effects, succinylcholine is still widely used in rapid sequence induction. The aim of the present study is to evaluate the effects of pretreat ment with magnesium and precurarization of vecuroni um on succinylcholine-induced fasciculation and subse quent tracheal intubation-induced hemodynamic changes during rapid sequence induction. Fifty-five patients were allocated to three groups by a blinded randomization: Group M received saline 100 ml with magnesium 40 mg x kg(-1) for 5 min at 6.5 min before induction and sub sequently administered saline 1-2 ml at 1.5 min before induction; Group V received saline 100 ml for 5 min at 6.5 min before induction and subsequently administered vecuronium 0.02 mg x kg(-1) at 1.5 min before induction; Group C received saline 100 ml for 5 min at 6.5 min before induction and then saline 1-2 ml at 1.5 min before induction. Fasciculation scores and mean percent changes of heart rate, systolic blood pressure and rate pressure product between baseline and after induction were significantly lower in group M than those in group C and group V. Pretreatment with magnesium is more effective to limit succinylcholine-induced fasciculation and subsequent tracheal intubation-induced hemody namic changes in rapid sequence induction compared with vecuronium pretreatment, although magnesium does not prevent the elevation of serum potassium con centration after induction.

Halothane-diethyl ether azeotrope anesthesia under primitive conditions: guidelines for neuromuscular blockade with vecuronium.

The dose-response relationship for vecuronium during anesthesia with the azeotropic mixture of halothane and diethyl ether (HE) (66 ml of halothane mixed with 34 ml of ether in the same bottle)/oxygen was compared with halothane/nitrous oxide/oxygen anesthesia. The HE is not explosive and has preserved many of the advantageous properties of diethyl ether, making it an attractive anesthetic drug under difficult circumstances such as war or civil disaster. If muscular relaxation is needed in military anesthesia, it is essential to use a short- to medium-acting compound that can be stored without refrigeration. Vecuronium bromide fulfills these criteria. The study was conducted on 20 young (mean 24 years, range 18-31), ASA I patients. Using adductor pollicis mechanomyography and a cumulative dose technique, individual dose-response curves for vecuronium were constructed. Care was taken to keep peripheral and core temperatures normal and the times of exposure to inhalational gases equal. The results were analyzed by the log-probit method. During HE anesthesia, ED50 for vecuronium was 22 +/- 2 micrograms kg-1 (mean +/- SE) and ED90 was 49 +/- 8 micrograms kg-1. During halothane/nitrous oxide anesthesia, ED50 for vecuronium was 18 +/- 2 micrograms kg-1 (mean +/- SE) and ED90 was 39 +/- 7 micrograms kg-1. The dose-response relationships for vecuronium were similar during HE anesthesia and halothane/nitrous oxide anesthesia.

Breathing and swallowing in normal man--effects of changes in body position, bolus types, and respiratory drive.

BACKGROUND: Coordination of breathing and swallowing is essential for airway protection and dyscoordination may cause morbidity and mortality. METHODS: Using a recently developed technique for high accuracy respiratory measurements of airflow during swallowing, we investigated the effects of body position (upright vs left lateral), bolus type (spontaneously swallowed saliva vs water), and respiratory drive (normo- vs hypercapnia) on coordination of breathing and swallowing in 32 healthy volunteers. KEY RESULTS: Swallows were in all cases (100%) proceded by expiration and 98% were also followed by expiration, regardless of body position, bolus type, or respiratory drive. While the endpoint of postswallow apnea correlated well to the endpoint of pharyngeal swallowing, duration of preswallow apnea was highly variable. In a small fraction of swallows followed by inspiration (3%), the expiratory phase before swallowing and duration of postswallow apnea was significantly longer. Body position and respiratory drive affected the increase in upper esophageal sphincter tone during inspiration. Increased respiratory drive also reduced swallowing frequency and shortened duration of preswallow apnea. Water swallows had longer duration of preswallow apnea. CONCLUSIONS & INFERENCES: Swallowing occurs during the expiratory phase of respiration, and the fraction of swallows preceded and followed by expiration approach 100% in healthy humans. This integration between breathing and swallowing remains unchanged regardless of body position, bolus characteristics, or respiratory drive. Our results provide a platform for future studies aiming at understanding how this integration is changed by aging, diseases, and drugs.

Swallowing and respiratory pattern in young healthy individuals recorded with high temporal resolution.

