A complement atlas identifies interleukin-6-dependent alternative pathway dysregulation as a key druggable feature of COVID-19.

Improvements in COVID-19 treatments, especially for the critically ill, require deeper understanding of the mechanisms driving disease pathology. The complement system is not only a crucial component of innate host defense but can also contribute to tissue injury. Although all complement pathways have been implicated in COVID-19 pathogenesis, the upstream drivers and downstream effects on tissue injury remain poorly defined. We demonstrate that complement activation is primarily mediated by the alternative pathway, and we provide a comprehensive atlas of the complement alterations around the time of respiratory deterioration. Proteomic and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal, and myeloid cells in complement production, in addition to liver-derived factors. We identify IL-6 and STAT1/3 signaling as an upstream driver of complement responses, linking complement dysregulation to approved COVID-19 therapies. Furthermore, an exploratory proteomic study indicates that inhibition of complement C5 decreases epithelial damage and markers of disease severity. Collectively, these results support complement dysregulation as a key druggable feature of COVID-19.

STE20 kinase TAOK3 regulates type 2 immunity and metabolism in obesity.

Healthy adipose tissue (AT) contains ST2+ Tregs, ILC2s, and alternatively activated macrophages that are lost in mice or humans on high caloric diet. Understanding how this form of type 2 immunity is regulated could improve treatment of obesity. The STE20 kinase Thousand And One amino acid Kinase-3 (TAOK3) has been linked to obesity in mice and humans, but its precise function is unknown. We found that ST2+ Tregs are upregulated in visceral epididymal white AT (eWAT) of Taok3-/- mice, dependent on IL-33 and the kinase activity of TAOK3. Upon high fat diet feeding, metabolic dysfunction was attenuated in Taok3-/- mice. ST2+ Tregs disappeared from eWAT in obese wild-type mice, but this was not the case in Taok3-/- mice. Mechanistically, AT Taok3-/- Tregs were intrinsically more responsive to IL-33, through higher expression of ST2, and expressed more PPARγ and type 2 cytokines. Thus, TAOK3 inhibits adipose tissue Tregs and regulates immunometabolism under excessive caloric intake.

Loss of GM-CSF-dependent instruction of alveolar macrophages in COVID-19 provides a rationale for inhaled GM-CSF treatment.

GM-CSF promotes myelopoiesis and inflammation, and GM-CSF blockade is being evaluated as a treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is, however, required for monocytes to differentiate into alveolar macrophages (AMs) that control alveolar homeostasis. By mapping cross-species AM development to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of pro-inflammatory macrophages. In a multi-center, open-label RCT in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides a rationale for further testing of inhaled GM-CSF as a non-invasive treatment to improve alveolar gas exchange and simultaneously boost antiviral immunity in COVID-19. This study is registered at ClinicalTrials.gov (NCT04326920) and EudraCT (2020-001254-22).

Efficacy and safety of the investigational complement C5 inhibitor zilucoplan in patients hospitalized with COVID-19: an open-label randomized controlled trial.

BACKGROUND: The efficacy and safety of complement inhibition in COVID-19 patients is unclear. METHODS: A multicenter randomized controlled, open-label trial. Hospitalized COVID-19 patients with signs of systemic inflammation and hypoxemia (PaO2/FiO2 below 350 mmHg) were randomized (2:1 ratio) to receive standard of care with or without the C5 inhibitor zilucoplan daily for 14 days, under antibiotic prophylaxis. The primary outcome was improvement in oxygenation at day 6 and 15. RESULTS: 81 patients were randomly assigned to zilucoplan (n = 55) or the control group (n = 26). 78 patients were included in the safety and primary analysis. Most were men (87%) and the median age was 63 years. The mean improvement in PaO2/FiO2 from baseline to day 6 was 56.4 mmHg in the zilucoplan group and 20.6 mmHg in the control group (mean difference + 35.8; 95% confidence interval (CI) - 9.4 to 80.9; p = 0.12), an effect also observed at day 15. Day 28 mortality was 9% in the zilucoplan and 21% in the control group (odds ratio 0.4; 95% CI 0.1 to 1.5). At long-term follow up, the distance walked in a 6-min test was 539.7 m in zilucoplan and 490.6 m in the control group (p = 0.18). Zilucoplan lowered serum C5b-9 (p < 0.001) and interleukin-8 (p = 0.03) concentration compared with control. No relevant safety differences between the zilucoplan and control group were identified. CONCLUSION: Administration of zilucoplan to COVID-19 patients in this proof-of-concept randomized trial was well tolerated under antibiotic prophylaxis. While not reaching statistical significance, indicators of respiratory function (PaO2/FiO2) and clinical outcome (mortality and 6-min walk test) suggest that C5 inhibition might be beneficial, although this requires further research in larger randomized studies.

