Presence and evolution of NET markers and DAMPS in critically ill COVID-19 patients

Resumen del trabajo presentado en el 4th European Congress on Thrombosis and Haemostasis, celebrado en Gante (Bélgica), los días 14 y 15 de octubre de 2021Background: The coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection presents with a wide range of disease symptoms. In the more severe patients, COVID-19 is associated with respiratory failure, neutrophil extracellular trap (NET) formation, and multiple organ failure (MOF). Aims: We investigated the presence and evolution of several damage associated molecular patterns (DAMPs) neutrophil markers and immune modulators in a group of 100 COVID-19-positive ICU patients. Methods: Citrated plasma was collected from adult patients with confirmed COVID-19 by PCR detection of SARS-CoV-2 E and N-genes in nasopharyngeal swabs admitted to the intensive care unit (ICU) at Uppsala University hospital, Sweden. Written informed consent was obtained from the patients, or next of kin if the patient was unable to give consent. The Declaration of Helsinki and its subsequent revisions were followed. Plasma concentration of cell free DNA (cfDNA), extracellular histone H3 (H3), neutrophil elastase (NE), myeloperoxidase (MPO) and the cfDNA-MPO complex, and the immune modulators GAS6, and sAXL were measured in all COVID-19-positive and in COVID-19-negative patients and healthy controls. We determined marker levels upon admission, of their evolution, and correlation with disease severity, organ failure, thromboembolic events, mortality, and other blood parameters. Results: The level of cfDNA, H3, NE, MPO, cfDNA-MPO complex, GAS6, and sAXL were all significantly increased in plasma of COVID-19 patients compared to controls. Importantly, a diminution of cfDNA and GAS6 levels over time was observed in patients surviving 30 days after ICU admission. Histone H3 levels were detected in 40% of the COVID-19 patient plasma at ICU admission and the presence of histone H3 during ICU stay was associated with an increased risk of thromboembolic events and secondary infection. Though NET markers were not predictive of 30-day mortality, they correlated with several parameters of tissue damage and neutrophil counts. Summary/Conclusion: The increased presence of cfDNA, H3 and NE, MPO, and MPO-DNA illustrates the severity of cellular damage and indicates activation of NETosis in severe COVID-19 ICU patients. The evolution of cfDNA and Gas6 is able to predict disease prognosis of severely ill COVID-19 patients, where GAS6 appears to be part of an early activated mechanism in response to COVID-19. These data support treatment aimed at the reduction of NET formation in severe COVID-19 patients

Markers of NETosis and DAMPs are altered in critically ill COVID-19 patients

Background Coronavirus disease 19 (COVID-19) is known to present with disease severities of varying degree. In its most severe form, infection may lead to respiratory failure and multi-organ dysfunction. Here we study the levels of extracellular histone H3 (H3), neutrophil elastase (NE) and cfDNA in relation to other plasma parameters, including the immune modulators GAS6 and AXL, ICU scoring systems and mortality in patients with severe COVID-19. Methods We measured plasma H3, NE, cfDNA, GAS6 and AXL concentration in plasma of 83 COVID-19-positive and 11 COVID-19-negative patients at admission to the Intensive Care Unit (ICU) at the Uppsala University hospital, a tertiary hospital in Sweden and a total of 333 samples obtained from these patients during the ICU-stay. We determined their correlation with disease severity, organ failure, mortality and other blood parameters. Results H3, NE, cfDNA, GAS6 and AXL were increased in plasma of COVID-19 patients compared to controls. cfDNA and GAS6 decreased in time in in patients surviving to 30 days post ICU admission. Plasma H3 was a common feature of COVID-19 patients, detected in 40% of the patients at ICU admission. Although these measures were not predictive of the final outcome of the disease, they correlated well with parameters of tissue damage (H3 and cfDNA) and neutrophil counts (NE). A subset of samples displayed H3 processing, possibly due to proteolysis. Conclusions Elevated H3 and cfDNA levels in COVID-19 patients illustrate the severity of the cellular damage observed in critically ill COVID-19 patients. The increase in NE indicates the important role of neutrophil response and the process of NETosis in the disease. GAS6 appears as part of an early activated mechanism of response in Covid-19.The study was supported through grants from the dedSciLifeLab/KAW national COVID-19 research program project grant (MH), by Scilifelab, the Knut and Alice Wallenberg Foundation and in part by the Swedish Research Council (RF, grant no 2014-02569 and 2014-07606), and the Netherlands Thrombosis Foundation (GN).N

Retrospective evaluation of the Dutch pre-newborn screening cohort for propionic acidemia and isolated methylmalonic acidemia:What to aim, expect, and evaluate from newborn screening?

