While several functions of the endogenous prion protein have been studied, the homeostatic function of prion protein is still debated. Notably, prion protein is highly expressed on mast cells, granular immune cells that regulate inflammation. When activated, mast cells shed prion protein, although the mechanism and consequences of this are not yet understood. First, we tested several mast cell lines and found that, while prion protein was almost always present, the total amount differed greatly. Activation of mast cells induced a cleavage of the N-terminal region of prion protein, and this was reduced by protease inhibitors. Exogenous mast cell proteases caused a similar loss of the prion protein N-terminus. Additionally, mast cells shed prion protein in an ADAM10-dependent fashion, even in the absence of activation. Our results suggest that prion protein is cleaved from resting mast cells by ADAM10 and from activated mast cells by mast cell proteases. Prion protein also appears to affect mast cell function, as Prnp-/- bone marrow-derived mast cells showed lower levels of degranulation and cytokine release, as well as lower levels of both Fc epsilon RI and CD117. Finally, we sought to provide clinical relevance by measuring the levels of prion protein in bodily fluids of asthmatic patients, a disease that involves the activation of mast cells. We found an N-terminal fragment of prion protein could be detected in human sputum and serum, and the amount of this prion protein fragment was decreased in the serum of patients with asthma. Loss of prion protein from mast cells occurs constitutively by ADAM10 and as a result of degranulation by mast cell granule proteases.

IgE Abs, best known for their role in allergic reactions, have only rarely been used in immunotherapies. Nevertheless, they offer a potential alternative to the more commonly used IgGs. The affinity of IgE Ag binding influences the type of response from mast cells, so any immunotherapies using IgEs must balance Ag affinity with desired therapeutic effect. One potential way to harness differential binding affinities of IgE is in protein aggregation diseases, where low-affinity binding of endogenous proteins is preferred, but enhanced binding of clusters of disease-associated aggregated proteins could target responses to the sites of disease. For this reason, we sought to create a low-affinity IgE against the prion protein (PrP), which exists in an endogenous monomeric state but can misfold into aggregated states during the development of prion disease. First, we determined that mast cell proteases tryptase and cathepsin G were capable of degrading PrP. Then we engineered a recombinant IgE Ab directed against PrP from the V region of a PrP-specific IgG and tested its activation of the human mast cell line LAD2. The αPrP IgE bound LAD2 through Fc receptors. Crosslinking receptor-bound αPrP IgE activated SYK and ERK phosphorylation, caused Fc receptor internalization, and resulted in degranulation. This work shows that a recombinant αPrP IgE can activate LAD2 cells to release enzymes that can degrade PrP, suggesting that IgE may be useful in targeting diseases that involve protein aggregation.

Mast cells (MC) synthesize and store proinflammatory mediators and are centrally important in atopic diseases such as asthma and atopic dermatitis. Quercetin a and resveratrol are plant derived polyphenolic compounds with anti-inflammatory properties that inhibit MC degranulation and mediator release. However, the underlying mechanism of these inhibitory effects on MC is poorly understood and it is unclear whether this is a general effect on all MC phenotypes. We have characterized and compared the effects of quercetin with resveratrol on human (LAD2) and mouse (MC/9 and BMMC) MC mediator release, receptor expression and FcεRI signaling to better understand the mechanisms involved in quercetin and resveratrol-mediated inhibition of MC activation. Quercetin significantly decreased the expression of FcεRI by BMMC and MC/9, although the effects on MC/9 were associated with a significant reduction in cell viability. Quercetin also inhibited antigen-stimulated TNF release by BMMC. Although neither quercetin nor resveratrol significantly altered antigen-stimulated BMMC degranulation or downstream signaling events such as phosphorylation of spleen tyrosine kinase (SYK) or extracellular signal-regulated kinase 1/2 (ERK), resveratrol inhibited ERK phosphorylation and FcεRI- stimulated degranulation in LAD2. Our data suggests that quercetin and resveratrol inhibit human and mouse MC differentially and that these effects are associated with modification of FcεRI expression, signaling (phosphorylation of SYK and ERK) and mediator release.

Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured. In principle, relative affinities can be ranked by combining CaR-ESI-MS data with relative concentrations established by hydrophilic interaction liquid chromatography (HILIC) performed on the fluorophore-labeled glycan library. To validate this approach, as well as the feasibility of performing CaR-ESI-MS directly on labeled glycans, libraries of labeled N-glycans extracted from the human monocytic U937 cells or intestinal tissues were labeled with 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA). The libraries were screened against plant and human GBPs with known specificities for α2-3- and α2-6-linked sialosides and quantified by HILIC. Dramatic differences, in some cases, were found for affinity rankings obtained with libraries labeled with different fluorophores, as well as those produced using the combined unlabeled/labeled library approach. The origin of these differences could be explained by differential glycan labeling efficiencies, the impact of specific labels on glycan affinities for the GBPs, and the relative efficiency of release of ligands from GBPs in CaR-ESI-MS. Overall, the results of this study suggest that the 2-AB(CaR-ESI-MS)/2-AB(HILIC) combination provides the most reliable description of the binding specificities of GBPs for N-glycans and is recommended for MS-SG applications.

Protease-resistant aggregates of prion proteins (PrP) have been associated with several fata neurodegenerative disease in both humans and animals. Since mast cells are located peripherally at musocal surfaces where prion proteins enter, mast cell responses to PrP may be relevant. To investigate these interactions, a chimeric IgE antibody specific for PrP was designed and used to activate human mast cells.

The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on the 25th January 2020. By March 2020, the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta, and British Columbia, and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease, and Parkinson's disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease, and chronic lung disease, and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming increasingly evident that underlying inflammatory disease contributes to the pathogenesis of SARS-CoV-2. Here, we review the mechanisms behind SARS-CoV-2 infection, and the host inflammatory responses that lead to resolution or progression to severe COVID-19 disease. Furthermore, we discuss the landscape of COVID-19 therapeutics that are currently in development in Canada.

Terahertz radiation (THz) technology is fast-growing, with applications in sensing, security, monitoring and pharmaceutical industries. Since it is non-invasive, THz has been used as a diagnostic and therapeutic tool in medicine but its specific effects on biological systems is still largely under-studied. THz has been used to image tissues and cells mainly because it allows for identification of morphological features without the need for fluorescent or radioactive labels, but the potential effects of high intensities of THz radiation are currently not well understood. One of the hypotheses that has been proposed for possible effects of THz on living cells, is that it disrupts the cell membrane and induces increased permeability. To test this hypothesis we exposed a rat basophilic leukemia cell line (RBL-2H3) to non-thermal intense THz radiation (duration, dose, etc) and observed the internalization of propidium iodide, a fluorescent intercalating agent that binds to DNA. We did not observe any changes in RBL-2H3 fluorescence following exposure to these intense THz pulses suggesting that exposure of RBL-2H3 to THz radiation may not increase their membrane permeability. These experiments were preliminary and further optimization and analysis is required before we can make definitive conclusions. However, our preliminary observations have set a baseline of RBL-2H3 internalization of propidium iodide, show that it is possible to expose RBL-2H3 cells to THz radiation using our configuration, and set the stage for future experiments.

Misfolded prion protein (PrP) is the causative infectious agent in prion diseases in both humans and animals. Despite this, the physiological role of PrP in its native conformation (PrPC) is still poorly understood. While best known for its presence on neurons, PrPC is also present on various immune cells. We determined whether mast cells also expressed surface PrPC and whether PrPC expression was linked to changes in mast cell function. Mast cells are tissue-resident immune cells that modulate many physiologic responses such as allergic inflammation, tissue remodeling, response to infection and neuroplasticity. When activated, mast cells degranulate rapidly, releasing several pre-formed signaling molecules and enzymes. We hypothesized that mast cells expressed PrP and that its expression was influenced by mast cell activation. Human (HMC-1 and LAD2) and rodent (BMMC) mast cell lines expressed surface PrPC with the highest expression in BMMC. Activation of mouse bone marrow derived mast cells (BMMC) with A23187 caused a 12 fold decrease in surface PrP expression after 30 min. Surprisingly, levels of PrP recovered to almost half of untreated levels 3 hrs post-stimulation, and reached similar levels to untreated by 48 hrs. BMMCs derived from PrP knockout mice activated by IgE/antigen released 15–25% less granule contents compared to wild type BMMC, suggesting that PrP is somehow involved in degranulation. There were no significant differences in cell surface expression of the IgE receptor FcɛRI in wild type and knockout BMMC. Our results indicate that PrP is highly expressed on human and rodent mast cells and that it may play an important role in degranulation and activation of mast cells, independent of FcɛRI expression.

The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on 25th January 2020. By March 2020 the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta and British Columbia and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease and Parkinson's disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease and chronic lung disease and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming increasingly evident that underlying inflammatory disease contributes to SARS-CoV-2 pathogenesis. Here, we review the mechanisms of SARS-CoV-2 infection and the host inflammatory responses that lead to resolution or progression to severe COVID-19 disease. Furthermore, we discuss the landscape of COVID-19 therapeutics that are currently in development in Canada.