The STE20 kinase TAOK3 controls the development of house dust mite-induced asthma in mice.

BACKGROUND: The most common endotype of asthma is type 2-high asthma, which is sometimes driven by adaptive allergen-specific TH2 lymphocytes that react to allergens presented by dendritic cells (DCs), or sometimes by an innate immune response dominated by type 2 innate lymphocytes (ILC2s). Understanding the underlying pathophysiology of asthma is essential to improve patient-tailored therapy. The STE20 kinase thousand-and-one kinase 3 (TAOK3) controls key features in the biology of DCs and lymphocytes, but to our knowledge, its potential usefulness as a target for asthma therapy has not yet been addressed. OBJECTIVE: We examined if and how loss of Taok3 affects the development of house dust mite (HDM)-driven allergic asthma in an in vivo mouse model. METHODS: Wild-type Taok3+/+ and gene-deficient Taok3-/- mice were sensitized and challenged with HDM, and bronchoalveolar lavage fluid composition, mediastinal lymph node cytokine production, lung histology, and bronchial hyperreactivity measured. Conditional Taok3fl/fl mice were crossed to tissue- and cell-specific specific deletor Cre mice to understand how Taok3 acted on asthma susceptibility. Kinase-dead (KD) Taok3KD mice were generated to probe for the druggability of this pathway. Activation of HDM-specific T cells was measured in adoptively transferred HDM-specific T-cell receptor-transgenic CD4+ T cells. ILC2 biology was assessed by in vivo and in vitro IL-33 stimulation assays in Taok3-/- and Taok3+/+, Taok3KD, and Red5-Cre Taok3fl/fl mice. RESULTS: Taok3-/- mice failed to mount salient features of asthma, including airway eosinophilia, TH2 cytokine production, IgE secretion, airway goblet cell metaplasia, and bronchial hyperreactivity compared to controls. This was due to intrinsic loss of Taok3 in hematopoietic and not epithelial cells. Loss of Taok3 resulted in hampered HDM-induced lung DC migration to the draining lymph nodes and defective priming of HDM-specific TH2 cells. Strikingly, HDM and IL-33-induced ILC2 proliferation and function were also severely affected in Taok3-deficient and Taok3KD mice. CONCLUSIONS: Absence of Taok3 or loss of its kinase activity protects from HDM-driven allergic asthma as a result of defects in both adaptive DC-mediated TH2 activation and innate ILC2 function. This identifies Taok3 as an interesting drug target, justifying further testing as a new treatment for type 2-high asthma.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

Effect of anti-interleukin drugs in patients with COVID-19 and signs of cytokine release syndrome (COV-AID): a factorial, randomised, controlled trial.

