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The proceedings contain 222 papers. The topics discussed include: acute respiratory distress syndrome;lung protective and acute respiratory distress syndrome ventilation strategies in the age of COVID19;natural history and trajectory of non-COVID-19 acute respiratory distress syndrome patients. an observational study for comparison to COVID-19 populations;evaluation of neutrophil microvesicles function in a novel tri-culture model of pulmonary vascular inflammation;adult pediatric critical care in low and middle-income countries;a review of re-intubations in a mixed general and neurosciences unit and the development of extubation check list;accidental tooth ingestion in the intensive care unit;life-threatening tracheobronchial obstruction with blood clot, managed using whole endotracheal tube suction;why can't we purchase kidneys? a ethical review of presumed consent organ donation compared against a regulated organ marketplace;brain death, organ donation and transplantation;and retrospective review of patients with out-of-hospital cardiac arrest: post-resuscitation care pathway drives improved patient survival.
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Aim and background: Cases of thrombotic thrombocytopenia induced by coronavirus disease 2019 (COVID-19) vaccines have been reported recently. Herein, we describe hemophagocytic lymphohistiocytosis (HLH) following COVID-19 vaccination. Case report: A 35-year-old male, chronic alcoholic, 3 years into abstinence received first dose Covishield vaccine. He started developing a fever, testicular pain, diminished sensorium requiring invasive ventilation, and decreased urine output 4 days after getting vaccinated. Initial workup for NCCT brain and HRCT chest was normal, tropical fever panel was negative, cultures for blood and endotracheal aspirate were sterile, liver and renal functions showed mild derangement, CSF study was normal. Ultrasound examination of the abdomen revealed mild hepatosplenomegaly, mild testicular swelling, and suprainguinal lymphadenopathy, with no focus of infection. Subsequently, he developed bicytopenia with haemoglobin 9.0 g/dL and platelet counts 50 × 109/L, ferritin 2130 μg/L, triglyceride 353 mg/dL, and decreased fibrinogen 1.41 g/L. Bone marrow as well as lymph node biopsy showed haemophagocytosis with engulfment of neutrophils, lymphocytes, and normoblasts making HLH a likely diagnosis. Soluble CD25 and NK cell function could not be performed. Extensive evaluation was done to look into the etiology of HLH. SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR) test was negative. RT-PCR test for Epstein-Barr virus (EBV), influenza A (H1N1, H3N2), influenza B, cytomegalovirus (CMV) performed from endotracheal aspirate (ETA) was negative. Similarly, the RT-PCR test from serum samples for EBV, Parvo B-19, CMV, and from CSF sample for EBV, Parvo B-19, CMV, and HSV-1 was negative. Hepatitis B, C, and HIV serologies were negative. Culture and sensitivity repeated from blood, ETA and urine was sterile. Autoimmune panel including complements levels were negative. Peripheral smear, bone marrow, and lymph node biopsy were normal and did not reveal abnormal or malignant cells. He had persistent fevers to 38.6°C during the first 6 days of his admission, with a rise in his ferritin to 1950 μg/L. The patient received steroids but not etoposide. By the 8th day, his fevers resolved, with improvement in his lethargy and malaise. Two weeks later, his ferritin had reduced to 510 μg/L, platelet count rose to 180 × 109/L, and repeat ultrasound abdomen demonstrated resolution of his splenomegaly. In our patient, there was no clear precipitant of HLH other than the Covishield vaccine. There was no evidence of an infection or malignancy. Due to our patient's clinical stability, resolution of symptoms, and improvement of HLH parameters he did not require HLH specific therapy. It is unclear if he had a pre-existing genetic predisposition to HLH as genetic testing is pending, however, it is unlikely as he has reached the age of 35 and suffered from previous viral infections without developing HLH.