Evidence for effectiveness of newborn screening (NBS) for propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is scarce. Prior to implementation in the Netherlands, we aim to estimate the expected health gain of NBS for PA and MMA. In this national retrospective cohort study, the clinical course of 76/83 Dutch PA and MMA patients, diagnosed between January 1979 and July 2019, was evaluated. Five clinical outcome parameters were defined: adverse outcome of the first symptomatic phase, frequency of acute metabolic decompensations (AMD), cognitive function, mitochondrial complications, and treatment-related complications. Outcomes of patients identified by family testing were compared with the outcomes of their index siblings. An adverse outcome due to the first symptomatic phase was recorded in 46% of the clinically diagnosed patients. Outcome of the first symptomatic phase was similar in 5/9 sibling pairs and better in 4/9 pairs. Based on the day of diagnosis of the clinically diagnosed patients and sibling pair analysis, a preliminary estimated reduction of adverse outcome due to the first symptomatic phase from 46% to 36%-38% was calculated. Among the sibling pairs, AMD frequency, cognitive function, mitochondrial, and treatment-related complications were comparable. These results suggest that the health gain of NBS for PA and MMA in overall outcome may be limited, as only a modest decrease of adverse outcomes due to the first symptomatic phase is expected. With current clinical practice, no reduced AMD frequency, improved cognitive function, or reduced frequency of mitochondrial or treatment-related complications can be expected

Correction:How the COVID-19 pandemic highlights the necessity of animal research (vol 30, pg R1014, 2020)

(Current Biology 30, R1014–R1018; September 21, 2020) As a result of an author oversight in the originally published version of this article, a number of errors were introduced in the author list and affiliations. First, the middle initials were omitted from the names of several authors. Second, the surname of Dr. van Dam was mistakenly written as “Dam.” Third, the first name of author Bernhard Englitz was misspelled as “Bernard” and the surname of author B.J.A. Pollux was misspelled as “Pullox.” Finally, Dr. Keijer's first name was abbreviated rather than written in full. These errors, as well as various errors in the author affiliations, have now been corrected online

CSTB NULL MUTATION ASSOCIATED WITH MICROCEPHALY, EARLY DEVELOPMENTAL DELAY, AND SEVERE DYSKINESIA

The CSTB gene encodes for cystatin B, an inhibitor of lysosomal cysteine protease (cathepsins B, H, L, and S).(CSTB)-C-1 mutations have been associated with type 1 progressive myoclonic epilepsy, also known as Unverricht-Lundborg (ULD) disease, or Baltic myoclonus.(2,3) A total of 90% of all disease alleles consists of an expansion of at least 30 times of an unstable 12-nucleotide stretch (dodecamer 5-CCCCGCCCCGCG-3) in the CSTB promoter region. Homozygosity for this expansion is considered the founder mutation in the Finnish population. Few other mutations have been described, among these the p.Arg68*, but until now only as compound heterozygous with the dodecamer expansion.(4-6) Expression of the p.Arg68* mutation in vitro indicates that the truncated protein is rapidly degraded, confirming that it is a null mutation.(7) Between the ages of 6 and 16 years, ULD begins with stimulus-sensitive myoclonus and generalized tonic-clonic seizures, which can be worsened by phenytoin, followed by ataxia and slow neurodegeneration. Here we report on the first 2 patients with a homozygous p.Arg68* null mutation

Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses

Fragile X mental retardation is caused by absence of the RNA-binding protein fragile X mental retardation protein (FMRP), encoded by the FMR1 gene. There is increasing evidence that FMRP regulates transport and modulates translation of some mRNAs. We studied neurotransmitter-activated synaptic protein synthesis in fmr1-knockout mice. Synaptoneurosomes from knockout mice did not manifest accelerated polyribosome assembly or protein synthesis as it occurs in wild-type mice upon stimulation of group I metabotropic glutamate receptors. Direct activation of protein kinase C did not compensate in the knockout mouse, indicating that the FMRP-dependent step is further along the signaling pathway. Visual cortices of young knockout mice exhibited a lower proportion of dendritic spine synapses containing polyribosomes than did the cortices of wild-type mice, corroborating this finding in vivo. This deficit in rapid neurotransmitter-controlled local translation of specific proteins may contribute to morphological and functional abnormalities observed in patients with fragile X syndrome

The Psychological Challenges of Replacing Conventional Karyotyping with Genomic SNP Array Analysis in Prenatal Testing

Pregnant couples tend to prefer a maximum of information about the health of their fetus. Therefore, we implemented whole genome microarray instead of conventional karyotyping (CK) for all indications for prenatal diagnosis (PND). The array detects more clinically relevant anomalies, including early onset disorders, not related to the indication and more genetic anomalies of yet unquantifiable risk, so-called susceptibility loci (SL) for mainly neurodevelopmental disorders. This manuscript highlights the psychological challenges in prenatal genetic counselling when using the array and provides counselling suggestions. First, we suggest that pre-test decision counselling should emphasize deliberation about what pregnant couples wish to learn about the future health of their fetus more than information about possible outcomes. Second, pregnant couples need support in dealing with SL. Therefore, in order to consider the SL in a proportionate perspective, the presence of phenotypes associated with SL in the family, the incidence of a particular SL in control populations and in postnatally ascertained patients needs highlighting during post-test genetic counselling. Finally, the decision that couples need to make about the course of their pregnancy is more complicated when the expected phenotype is variable and not quantifiable. Therefore, during post-test psychological counseling, couples should concretize the options of continuing and ending their pregnancy; all underlying feelings and thoughts should be made explicit, as well as the couple’s resources, in order to attain adequate decision-making. As such, pre- and post-test counselling aids pregnant couples in handling the uncertainties that may accompany offering a broader scope of genetic PND using the array