Evaluation of serological lateral flow assays for severe acute respiratory syndrome coronavirus-2.

BACKGROUND: COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. METHODS: We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays' performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. RESULTS: Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 µg/mL), followed by a similar LOD of 1.5 µg/mL for CareHealth, Cellex, KHB, and Vivachek. CONCLUSION: We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

RATIONALE: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown. OBJECTIVE: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene. METHODS AND RESULTS: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions. CONCLUSIONS: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.

Lipodystrophy, Diabetes and Normal Serum Insulin in PPARγ-Deficient Neonatal Mice.

Peroxisome proliferator activated receptor gamma (PPARγ) is a pleiotropic ligand activated transcription factor that acts in several tissues to regulate adipocyte differentiation, lipid metabolism, insulin sensitivity and glucose homeostasis. PPARγ also regulates cardiomyocyte homeostasis and by virtue of its obligate role in placental development is required for embryonic survival. To determine the postnatal functions of PPARγ in vivo we studied globally deficient neonatal mice produced by epiblast-restricted elimination of PPARγ. PPARγ-rescued placentas support development of PPARγ-deficient embryos that are viable and born in near normal numbers. However, PPARγ-deficient neonatal mice show severe lipodystrophy, lipemia, hepatic steatosis with focal hepatitis, relative insulin deficiency and diabetes beginning soon after birth and culminating in failure to thrive and neonatal lethality between 4 and 10 days of age. These abnormalities are not observed with selective PPARγ2 deficiency or with deficiency restricted to hepatocytes, skeletal muscle, adipocytes, cardiomyocytes, endothelium or pancreatic beta cells. These observations suggest important but previously unappreciated functions for PPARγ1 in the neonatal period either alone or in combination with PPARγ2 in lipid metabolism, glucose homeostasis and insulin sensitivity.

PPARgamma regulates trophoblast proliferation and promotes labyrinthine trilineage differentiation.

BACKGROUND: Abnormal trophoblast differentiation and function is the basis of many placenta-based pregnancy disorders, including pre-eclampsia and fetal growth restriction. PPARgamma, a ligand-activated nuclear receptor, plays essential roles in placental development; null murine embryos die at midgestation due to abnormalities in all placental layers, in particular, small labyrinth and expanded giant cell layer. Previous studies have focused mostly on the role of PPARgamma in trophoblast invasion. Based on the previously reported role of PPARgamma in preadipocyte differentiation, we hypothesized that PPARgamma also plays a pivotal role in trophoblast differentiation. To test this hypothesis, we report derivation of wild-type and PPARgamma-null trophoblast stem (TS) cells. METHODOLOGY/PRINCIPAL FINDINGS: PPARgamma-null TS cells showed defects in both proliferation and differentiation, specifically into labyrinthine trophoblast. Detailed marker analysis and functional studies revealed reduced differentiation of all three labyrinthine lineages, and enhanced giant cell differentiation, particularly the invasive subtypes. In addition, rosiglitazone, a specific PPARgamma agonist, reduced giant cell differentiation, while inducing Gcm1, a key regulator in labyrinth. Finally, reintroducing PPARgamma into null TS cells, using an adenovirus, normalized invasion and partially reversed defective labyrinthine differentiation, as assessed both by morphology and marker analysis. CONCLUSIONS/SIGNIFICANCE: In addition to regulating trophoblast invasion, PPARgamma plays a predominant role in differentiation of labyrinthine trophoblast lineages, which, along with fetal endothelium, form the vascular exchange interface with maternal blood. Elucidating cellular and molecular mechanisms mediating PPARgamma action will help determine if modulating PPARgamma activity, for which clinical pharmacologic agonists already exist, might modify the course of pregnancy disorders associated with placental dysfunction.

E-selectin is required for the antiangiogenic activity of endostatin.

Endostatin, a 20-kDa fragment of collagen XVIII, is a potent angiogenesis inhibitor. E-selectin, an inducible leukocyte adhesion molecule specifically expressed by endothelial cells, has also been implicated in angiogenesis. By using in vivo, ex vivo, and in vitro angiogenic assays, we investigated the functional relationship between endostatin and E-selectin. In corneal micropocket assays, recombinant endostatin administered i.p. by osmotic pump inhibited basic fibroblast growth factor-induced angiogenesis in WT, but not E-selectin-deficient, mice. Similarly, endostatin inhibited vascular endothelial growth factor-stimulated endothelial sprout formation from aortic rings dissected from WT but not from E-selectin-deficient mice. To further explore this apparent requirement for E-selectin in endostatin action, we manipulated E-selectin expression in cultured human endothelial cells. When E-selectin was induced by IL-1beta, or lipopolysaccharide, human umbilical vein endothelial cells and human dermal microvascular endothelial cells each became markedly more sensitive to inhibition by endostatin in a vascular endothelial growth factor-induced cell migration assay. To dissociate E-selectin expression from other consequences of endothelial activation, human umbilical vein endothelial cells were transduced with an adenoviral human E-selectin expression construct; these cells also showed increased sensitivity to endostatin, and this effect required the E-selectin cytoplasmic domain. Taken together, these results indicate that E-selectin is required for the antiangiogenic activity of endostatin in vivo and ex vivo and confers endostatin sensitivity to nonresponsive human endothelial cells in vitro. E-selectin may be a useful predictor and modulator of endostatin efficacy in antiangiogenic therapy.