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Endothelial cells (ECs) lining the blood vessels of all organs express the SARS-CoV2 receptor. In the absence of preexisting tissue damage, the virus would need to pass through the ECs to blood vessels to infect other tissues. Thus, EC are a target for SARS-CoV-2 infection and a conduit for viral dissemination to distant organs. We hypothesized that ECs infection and/ or injury are the mechanisms of COVID-19 pathology and multi-organ dissemination and injury. Methods: Human studies: We used lung, heart, kidney, and small bowel specimens obtained during autopsies (n=5) from COVID-19 patients and uninfected subjects. Studies: 1) histologic evaluation of endothelial damage and endotheliitis, 2) immunohistochemistry for vWF, PAI-1, VCAM-1, & ICAM-1. Studies in cultured human microvascular ECs (HMVECs): We cultured lung and cardiac HMVECs in the presence or absence of SARSCoV- 2 S1 and/or S2 protein (10 ng/ml) for 0 - 24 hr. Studies:1) cell viability and proliferation;2) angiogenesis on Matrigel and cell migration;3) mitochondrial membrane potential (MMP);4) RNA seq analysis;5) Western blotting for vWF, PAI-1, VCAM-1, and ICAM-1. We examined the protective effect of melatonin, Coenzyme Q10 and nerve growth factor on S1/S2 protein induced HMVEC cell damage. Results: Histopathologic examination revealed presence of endothelial abnormalities and endotheliitis with marked presence of inflammatory cells in vessel wall & lumen, and fibrinous microthrombi) in lung, heart & kidney in autopsy specimens of COVID-19 patients. Immunostaining visualized increased vWF, PAI-1, VCAM- 1, & ICAM-1 in COVID-19. In in vitro study, S1 and S2 proteins induced endothelial injury, reduced angiogenesis and phosphorylated/activated Erk and Akt proteins in cultured HMVECs. Treatment of HMVECs for 1 & 4 hours with S2 but not S1 protein increased ICAM-1 levels by 1.4- to 1.8-fold (P < 0.001). RNA Seq analysis showed that treatment of HMVECs with S1 and S2 proteins upregulated VCAM-1, ICAM-1 and E-selectin mRNA in cultured HMVECs. Melatonin, Coenzyme Q10 and NGF stimulated angiogenesis in HMVECs by 2.4-, 1.3-&1.4-fold (all P < 0.001). Conclusions: 1) Significant endothelial abnormalities, blood vessel damage and endotheliitis are present in lung, heart and kidney autopsy specimens of COVID-19 patients, 2) There is increased expression of vWF, PAI-1, VCAM-1, and ICAM- 1 in lung, heart, and kidney specimens of COVID-19 patients, 3) Treatment of cultured HMVECs with SARS-CoV-2 S1 and S2 proteins upregulates VCAM-1, ICAM-1 and Eselectin expression, 4) SARS-CoV-2 S1 and S2 proteins induce endothelial injury in cultured HMVECs, and 5) melatonin, Coenzyme Q10 and NGF stimulated EC function. These studies uncovered novel mechanism – endothelial dysfunction underlying SARS-CoV-2 and identified melatonin, Coenzyme Q10 and NGF as potential drugs for treatment of COVID- 19-induced EC injury
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RATIONALE: Treatments for the coronavirus disease of 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through the interactions of its spike (S) protein with ACE2 and TMPRSS2 on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. Our finding that the airway administration of OM-85 inhibits Ace2 and Tmprss2 transcription in mouse lungs prompted us to investigate whether and how OM-85 may protect non-human primate and human epithelial cells against SARS-CoV-2 infection. METHODS: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein-pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2- transfected HEK293T cells treated with OM-85 in vitro. RESULTS: OM-85 significantly downregulated ACE2 and TMPRSS2 mRNA in epithelial cell lines and primary bronchial epithelial cells, and strongly inhibited SARS-CoV-2 S protein binding to, SARS-CoV-2 S proteinpseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on the downregulation of SARS-CoV-2 receptor expression. CONCLUSIONS: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of COVID-19.
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Nature efficiently self-organizes cells and tissues into complex fractal forms. Whether fractal patterning contributes functionally to maturation, and how cells sense and interpret such shape cues, is not well understood. Using kidney podocytes as a model system, bioinspired templating of glomerular histology was leveraged to design controlled fractal 21/2 -D surfaces for cell culture. Microcurvature was associated with charge density gradients in space, found to direct extracellular matrix protein organization resulting in hierarchical assembly of cell structures and fractally-branching podocyte morphology in vitro, outlined with a novel fluorescent assaying technique. Shape simulation was uniquely associated with mature-like foot processes on an organized ECM. In applications of drug testing, coronavirus infection, and a cells-as-sensors approach to patient serum diagnostics, fractally stimulated cells were more responsive than flat cultures. Fractal frameworks may thus provide a functional role in podocyte maturation and could serve to advance other bioengineered systems.