BACKGROUND: Infections with SARS-CoV-2 continue to cause significant morbidity and mortality. Interleukin (IL)-1 and IL-6 blockade have been proposed as therapeutic strategies in COVID-19, but study outcomes have been conflicting. We sought to study whether blockade of the IL-6 or IL-1 pathway shortened the time to clinical improvement in patients with COVID-19, hypoxic respiratory failure, and signs of systemic cytokine release syndrome. METHODS: We did a prospective, multicentre, open-label, randomised, controlled trial, in hospitalised patients with COVID-19, hypoxia, and signs of a cytokine release syndrome across 16 hospitals in Belgium. Eligible patients had a proven diagnosis of COVID-19 with symptoms between 6 and 16 days, a ratio of the partial pressure of oxygen to the fraction of inspired oxygen (PaO2:FiO2) of less than 350 mm Hg on room air or less than 280 mm Hg on supplemental oxygen, and signs of a cytokine release syndrome in their serum (either a single ferritin measurement of more than 2000 µg/L and immediately requiring high flow oxygen or mechanical ventilation, or a ferritin concentration of more than 1000 µg/L, which had been increasing over the previous 24 h, or lymphopenia below 800/mL with two of the following criteria: an increasing ferritin concentration of more than 700 µg/L, an increasing lactate dehydrogenase concentration of more than 300 international units per L, an increasing C-reactive protein concentration of more than 70 mg/L, or an increasing D-dimers concentration of more than 1000 ng/mL). The COV-AID trial has a 2 × 2 factorial design to evaluate IL-1 blockade versus no IL-1 blockade and IL-6 blockade versus no IL-6 blockade. Patients were randomly assigned by means of permuted block randomisation with varying block size and stratification by centre. In a first randomisation, patients were assigned to receive subcutaneous anakinra once daily (100 mg) for 28 days or until discharge, or to receive no IL-1 blockade (1:2). In a second randomisation step, patients were allocated to receive a single dose of siltuximab (11 mg/kg) intravenously, or a single dose of tocilizumab (8 mg/kg) intravenously, or to receive no IL-6 blockade (1:1:1). The primary outcome was the time to clinical improvement, defined as time from randomisation to an increase of at least two points on a 6-category ordinal scale or to discharge from hospital alive. The primary and supportive efficacy endpoints were assessed in the intention-to-treat population. Safety was assessed in the safety population. This study is registered online with ClinicalTrials.gov (NCT04330638) and EudraCT (2020-001500-41) and is complete. FINDINGS: Between April 4, and Dec 6, 2020, 342 patients were randomly assigned to IL-1 blockade (n=112) or no IL-1 blockade (n=230) and simultaneously randomly assigned to IL-6 blockade (n=227; 114 for tocilizumab and 113 for siltuximab) or no IL-6 blockade (n=115). Most patients were male (265 [77%] of 342), median age was 65 years (IQR 54-73), and median Systematic Organ Failure Assessment (SOFA) score at randomisation was 3 (2-4). All 342 patients were included in the primary intention-to-treat analysis. The estimated median time to clinical improvement was 12 days (95% CI 10-16) in the IL-1 blockade group versus 12 days (10-15) in the no IL-1 blockade group (hazard ratio [HR] 0·94 [95% CI 0·73-1·21]). For the IL-6 blockade group, the estimated median time to clinical improvement was 11 days (95% CI 10-16) versus 12 days (11-16) in the no IL-6 blockade group (HR 1·00 [0·78-1·29]). 55 patients died during the study, but no evidence for differences in mortality between treatment groups was found. The incidence of serious adverse events and serious infections was similar across study groups. INTERPRETATION: Drugs targeting IL-1 or IL-6 did not shorten the time to clinical improvement in this sample of patients with COVID-19, hypoxic respiratory failure, low SOFA score, and low baseline mortality risk. FUNDING: Belgian Health Care Knowledge Center and VIB Grand Challenges program.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Case Report: Convalescent Plasma, a Targeted Therapy for Patients with CVID and Severe COVID-19.

The disease course of COVID-19 in patients with immunodeficiencies is unclear, as well as the optimal therapeutic strategy. We report a case of a 37-year old male with common variable immunodeficiency disorder and a severe SARS-CoV-2 infection. After administration of convalescent plasma, the patient's condition improved rapidly. Despite clinical recovery, viral RNA remained detectable up to 60 days after onset of symptoms. We propose that convalescent plasma might be considered as a treatment option in patients with CVID and severe COVID-19. In addition, in patients with immunodeficiencies, a different clinical course is possible, with prolonged viral shedding.