The coordination of swallowing and respiration is essential for a safe swallow. Swallowing consists of several subsecond events. To study this, it is important to use modalities with high temporal resolution. In this study, we have examined young healthy individuals with simultaneous videofluoroscopy, videomanometry and respiratory recording, all with high temporal resolution. The onset of 13 predetermined swallowing and respiratory events and the surrounding respiratory phase pattern were studied in different body positions and during different respiratory drives. An increased respiratory drive was induced by breathing 5% CO(2). The results demonstrated a highly repeatable and fixed temporal coordination of the swallowing pattern despite body position and respiratory drive. Previous studies have demonstrated a period of centrally controlled apnoea during swallowing. This apnoea period has a variable length, varying from 1 to 5 s. During increased respiratory drive, we could demonstrate a significantly shorter period of apnoea during swallowing, mainly due to an earlier resumption of respiration. The high temporal recordings in this study have revealed that swallowing during expiration is present basically in all healthy individuals. This swallowing respiratory pattern seems to be appropriate for a safe swallow. This knowledge will be used as a reference for future studies on how swallowing and respiratory coordination might be altered due to ageing and diseases.

Good clinical research practice in pharmacodynamic studies of neuromuscular blocking agents II: the Stockholm revision.

The set of guidelines for good clinical research practice (GCRP) in pharmacodynamic studies of neuromuscular blocking agents, which was developed following an international consensus conference in Copenhagen, has been revised and updated following the second consensus conference in Stockholm in 2005. It is hoped that these guidelines will continue to help researchers in the field and assist the pharmaceutical industry and equipment manufacturers in enhancing the standards of the studies they sponsor.

Scandinavian Clinical Practice Guidelines on the diagnosis, management and follow-up of anaphylaxis during anaesthesia.

The present approach to the diagnosis, management and follow-up of anaphylaxis during anaesthesia varies in the Scandinavian countries. The main purpose of these Scandinavian Clinical Practice Guidelines is to increase the awareness about anaphylaxis during anaesthesia amongst anaesthesiologists. It is hoped that increased focus on the subject will lead to prompt diagnosis, rapid and correct treatment, and standardised management of patients with anaphylactic reactions during anaesthesia across Scandinavia. The recommendations are based on the best available evidence in the literature, which, owing to the rare and unforeseeable nature of anaphylaxis, mainly includes case series and expert opinion (grade of evidence IV and V). These guidelines include an overview of the epidemiology of anaphylactic reactions during anaesthesia. A treatment algorithm is suggested, with emphasis on the incremental titration of adrenaline (epinephrine) and fluid therapy as first-line treatment. Recommendations for primary and secondary follow-up are given, bearing in mind that there are variations in geography and resources in the different countries. A list of National Centres from which anaesthesiologists can seek advice concerning follow-up procedures is provided. In addition, an algorithm is included with advice on how to manage patients with previous suspected anaphylaxis during anaesthesia. Lastly, Appendix 2 provides an overview of the incidence, mechanisms and possibilities for follow-up for some common drug groups.

Midazolam depresses carotid body chemoreceptor activity.

BACKGROUND: Although the contribution of the gamma-aminobutyric acid (GABA) receptor system in peripheral chemosensation is unclear, immunohistochemistry has demonstrated the presence of GABA-ergic receptors in mammalian carotid bodies. We hypothesized that an activation of the carotid body GABA receptors would counteract the depolarizing effect of hypoxia. METHODS: The carotid body with arterial supply and the carotid sinus nerve was removed en bloc from New Zealand White rabbits and placed in a perfusion chamber. The carotid body preparation was perfused via the cut common carotid artery with a modified Tyrode's solution at a rate of 3.5-4.5 ml/min with a constant pressure of 45 cmH2O. The carotid sinus nerve firing frequency (Hz) was recorded at two different oxygen tension levels during perfusion with midazolam of 1, 10 and 100 microg/l. RESULTS: The frequency was decreased by midazolam in a dose-dependent manner (n = 8). Firing frequencies (mean +/- SEM) at the low oxygen tension level decreased from 643.13 +/- 67.2 Hz in the control to 554.5 +/- 67.7 Hz (P = 0.054 vs. control), 509.01 +/- 100.5 Hz (P < 0.012 vs. control) and 422.6 +/- 77.3 Hz (P < 0.001 vs. control) during perfusion with midazolam of 1, 10 and 100 microg/l, respectively. CONCLUSION: Midazolam depresses carotid body chemoreceptor activity in a dose-dependent manner.

A porcine model of acute quadriplegic myopathy: a feasibility study.

BACKGROUND: The mechanisms underlying acute quadriplegic myopathy (AQM) are poorly understood, partly as a result of the fact that patients are generally diagnosed at a late stage of the disease. Accordingly, there is a need for relevant experimental animal models aimed at identifying underlying mechanisms. METHODS: Pigs were mechanically ventilated and exposed to various combinations of agents, i.e. pharmacological neuromuscular blockade, corticosteroids and/or sepsis, for a period of 5 days. Electromyography and myofibrillar protein and mRNA expression were analysed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), confocal microscopy, histochemistry and real-time polymerase chain reaction (PCR). RESULTS: A decreased compound muscle action potential, normal motor nerve conduction velocities, and intact sensory nerve function were observed. Messenger RNA expression, determined by real-time PCR, of the myofibrillar proteins myosin and actin decreased in spinal and cranial nerve innervated muscles, suggesting that the loss of myosin observed in AQM patients is not solely the result of myofibrillar protein degradation. CONCLUSION: The present porcine AQM model demonstrated findings largely in accordance with results previously reported in patients and offers a feasible approach to future mechanistic studies aimed at identifying underlying mechanisms and developing improved diagnostic tests and intervention strategies.

