ABSTRACT
Purpose of Study To investigate potential evidence of in utero transmission of SARS-CoV-2 in the stool of newborns born to mothers with COVID-19 infection during pregnancy. Methods Used We investigated stool from 1 day to 2 months of age from 14 newborns born at 25-41 weeks whose mothers had COVID-19 during pregnancy. Newborns were admitted at delivery to the NICU or newborn nursery of our urban academic hospital from July 2020 to May 2021. A comparison group of 30 newborns had similar GAs and were born to mothers without COVID-19 during pregnancy. SARS-CoV-2 RNA was quantified with quantitative PCR using primers against SARS-CoV-2 envelope protein and non-structural protein 14 (NSP-14), spike protein with enzyme-linked immunosorbent assay (ELISA), and inflammatory cytokines interleukin- 6 (IL-6) and interferon-g (IFN-γ) elicited by stool homogenates in mouse bone marrow macrophages. This study was IRB approved with parental consent. Summary of Results Despite negative nasal PCRs from all newborns, viral RNAs and spike protein were detected in the stool of 11 out of 14 newborns as early as the first day of life (range 0-2 months, figure 2A and 2B). Stool RNA and spike protein levels increased over time in 2 and 4 newborns, respectively. Stool homogenates from all newborns elicited elevated inflammatory cytokines, IL-6 and IFN-γ, from mouse macrophages (figure 1). Most newborns were clinically well except for one death from gestational autoimmune liver disease and one with necrotizing enterocolitis. Conclusions These novel findings suggest risk of in utero SARS-CoV-2 transmission to the premature and term fetal intestine during gestation despite negative postnatal nasal PCRs. It is unclear if the presence of viral RNA and protein within the gut microbiome represents active virus in newborns with clinical hospital courses typical of their gestational age in 12 out of 14 cases. However, increasing levels of viral RNA and protein over time suggest replication in some infants, and their gut microbiome induced inflammation in mouse models. The presence of SARS-CoV-2 RNA and spike protein in the intestines of newborns may potentially impact development of the gut microbiome and the immune system and should be further investigated. (Figure Presented).
ABSTRACT
Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.
ABSTRACT
Background: Modest weight loss (5-7%) remains the goldstandard of treatment for nonalcoholic fatty liver disease (NAFLD), though patients rarely achieve this independently The YMCA's Diabetes Prevention Program (DPP) is a nationwide structured lifestyle intervention program that has demonstrated weight loss within this range The goal of this study is to determine if the DPP can be repurposed to induce weight loss, decrease liver enzymes, and improve hepatic steatosis in NAFLD Methods: Eligible patients had NAFLD (defined by Fibroscan controlled attenuation parameter (CAP) score >238dB/m) and elevated liver enzymes (ALT>25U/L in females, >33U/L in males) without competing etiologies for liver disease Patients with cirrhosis were excluded A retrospective cohort that received standard of care recommendations was analyzed for change in ALT Two sequential prospective cohorts of 20 patients were enrolled in the experimental YMCA DPP Classroom sessions occurred weekly for 16 weeks, bi-weekly for 4 weeks, and then monthly for 48 weeks total Primary endpoints included change in BMI, ALT, and Fibroscan score. Completers were defined as those attending at least 9 of the first 16 weekly sessions. Changes in variables were analyzed using paired t-test End of study data was not completed for cohort 2 due to COVID19 and therefore aggregate data is presented for week 16 and end of study data (week 48) is presented for only cohort 1 Results: A total of 20 patients from both cohorts were completers 16% were female (n=12) 16 patients had interim study data and were included in the week 16 analysis All but one patient lost weight Baseline mean BMI was 34 3 and decreased to 33 6 by week 16 (p=0 02) Baseline mean ALT was 91 8U/L and decreased to 55 1U/L by week 16 (p=0 005) Mean Fibroscan fibrosis score improved from 6.7kPa to 5.5kPa and CAP score improved from 321dB/m to 291dB/m by week 16 (p=0 008 and p=0 12, respectively) 9 patients from cohort 1 were included in the week 48 analysis Mean BMI was 35 4 at baseline and decreased to 33 6 by week 48 (p=0 002) Mean ALT was 85 0U/L at baseline decreased to 41 6U/L (p=0 01) Mean Fibroscan score improved from 6 9kPa to 5 9kPa (p=0 07) Other metabolic parameters also trended towards significant improvement at week 16 and 48 (Table 1). The retrospective cohort (N=31) had a mean baseline ALT of 69U/L with no significant change at 6-month follow-up (mean ALT 60U/L, p=0 24) Conclusion: Standard of care lifestyle recommendations are ineffective in inducing weight loss and improvement in ALT in NAFLD The YMCA DPP repurposed to treat NAFLD resulted in statistically significant improvements in BMI and ALT in patients with NAFLD Fibroscan scores and other metabolic parameters also improved, though the sample size is likely too small to see a statistically significant improvement The existing infrastructure established by the YMCA throughout the U S could emerge as a leading treatment option for NAFLD patients.
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Background: COVID-19 was declared a pandemic by the World Health Organization, caused by severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) Respiratory failure is the most common mortality outcome in COVID-19 patients, yet serious and even fatal manifestations are seen across multiple organ systems Emerging clinical data from our own hospital revealed a high prevalence of initial presentations with GI manifestations Almost one third of patients presenting to our hospital reported at least one gastrointestinal manifestation including nausea, vomiting, diarrhea, or abdominal pain 62% of patients presented with biochemical evidence of liver injury Moreover, the presence of liver injury on presentation was associated with a significantly higher risk of ICU admission and death As this is a new and novel clinical entity, robust in vitro models that phenocopy SARS-CoV-2 infection and human COVID-19 disease are limited Current in vitro (e g Vero cells) and in vivo models (mouse models engineered with ACE2) are so distinct from human infection that they may not capture key components of viral infection or virus-host interactions Therefore, the development of robust human models of COVID-19 infection will be essential for the study of SARS-Cov-2 viral infection and to identify robust SARS-CoV-2 therapeutics Methods: Human pluripotent stem cells (hPSCs), including human embryonic stem cells hESCs) and induced pluripotent stem cells (hiPSCs), can be used to derive functional human cells/tissues/organoids for modeling human disease and drug discovery, including for infectious diseases Here we leveraged several stem cell platforms (e g endodermal lineages including hepatocyte and cholangiocyte) along with primary human hepatocytes and cholangiocyte organoid systems to study SARS-CoV-2 infection SARS-CoV-2 pseudoparticles were used to study SARS-CoV-2 viral entry SARS-CoV-2 (USA-WA1/2020) was used to validate viral infection and to study cellular response Autopsy liver samples from COVID-19 patients were obtained and compared to SARS-CoV-2 infected liver models Results: Adult hepatocyte and cholangiocyte organoids along with PSC derived hepatocytes and cholangiocytes are Permissive to SARS-CoV-2 virus infection and show similar transcriptome changes and chemokine responses for SARSCoV- 2 infection as seen in autopsy samples from COVID-19 Patients Conclusion: We report here the development of robust models of SARS-CoV-2 infection in primary and PSC derived hepatocyte and cholangiocytes which phenocopy COVID-19 hepatic disease These disease-relevant human cell/organoid-based platforms can be directly applied for drug screening and the evaluation of prospective antiviral therapeutics as well be used to delineate molecular mechanisms underlying COVID-19 disease.