Zilucoplan in patients with acute hypoxic respiratory failure due to COVID-19 (ZILU-COV): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Zilucoplan (complement C5 inhibitor) has profound effects on inhibiting acute lung injury post COVID-19, and can promote lung repair mechanisms that lead to improvement in lung oxygenation parameters. The purpose of this study is to investigate the efficacy and safety of Zilucoplan in improving oxygenation and short- and long-term outcome of COVID-19 patients with acute hypoxic respiratory failure. TRIAL DESIGN: This is a phase 2 academic, prospective, 2:1 randomized, open-label, multi-center interventional study. PARTICIPANTS: Adult patients (≥18y old) will be recruited at specialized COVID-19 units and ICUs at 9 Belgian hospitals. The main eligibility criteria are as follows: 1) Inclusion criteria: a. Recent (≥6 days and ≤16 days) SARS-CoV-2 infection. b. Chest CT scan showing bilateral infiltrates within the last 2 days prior to randomisation. c. Acute hypoxia (defined as PaO2/FiO2 below 350 mmHg or SpO2 below 93% on minimal 2 L/min supplemental oxygen). d. Signs of cytokine release syndrome characterized by either high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those. 2) Exclusion criteria: e. Mechanical ventilation for more than 24 hours prior to randomisation. f. Active bacterial or fungal infection. g. History of meningococcal disease (due to the known high predisposition to invasive, often recurrent meningococcal infections of individuals deficient in components of the alternative and terminal complement pathways). INTERVENTION AND COMPARATOR: Patients in the experimental arm will receive daily 32,4 mg Zilucoplan subcutaneously and a daily IV infusion of 2g of the antibiotic ceftriaxone for 14 days (or until hospital discharge, whichever comes first) in addition to standard of care. These patients will receive additional prophylactic antibiotics until 14 days after the last Zilucoplan dose: hospitalized patients will receive a daily IV infusion of 2g of ceftriaxone, discharged patients will switch to daily 500 mg of oral ciprofloxacin. The control group will receive standard of care and a daily IV infusion of 2g of ceftriaxone for 1 week (or until hospital discharge, whichever comes first), to control for the effects of antibiotics on the clinical course of COVID-19. MAIN OUTCOMES: The primary endpoint is the improvement of oxygenation as measured by mean and/or median change from pre-treatment (day 1) to post-treatment (day 6 and 15 or at discharge, whichever comes first) in PaO2/FiO2 ratio, P(A-a)O2 gradient and a/A PO2 ratio. (PAO2= Partial alveolar pressure of oxygen, PaO2=partial arterial pressure of oxygen, FiO2=Fraction of inspired oxygen). RANDOMISATION: Patients will be randomized in a 2:1 ratio (Zilucoplan: control). Randomization will be done using an Interactive Web Response System (REDCap). BLINDING (MASKING): In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 81 patients will be enrolled: 54 patients will be randomized to the experimental arm and 27 patients to the control arm. TRIAL STATUS: ZILU-COV protocol Version 4.0 (June 10 2020). Participant recruitment started on June 23 2020 and is ongoing. Given the uncertainty of the pandemic, it is difficult to predict the anticipated end date. TRIAL REGISTRATION: The trial was registered on Clinical Trials.gov on May 11th, 2020 (ClinicalTrials.gov Identifier: NCT04382755 ) and on EudraCT (Identifier: 2020-002130-33 ). FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

TAO-kinase 3 governs the terminal differentiation of NOTCH2-dependent splenic conventional dendritic cells.

Antigen-presenting conventional dendritic cells (cDCs) are broadly divided into type 1 and type 2 subsets that further adapt their phenotype and function to perform specialized tasks in the immune system. The precise signals controlling tissue-specific adaptation and differentiation of cDCs are currently poorly understood. We found that mice deficient in the Ste20 kinase Thousand and One Kinase 3 (TAOK3) lacked terminally differentiated ESAM+ CD4+ cDC2s in the spleen and failed to prime CD4+ T cells in response to allogeneic red-blood-cell transfusion. These NOTCH2- and ADAM10-dependent cDC2s were absent selectively in the spleen, but not in the intestine of Taok3-/- and CD11c-cre Taok3fl/fl mice. The loss of splenic ESAM+ cDC2s was cell-intrinsic and could be rescued by conditional overexpression of the constitutively active NOTCH intracellular domain in CD11c-expressing cells. Therefore, TAOK3 controls the terminal differentiation of NOTCH2-dependent splenic cDC2s.

