Corey R. Arnold

The coronavirus disease 2019 (COVID-19) pandemic has affected all Canadian families, with some impacted differently than others. Our study aims to: (1) determine the prevalence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among Canadian families, (2) identify predictors of infection susceptibility and severity of SARS-CoV-2, and (3) identify health and psychosocial impacts of the COVID-19 pandemic. This study builds upon the CHILD Cohort Study, an ongoing multi-ethnic general population prospective cohort consisting of 3,454 Canadian families with children born in Vancouver, Edmonton, Manitoba, and Toronto between 2009 and 2012. During the pandemic, CHILD households were invited to participate in the CHILD COVID-19 Add-On Study involving: (1) brief biweekly surveys about COVID-19 symptoms and testing; (2) quarterly questionnaires assessing COVID-19 exposure and testing, vaccination status, physical and mental health, and pandemic-driven life changes; and (3) in-home biological sampling kits to collect blood and stool. In total, 1,462 households (5,378 participants) consented to the CHILD COVID-19 Add-On Study: 2,803 children (mean±standard deviation [SD], 9.0±2.7 years; range, 0-17 years) and 2,576 adults (mean±SD, 43.0±6.5 years; range, 18-85 years). We will leverage the wealth of pre-pandemic CHILD data to identify risk and resilience factors for susceptibility and severity to the direct and indirect pandemic effects. Our short-term findings will inform key stakeholders and knowledge users to shape current and future pandemic responses. Additionally, this study provides a unique resource to study the long-term impacts of the pandemic as the CHILD Cohort Study continues.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage‐like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation—a signature cellular process of the macrophage—many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation‐competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage‐like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow‐derived macrophages. To supplement the existing data, mass spectrometry‐based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow‐derived macrophages and the macrophage‐like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1 , but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2 , functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2 , thereby modulating stroke risk.

BACKGROUND Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable “Made-in-Canada” solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The “Made-in-Canada” set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

Historically an enzyme of the immune system, the lysosomal reductase GILT serves a unique homeostatic role in bone resorption.