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral co-receptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Mast cells are long-lived, granular, myeloid-derived leukocytes that have significant protective and repair functions in tissues. Mast cells sense disruptions in the local microenvironment and are first responders to physical, chemical and biological insults. When activated, mast cells release growth factors, proteases, chemotactic proteins and cytokines thereby mobilizing and amplifying the reactions of the innate and adaptive immune system. Mast cells are therefore significant regulators of homeostatic functions and may be essential in microenvironmental changes during pathogen invasion and disease. During infection by helminths, bacteria and viruses, mast cells release antimicrobial factors to facilitate pathogen expulsion and eradication. Mast cell-derived proteases and growth factors protect tissues from insect/snake bites and exposure to ultraviolet radiation. Finally, mast cells release mediators that promote wound healing in the inflammatory, proliferative and remodelling stages. Since mast cells have such a powerful repertoire of functions, targeting mast cells may be an effective new strategy for immunotherapy of disease and design of novel vaccine adjuvants. In this review, we will examine how certain strategies that specifically target and activate mast cells can be used to treat and resolve infections, augment vaccines and heal wounds. Although these strategies may be protective in certain circumstances, mast cells activation may be deleterious if not carefully controlled and any therapeutic strategy using mast cell activators must be carefully explored.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a highly contagious respiratory disease referred to as COVID-19. However, emerging evidence indicates that a small but growing number of COVID-19 patients also manifest neurological symptoms, suggesting that SARS-CoV-2 may infect the nervous system under some circumstances. SARS-CoV-2 primarily enters the body through the epithelial lining of the respiratory and gastrointestinal tracts, but under certain conditions this pleiotropic virus may also infect peripheral nerves and gain entry into the central nervous system (CNS). The brain is shielded by various anatomical and physiological barriers, most notably the blood-brain barrier (BBB) which functions to prevent harmful substances, including pathogens and pro-inflammatory mediators, from entering the brain. The BBB is composed of highly specialized endothelial cells, pericytes, mast cells and astrocytes that form the neurovascular unit, which regulates BBB permeability and maintains the integrity of the CNS. In this review, potential routes of viral entry and the possible mechanisms utilized by SARS-CoV-2 to penetrate the CNS, either by disrupting the BBB or infecting the peripheral nerves and using the neuronal network to initiate neuroinflammation, are briefly discussed. Furthermore, the long-term effects of SARS-CoV-2 infection on the brain and in the progression of neurodegenerative diseases known to be associated with other human coronaviruses are considered. Although the mechanisms of SARS-CoV-2 entry into the CNS and neurovirulence are currently unknown, the potential pathways described here might pave the way for future research in this area and enable the development of better therapeutic strategies.

Understanding the heterogeneity of dysregulated pathways associated with the development of hepatocellular carcinoma (HCC) may provide prognostic and therapeutic avenues for disease management. As HCC involves a complex process of genetic and epigenetic modifications, we evaluated expression of both polyadenylated transcripts and microRNAs from HCC and liver samples derived from two cohorts of patients undergoing either partial hepatic resection or liver transplantation. Copy number variants were inferred from whole genome low-pass sequencing data, and a set of 56 cancer-related genes were screened using an oncology panel assay. HCC was associated with marked transcriptional deregulation of hundreds of protein-coding genes. In the partially resected livers, diminished transcriptional activity was observed in genes associated with drug catabolism and increased expression in genes related to inflammatory responses and cell proliferation. Moreover, several long noncoding RNAs and microRNAs not previously linked with HCC were found to be deregulated. In liver transplant recipients, down-regulation of genes involved in energy production and up-regulation of genes associated with glycolysis were detected. Numerous copy number variants events were observed, with hotspots on chromosomes 1 and 17. Amplifications were more common than deletions and spanned regions containing genes potentially involved in tumorigenesis. Colony stimulating factor 1 receptor (CSF1R), fibroblast growth factor receptor 3 (FGFR3), fms-like tyrosine kinase 3 (FLT3), nucleolar phosphoprotein B23 (NPM1), platelet-derived growth factor receptor alpha polypeptide (PDGFRA), phosphatase and tensin homolog (PTEN), G-protein-coupled receptors-like receptor Smoothened (SMO), and tumor protein P53 (TP53) were mutated in all tumors; another 26 cancer-related genes were mutated with variable penetrance. Conclusion: Our results underscore the marked molecular heterogeneity between HCC tumors and reinforce the notion that precision medicine approaches are needed for management of individual HCC. These data will serve as a resource to generate hypotheses for further research to improve our understanding of HCC biology. (Hepatology Communications 2018; 00:000-000).