Effect of prolonged mechanical ventilation on diaphragm muscle mitochondria in piglets.

BACKGROUND: Respiratory muscle weakness is a common problem in the intensive care unit and could be involved in difficulties in weaning from the ventilator after prolonged mechanical ventilation. Animal models have shown that mechanical ventilation itself impairs diaphragm muscle function. In this study we investigated whether diaphragm contractile impairment caused by mechanical ventilation and immobilization in piglets is associated with a derangement in diaphragm mitochondria. METHODS: Seven piglets received controlled mechanical ventilation during 5 days. A control group of eight piglets were anaesthetized and surgically manipulated in the same way, but were mechanically ventilated for 4-6 h. After mechanical ventilation, diaphragm muscle biopsies were taken for measurements of mitochondria content, mitochondrial respiratory enzymes and markers of oxidative stress. RESULTS: Diaphragm mitochondrial content, as assessed by citrate synthase activities and volume density, was not different between the control and ventilated piglets. Activity of complex IV of the mitochondrial respiratory chain decreased by 21% (P=0.02) when expressed per muscle weight and by 11% (P=0.03) when expressed per citrate synthase activity. There were no changes in the markers of oxidative stress between the two groups. CONCLUSION: Five days of mechanical ventilation and immobilization decreased the activity of complex IV of the mitochondrial respiratory chain in the diaphragm muscle of the piglets.

Pre-operative fasting guidelines: an update.

Liberal pre-operative fasting routines have been implemented in most countries. In general, clear fluids are allowed up to 2 h before anaesthesia, and light meals up to 6 h. The same recommendations apply for children and pregnant women not in labour. In children <6 months, most recommendations now allow breast- or formula milk feeding up to 4 h before anaesthesia. Recently, the concept of pre-operative oral nutrition using a special carbohydrate-rich beverage has also gained support and been shown not to increase gastric fluid volume or acidity. Based on the available literature, our Task Force has produced new consensus-based Scandinavian guidelines for pre-operative fasting. What is still not clear is to what extent the new liberal fasting routines should apply to patients with functional dyspepsia or systematic diseases such as diabetes mellitus. Other still controversial areas include the need for and effect of fasting in emergency patients, women in labour and in association with procedures done under 'deep sedation'. We think more research on the effect of various fasting regimes in subpopulations of patients is needed before we can move one step further towards completely evidence-based pre-operative fasting guidelines.

Ventilation and neuromuscular blocking drugs.

Ventilatory failure after administration of neuromuscular blocking agents is an important factor in anaesthesia-related perioperative morbidity and mortality. Improved knowledge and new monitoring methods may avoid ventilatory failure caused by incomplete recovery of neuromuscular function in the postoperative period. Central respiratory muscles are less sensitive than, and their time course of neuromuscular block is different from those of, pharyngeal muscles and those of the upper airway. Differences in potency and time course of neuromuscular block may lead to incorrect assessment of ventilatory function during onset and recovery. Even if recovery of the mechanical adductor pollicis train-of-four (TOF) response to a ratio of 0.70 has previously been associated with adequate ventilatory capacity, it is now shown that hypoxic ventilatory responses may be markedly reduced despite adequate respiratory force at a TOF ratio of 0.70. Hence, partial paralysis may interfere with ventilatory regulation in hypoxaemia. Consequently, monitoring neuromuscular function by peripheral nerve stimulation in one muscle yields limited information about total ventilatory capacity, especially the function of the upper airway and ventilatory regulation. Therefore, neuromuscular monitoring should be used with caution during recovery and should always be combined with bedside clinical tests if possible.

Reduced hypoxic chemosensitivity in partially paralysed man. A new property of muscle relaxants?

BACKGROUND: It was hypothesized that non-depolarizing neuromuscular blocking agents impair hypoxic chemosensitivity in man. METHODS: In thirty randomly allocated male volunteers the hypoxic and hypercarbic ventilatory responses were measured during partial paralysis (TOF ratio 0.70) due to either atracurium (n = 10), pancuronium (n = 10) or vecuronium (n = 10). RESULTS: Hypoxic ventilatory responses were depressed by 306, 287 and 296% (mean SD) at steady-state infusion of atracurium, pancuronium and vecuronium, respectively. At a TOF ratio of > 0.90, the HVR was not different from control measurements. CONCLUSION: It is concluded that non-depolarizing neuromuscular blocking agents impair hypoxic ventilatory regulation. Further experimental studies are warranted to fully describe the mechanism(s) responsible for this interaction.