Treatment of severely ill COVID-19 patients with anti-interleukin drugs (COV-AID): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The purpose of this study is to test the safety and effectiveness of individually or simultaneously blocking IL-6, IL-6 receptor and IL-1 versus standard of care on blood oxygenation and systemic cytokine release syndrome in patients with COVID-19 coronavirus infection and acute hypoxic respiratory failure and systemic cytokine release syndrome. TRIAL DESIGN: A phase 3 prospective, multi-center, interventional, open label, 6-arm 2x2 factorial design study. PARTICIPANTS: Subjects will be recruited at the specialized COVID-19 wards and/or ICUs at 16 Belgian participating hospitals. Only adult (≥18y old) patients will be recruited with recent (≤16 days) COVID-19 infection and acute hypoxia (defined as PaO2/FiO2 below 350mmHg or PaO2/FiO2 below 280 on supplemental oxygen and immediately requiring high flow oxygen device or mechanical ventilation) and signs of systemic cytokine release syndrome characterized by high serum ferritin, or high D-dimers, or high LDH or deep lymphopenia or a combination of those, who have not been on mechanical ventilation for more than 24 hours before randomisation. Patients should have had a chest X-ray and/or CT scan showing bilateral infiltrates within the last 2 days before randomisation. Patients with active bacterial or fungal infection will be excluded. INTERVENTION AND COMPARATOR: Patients will be randomized to 1 of 5 experimental arms versus usual care. The experimental arms consist of Anakinra alone (anti-IL-1 binding the IL-1 receptor), Siltuximab alone (anti-IL-6 chimeric antibody), a combination of Siltuximab and Anakinra, Tocilizumab alone (humanised anti-IL-6 receptor antibody) or a combination of Anakinra with Tocilizumab in addition to standard care. Patients treated with Anakinra will receive a daily subcutaneous injection of 100mg for a maximum of 28 days or until hospital discharge, whichever comes first. Siltuximab (11mg/kg) or Tocilizumab (8mg/kg, with a maximum dose of 800mg) are administered as a single intravenous injection immediately after randomization. MAIN OUTCOMES: The primary end point is the time to clinical improvement defined as the time from randomization to either an improvement of two points on a six-category ordinal scale measured daily till day 28 or discharge from the hospital or death. This ordinal scale is composed of (1) Death; (2) Hospitalized, on invasive mechanical ventilation or ECMO; (3) Hospitalized, on non-invasive ventilation or high flow oxygen devices; (4) Hospitalized, requiring supplemental oxygen; (5) Hospitalized, not requiring supplemental oxygen; (6) Not hospitalized. RANDOMISATION: Patients will be randomized using an Interactive Web Response System (REDCap). A 2x2 factorial design was selected with a 2:1 randomization regarding the IL-1 blockade (Anakinra) and a 1:2 randomization regarding the IL-6 blockade (Siltuximab and Tocilizumab). BLINDING (MASKING): In this open-label trial neither participants, caregivers, nor those assessing the outcomes are blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 342 participants will be enrolled: 76 patients will receive usual care, 76 patients will receive Siltuximab alone, 76 patients will receive Tocilizumab alone, 38 will receive Anakinra alone, 38 patients will receive Anakinra and Siltuximab and 38 patients will receive Anakinra and Tocilizumab. TRIAL STATUS: COV-AID protocol version 3.0 (15 Apr 2020). Participant recruitment is ongoing and started on April 4th 2020. Given the current decline of the COVID-19 pandemic in Belgium, it is difficult to anticipate the rate of participant recruitment. TRIAL REGISTRATION: The trial was registered on Clinical Trials.gov on April 1st, 2020 (ClinicalTrials.gov Identifier: NCT04330638) and on EudraCT on April 3rd 2020 (Identifier: 2020-001500-41). FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Correction to: Treatment of severely ill COVID-19 patients with anti-interleukin drugs (COV-AID): A structured summary of a study protocol for a randomised controlled trial.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: Sargramostim to treat patients with acute hypoxic respiratory failure due to COVID-19 (SARPAC): A structured summary of a study protocol for a randomised controlled trial.

An amendment to this paper has been published and can be accessed via the original article.

