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BACKGROUND: Tracheostomies are highly aerosolizing procedures yet are often indicated in patients with COVID-19 who require prolonged intubation. Robust investigations of the safety of tracheostomy protocols and provider adherence and evaluations are limited. OBJECTIVES: To determine the rate of COVID-19 infection of health care personnel involved in COVID-19 tracheostomies under a multidisciplinary safety protocol and to investigate health care personnel's attitudes and suggested areas for improvement concerning the protocol. METHODS: All health care personnel involved in tracheostomies in COVID-19-positive patients from April 9 through July 11, 2020, were sent a 22-item electronic survey. RESULTS: Among 107 health care personnel (80.5%) who responded to the survey, 5 reported a positive COVID-19 test result (n = 2) or symptoms of COVID-19 (n = 3) within 21 days of the tracheostomy. Respondents reported 100% adherence to use of adequate personal protective equipment. Most (91%) were familiar with the tracheostomy protocol and felt safe (92%) while performing tracheostomy. Suggested improvements included creating dedicated tracheostomy teams and increasing provider choices surrounding personal protective equipment. CONCLUSIONS: Multidisciplinary engagement in the development and implementation of a COVID-19 tracheostomy protocol is associated with acceptable safety for all members of the care team.

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Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of acute lung injury and sepsis that may result in a pathophysiological condition characterized by alveolar flooding, pulmonary edema, and subsequent hypoxemia. In lung ECs, activation of Rho-associated kinase-1 (ROCK1) phosphorylates myosin light chain (MLC)-associated phosphatase at its inhibitory site, which favors phosphorylation of MLC, stress fiber formation, and hyperpermeability during acute lung injury. The role of microRNA-144 (miR-144) has been well investigated in many human diseases, including cardiac ischemia/reperfusion-induced injury, lung cancer, and lung viral infection; however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated the miR-144-mediated mechanism in the protection of endothelial barrier function in an LPS-induced lung injury model. By using transendothelial electrical resistance and transwell permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR-144 effectively blocked lung EC barrier disruption and hyperpermeability in response to proinflammatory agents. Furthermore, using a gain-and-loss-of-function strategy, overexpression of miR-144 significantly decreased ROCK1 expression. Concomitantly, miR-144 inhibits ROCK1-mediated phosphorylation of MLC phosphataseThr853 and thus phosphorylation of MLCThr18/Ser19 to counteract stress fiber formation in LPS-activated EC. Finally, in LPS-challenged mice, intranasal delivery of miR-144 mimic via liposomes attenuated endotoxemia-induced increases in lung wet/dry ratio, vascular permeability, and inflammation. In conclusion, these data suggest that miR-144-attenuated activation of inflammatory ROCK1/MLC pathway in vascular ECs is a promising therapeutic strategy to counter inflammatory lung injury.

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Extracorporeal life support (ECLS) is a widely used lifesaving technology. Whether ECLS results in immune dysregulation is unclear. This study's aim was to examine whether ECLS affected innate immune response. All patients placed on ECLS were eligible. Blood was obtained before, during, and after ECLS. Function of the innate immune system was measured by ex vivo lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and plasma cytokine levels (interleukin [IL]-6, IL-8, IL-10, and TNF-α). Immunoparalysis was defined as ex vivo TNF-α levels less than 200 pg/ml. Nineteen patients were enrolled with twelve <18 years old. Median ECLS duration was 10 days (range: 3-108); nine patients died. After stratifying the cohort by the presence of immunoparalysis before ECLS, those immunoparalyzed showed increased response to LPS on days 1 and 3 (p = 0.016). Those without pre-ECLS immunoparalysis showed a transient decrease in response on day 3 (p = 0.008). Plasma IL-10 levels were elevated in those with pre-ECLS immunoparalysis and dropped significantly by day 1 (p = 0.031). The number treated with steroids was similar in the two groups. In conclusion, patients with immunoparalysis before ECLS showed a gradual increase in immune function during ECLS, whereas those without immunoparalysis had a transient decrease in responsiveness on day 3.

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Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2Afl/fl) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2Afl/fl mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/ß, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/ß hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-ß/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/ß production compared with control BMDMs. Serum IFN-ß levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-ß-dependent pathways.

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