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Background. Fungal blood cultures (fungal isolators) should be used, if at all, primarily for identification of mold infections. At our institution we noted patients having fungal blood cultures drawn in many other situations, including when the primary team was concerned for candida bloodstream infection. We sought to describe the utility of this practice and of fungal blood cultures in general. Methods. We retrospectively reviewed the results of fungal blood cultures for 2 years, from 3/1/2019-3/1/2021. We evaluated the number of episodes, culture results, whether there was a had prior bloodstream infection, and risk factors for fungal infection including renal replacement (RRT), ECMO, and immunosuppression (IS). Immunosuppression was defined as chronic systemic steroid use, recent receipt of high dose steroids within 2 weeks, history of organ transplantation, history of stem cell transplantation, hematologic malignancies, or receipt of a biologic agent. Results. 187 fungal blood cultures were drawn in 143 patients - 80 cultures in 70 patients from 3/2019-3/2020 and 107 cultures in 73 patients from 3/2020-3/2021. Only 3 patients had positive fungal blood cultures:1 (Candida krusei) from 3/2019-3/2020 and 2 (Candida albicans and Cyrptococcus neoformans) from 3/2020-3/2021;in all 3 cases the organism also grew from standard blood culture isolators. From 3/2019-3/2020, 1/80 cultures were drawn from an individual on ECMO while 15/80 were drawn from individuals on RRT, and 32/80 were in a IS individuals. From 3/2020-3/2021, 45/107 cultures were drawn from an individual on ECMO, 24/107 were drawn in an individual on RRT, and 73/107 were drawn in a IS individuals. The majority of individuals in whom a fungal blood culture was drawn during 3/2020-3/2021 were individuals with COVID-19. Upon chart review most of the cultures were drawn due to concern for candidemia. Conclusion. Fungal blood cultures have an extremely low yield at our institution, with a 1.6% positivity rate over a 2 year period, and all of those cultures were detected by standard blood culture isolators. Most of these cultures were drawn in situations where this test has no utility. Furthermore, the test has limited utility to detect dimorphic and mold bloodstream infections. Restriction of this test may limit inappropriate use.
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Background/Introduction: SARS-CoV-2 causes life threatening COVID- 19 complications including acute coronary syndrome, venous thromboembolism, hyperinflammation and damage in multiple tissues. The SARSCoV- 2 spike protein binds cell surface receptors including angiotensinconverting enzyme 2 (ACE2) for entry into host cells to initiate infection. Host cell dipeptidyl peptidase-4 (DPP4 / CD26) is implicated as a cofactor in uptake. Recent evidence indicates expression of factors involved in SARS-CoV-2 uptake into host cells is regulated by BET proteins, epigenetic readers modulating gene expression. Apabetalone, the most clinically advanced BET inhibitor (BETi), is in phase 3 trials for cardiovascular disease (CVD) (a, b). In cultured human cardiomyocytes, apabetalone suppressed infection with SARS-CoV-2 and prevented dysfunction of cardiac organoids induced by the cytokine-storm that arises in patients with severe symptoms (c). However, anti-viral properties of apabetalone in other cell types are not known. Purpose: To examine effects of apabetalone on SARS-CoV-2 infection in cell culture via downregulated expression of cell surface receptors involved in viral entry. Cell systems used mimic initial sites of infection in the lung as well as cell types contributing to complications in late stages of infection. Methods: Gene expression was measured by real-time PCR, protein levels by immunoblot or flow cytometry, and binding of recombinant SARSCoV- 2 spike protein by flow cytometry. Infection with SARS-CoV-2 was determined in a BSL3 facility. Infectivity was quantified by determining levels of viral spike protein amongst total cells via imaging on an Operetta CLS. Results: In Calu-3, a human bronchial epithelial cell line, apabetalone dose-dependently downregulated ACE2 gene expression (up to 98%), reduced ACE2 protein levels (up to 84%) and diminished binding of SARSCoV- 2 spike protein (up to 77%, p<0.001 for all parameters). Further, apabetalone abolished infection of Calu-3 cells with live SARS-CoV-2, which was comparable to other antiviral agents. Apabetalone-driven ACE2 downregulation was also observed in extrapulmonary cell types including HepG2, Huh-7 or primary hepatocytes (up to 90%, p<0.001 for all cell types), and Vero E6, a monkey kidney epithelial cell line (up to 38%, p<0.05). DPP4/CD26, a potential cofactor for SARS-CoV-2 uptake, was also downregulated by apabetalone in Calu-3 cells (mRNA ∼65% and protein ∼40%, p<0.001), which may be synergistic with ACE2 reductions to impede SARS-CoV-2 infection. Conclusions: Apabetalone, an investigational drug for CVD, reduced cell surface receptors (ACE2 and DPP4) involved in SARS-CoV-2 uptake into host cells and dramatically attenuated SARS-CoV-2 infection/propagation in vitro. Our results suggest apabetalone can mitigate SARS-CoV-2 replication in multiple organs, which together with an established safety profile supports clinical evaluation of apabetalone to treat.