Sargramostim to treat patients with acute hypoxic respiratory failure due to COVID-19 (SARPAC): A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The hypothesis of the proposed intervention is that Granulocyte-macrophage colony-stimulating factor (GM-CSF) has profound effects on antiviral immunity, and can provide the stimulus to restore immune homeostasis in the lung with acute lung injury post COVID-19, and can promote lung repair mechanisms, that lead to a 25% improvement in lung oxygenation parameters. Sargramostim is a man-made form of the naturally-occurring protein GM-CSF. TRIAL DESIGN: A phase 4 academic, prospective, 2 arm (1:1 ratio), randomized, open-label, controlled trial. PARTICIPANTS: Patients aged 18-80 years admitted to specialized COVID-19 wards in 5 Belgian hospitals with recent (< 2 weeks prior to randomization) confirmed COVID-19 infection and acute respiratory failure defined as a PaO2/FiO2 below 350 mmHg or SpO2 below 93% on minimal 2 L/min supplemental oxygen. Patients were excluded from the trial in case of (1) known serious allergic reactions to yeast-derived products, (2) lithium carbonate therapy, (3) mechanical ventilation prior to randomization, (4) peripheral white blood cell count above 25.000/µL and/or active myeloid malignancy, (5) high dose systemic steroid therapy (> 20 mg methylprednisolone or equivalent), (6) enrolment in another investigational study, (7) pregnant or breastfeeding or (8) ferritin levels > 2000 µg/mL. INTERVENTION AND COMPARATOR: Inhaled sargramostim 125 µg twice daily for 5 days in addition to standard care. Upon progression of disease requiring mechanical ventilation or to acute respiratory distress syndrome (ARDS) and initiation of mechanical ventilator support within the 5 day period, inhaled sargramostim will be replaced by intravenous sargramostim 125 µg/m2 body surface area once daily until the 5 day period is reached. From day 6 onwards, progressive patients in the active group will have the option to receive an additional 5 days of IV sargramostim, based on the treating physician's assessment. Intervention will be compared to standard of care. Subjects progressing to ARDS and requiring invasive mechanical ventilatory support, from day 6 onwards in the standard of care group will have the option (clinician's decision) to initiate IV sargramostim 125m µg/m2 body surface area once daily for 5 days. MAIN OUTCOMES: The primary endpoint of this intervention is measuring oxygenation after 5 days of inhaled (and intravenous) treatment through assessment of a change in pretreatment and post-treatment ratio of PaO2/FiO2 and through measurement of the P(A-a)O2 gradient (PAO2= Partial alveolar pressure of oxygen, PaO2=Partial arterial pressure of oxygen; FiO2= Fraction of inspired oxygen). RANDOMISATION: Patients will be randomized in a 1:1 ratio. Randomization will be done using REDCap (electronic IWRS system). BLINDING (MASKING): In this open-label trial neither participants, caregivers, nor those assessing the outcomes will be blinded to group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A total of 80 patients with confirmed COVID-19 and acute hypoxic respiratory failure will be enrolled, 40 in the active and 40 in the control group. TRIAL STATUS: SARPAC protocol Version 2.0 (April 15 2020). Participant recruitment is ongoing in 5 Belgian Hospitals (i.e. University Hospital Ghent, AZ Sint-Jan Bruges, AZ Delta Roeselare, University Hospital Brussels and ZNA Middelheim Antwerp). Participant recruitment started on March 26th 2020. Given the current decline of the COVID-19 pandemic in Belgium, it is difficult to anticipate the rate of participant recruitment. TRIAL REGISTRATION: The trial was registered on Clinical Trials.gov on March 30th, 2020 (ClinicalTrials.gov Identifier: NCT04326920) - retrospectively registered; https://clinicaltrials.gov/ct2/show/NCT04326920?term=sarpac&recrs=ab&draw=2&rank=1 and on EudraCT on March 24th, 2020 (Identifier: 2020-001254-22). FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Adverse outcomes of frailty in the elderly: the Rotterdam Study.

To investigate the prevalence of frailty in a Dutch elderly population and to identify adverse health outcomes associated with the frailty phenotype independent of the comorbidities. Cross-sectional and longitudinal analyses within the Rotterdam Study (the Netherlands), a prospective population-based cohort study in persons aged ≥55 years. Frailty was defined as meeting three or more of five established criteria for frailty, evaluating nutritional status, physical activity, mobility, grip strength and exhaustion. Intermediate frailty was defined as meeting one or two frailty criteria. Comorbidities were objectively measured. Health outcomes were assessed by means of questionnaires, physical examinations and continuous follow-up through general practitioners and municipal health authorities for mortality. Of 2,833 participants (median age 74.0 years, inter quartile range 9) with sufficiently evaluated frailty criteria, 163 (5.8 %) participants were frail and 1,454 (51.3 %) intermediate frail. Frail elderly were more likely to be older and female, to have an impaired quality of life and to have fallen or to have been hospitalized. 108 (72.0 %) frail participants had ≥2 comorbidities, compared to 777 (54.4 %) intermediate frail and 522 (44.8 %) non-frail participants. Adjusted for age, sex and comorbidities, frail elderly had a significantly increased risk of dying within 3 years (HR 3.4; 95 % CI 1.9-6.4), compared to the non-frail elderly. This study in a general Dutch population of community-dwelling elderly able to perform the frailty tests, demonstrates that frailty is common and that frail elderly are at increased risk of death independent of comorbidities.

Range of motion in femoroacetabular impingement.

Recent epidemiological studies have demonstrated that radiographic features specific to femoroacetabular impingement appear far more frequently in healthy and asymptomatic cohorts than previously anticipated. It remains unclear how incidental findings should be interpreted clinically. In addition, several authors have suggested that a decreased range of motion is part of the clinical presentation of femoroacetabular impingement. The purpose of the present study was to describe and analyze differences in range of motion between femoroacetabular impingement patients, asymptomatic individuals with incidental radiographic findings and healthy controls, using a validated electromagnetic tracking system. Furthermore, it was evaluated which motions were clinically relevant and could be used to differentiate between these three groups. We found all evaluated motions to differ significantly between patients and controls. The anterior impingement test showed a significant difference between patients and asymptomatic cases. In conclusion, functional evaluation of the range of motion appeared in this study as a useful tool in the diagnostic work-up of femoracetabular impingement.