Hepatocellular carcinoma (HCC) is influenced by numerous factors, which results in diverse genetic, epigenetic and transcriptional scenarios, thus posing obvious challenges for disease management. We scrutinized the molecular heterogeneity of HCC with a multi-omics approach in two small cohorts of resected and explanted livers. Whole-genome transcriptomics was conducted, including polyadenylated transcripts and micro (mi)-RNAs. Copy number variants (CNV) were inferred from whole genome low-pass sequencing data. Fifty-six cancer-related genes were screened using an oncology panel assay. HCC was associated with a dramatic transcriptional deregulation of hundreds of protein-coding genes suggesting downregulation of drugs catabolism, induction of inflammatory responses, and increased cell proliferation in resected livers. Moreover, several long non-coding RNAs and miRNAs not reported previously in the context of HCC were found deregulated. In explanted livers, downregulation of genes involved in energy-producing processes and upregulation of genes aiding in glycolysis were detected. Numerous CNV events were observed, with conspicuous hotspots on chromosomes 1 and 17. Amplifications were more common than deletions, and spanned regions containing genes potentially involved in tumorigenesis. CSF1R, FGFR3, FLT3, NPM1, PDGFRA, PTEN, SMO and TP53 were mutated in all tumors, while other 26 cancer-related genes were mutated with variable penetrance. Our results highlight a remarkable molecular heterogeneity between HCC tumors and reinforce the notion that precision medicine approaches are urgently needed for cancer treatment. We expect that our results will serve as a valuable dataset that will generate hypotheses for us or other researchers to evaluate to ultimately improve our understanding of HCC biology.

Rubella virus (RV), a member of Togaviridae, is an important human pathogen that can cause severe defects in the developing fetus. Compared to other togaviruses, RV replicates very slowly suggesting that it must employ effective mechanisms to delay the innate immune response. A recent study by our laboratory revealed that the capsid protein of RV is a potent inhibitor of apoptosis. A primary mechanism by which RV capsid interferes with programmed cell death appears to be through interaction with the pro-apoptotic Bcl-2 family member Bax. In the present study, we report that the capsid protein also blocks IRF3-dependent apoptosis induced by the double-strand RNA mimic polyinosinic-polycytidylic acid. In addition, analyses of cis-acting elements revealed that phosphorylation and membrane association are important for its anti-apoptotic function. Finally, the observation that hypo-phosphorylated capsid binds Bax just as well as wild-type capsid protein suggests that interaction with this pro-apoptotic host protein in and of itself is not sufficient to block programmed cell death. This provides additional evidence that this viral protein inhibits apoptosis through multiple mechanisms.

Capsid proteins are obligatory components of infectious virions. Their primary structural function is to protect viral genomes during entry and exit from host cells. Evidence suggests that these proteins can also modulate the activity and specificity of viral replication complexes. More recently, it has become apparent that they play critical roles at the virus–host interface. Here, we discuss how capsid proteins of RNA viruses interact with key host cell proteins and pathways to modulate cell physiology in order to benefit virus replication. Capsid–host cell interactions may also have implications for viral disease. Understanding how capsids regulate virus–host interactions may lead to the development of novel antiviral therapies based on targeting the activities of cellular proteins.

Rubella virus (RV) is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, RV infection leads to severe damage in fetuses. Despite its medical importance, compared with the related alphaviruses, very little is known about the structure of RV. The RV capsid protein is an essential structural component of virions as well as a key factor in virus-host interactions. Here we describe three crystal structures of the structural domain of the RV capsid protein. The polypeptide fold of the RV capsid protomer has not been observed previously. Combining the atomic structure of the RV capsid protein with the cryoelectron tomograms of RV particles established a low-resolution structure of the virion. Mutational studies based on this structure confirmed the role of amino acid residues in the capsid that function in the assembly of infectious virions.

Virus replication occurs in the midst of a life or death struggle between the virus and the infected host cell. To limit virus replication, host cells can activate a number of antiviral pathways, the most drastic of which is programmed cell death. Whereas large DNA viruses have the luxury of encoding accessory proteins whose main function is to interfere with host cell defences, the genomes of RNA viruses are not large enough to encode proteins of this type. Recent studies have revealed that proteins encoded by RNA viruses often play multiple roles in the battles between viruses and host cells. In this article, we discuss the many functions of the rubella virus capsid protein. This protein has well-defined roles in virus assembly, but recent research suggests that it also functions to modulate virus replication and block host cell defences.