Hybrid immunity from SARS-CoV-2 infection and vaccination in Canadian adults: cohort study.

Background: Few national-level studies have evaluated the impact of 'hybrid' immunity (vaccination coupled with recovery from infection) from the Omicron variants of SARS-CoV-2.Methods: From May 2020 to December 2022, we conducted serial assessments (each of ~4000-9000 adults) examining SARS-CoV-2 antibodies within a mostly representative Canadian cohort drawn from a national online polling platform. Adults, most of whom were vaccinated, reported viral test-confirmed infections and mailed self-collected dried blood spots to a central lab. Samples underwent highly sensitive and specific antibody assays to spike and nucleocapsid protein antigens, the latter triggered only by infection. We estimated cumulative SARS-CoV-2 incidence prior to the Omicron period and during the BA.1/1.1 and BA.2/5 waves. We assessed changes in antibody levels and in age-specific active immunity levels.Results: Spike levels were higher in infected than in uninfected adults, regardless of vaccination doses. Among adults vaccinated at least thrice and infected more than six months earlier, spike levels fell notably and continuously for the nine months post-vaccination. By contrast, among adults infected within six months, spike levels declined gradually. Declines were similar by sex, age group, and ethnicity. Recent vaccination attenuated declines in spike levels from older infections. In a convenience sample, spike antibody and cellular responses were correlated. Near the end of 2022, about 35% of adults above age 60 had their last vaccine dose more than six months ago, and about 25% remained uninfected. The cumulative incidence of SARS-CoV-2 infection rose from 13% (95% CI 11-14%) before omicron to 78% (76-80%) by December 2022, equating to 25 million infected adults cumulatively. However, the COVID-19 weekly death rate during the BA.2/5 waves was less than half of that during the BA.1/1.1 wave, implying a protective role for hybrid immunity.Conclusions: Strategies to maintain population-level hybrid immunity require up-to-date vaccination coverage, including among those recovering from infection. Population-based, self-collected dried blood spots are a practicable biological surveillance platform.Funding: Funding was provided by the COVID-19 Immunity Task Force, Canadian Institutes of Health Research, Pfizer Global Medical Grants, and St. Michael's Hospital Foundation. PJ and ACG are funded by the Canada Research Chairs Program.

A Significant Contribution of the Classical Pathway of Complement in SARS-CoV-2 Neutralization of Convalescent and Vaccinee Sera.

Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals.

The relationship between serum astroglial and neuronal markers and AQP4 and MOG autoantibodies.

BACKGROUND: Certain demyelinating disorders, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) exhibit serum autoantibodies against aquaporin-4 (αAQP4) and myelin oligodendrocyte glycoprotein (αMOG). The variability of the autoantibody presentation warrants further research into subtyping each case. METHODS: To elucidate the relationship between astroglial and neuronal protein concentrations in the peripheral circulation with occurrence of these autoantibodies, 86 serum samples were analyzed using immunoassays. The protein concentration of glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL) and tau protein was measured in 3 groups of subcategories of suspected NMOSD: αAQP4 positive (n = 20), αMOG positive (n = 32) and αMOG/αAQP4 seronegative (n = 34). Kruskal-Wallis analysis, univariate predictor analysis, and multivariate logistic regression with ROC curves were performed. RESULTS: GFAP and NFL concentrations were significantly elevated in the αAQP4 positive group (p = 0.003; p = 0.042, respectively), and tau was elevated in the αMOG/αAQP4 seronegative group (p < 0.001). A logistic regression model to classify serostatus was able to separate αAQP4 seropositivity using GFAP + tau, and αMOG seropositivity using tau. The areas under the ROC curves (AUCs) were 0.77 and 0.72, respectively. Finally, a combined seropositivity versus negative status logistic regression model was generated, with AUC = 0.80. CONCLUSION: The 3 markers can univariately and multivariately classify with moderate accuracy the samples with seropositivity and seronegativity for αAQP4 and αMOG.

The relationship between serum astroglial and neuronal markers and AQP4 and MOG autoantibodies.

Background: Certain demyelinating disorders, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) exhibit serum autoantibodies against aquaporin-4 (αAQP4) and myelin oligodendrocyte glycoprotein (αMOG). The variability of the autoantibody presentation warrants further research into subtyping each case. Methods: To elucidate the relationship between astroglial and neuronal protein concentrations in the peripheral circulation with occurrence of these autoantibodies, 86 serum samples were analyzed using immunoassays. The protein concentration of glial fibrillary acidic protein (GFAP), neurofilament light chain (NFL) and tau protein was measured in 3 groups of subcategories of suspected NMOSD: αAQP4 positive (n = 20), αMOG positive (n = 32) and αMOG/αAQP4 seronegative (n = 34). Kruskal-Wallis analysis, univariate predictor analysis, and multivariate logistic regression with ROC curves were performed. Results: GFAP and NFL concentrations were significantly elevated in the αAQP4 positive group (p = 0.003; p = 0.042, respectively), and tau was elevated in the αMOG/αAQP4 seronegative group (p < 0.001). A logistic regression model to classify serostatus was able to separate αAQP4 seropositivity using GFAP + tau, and αMOG seropositivity using tau. The areas under the ROC curves (AUCs) were 0.77 and 0.72, respectively. Finally, a combined seropositivity versus negative status logistic regression model was generated, with AUC = 0.80. Conclusion: The 3 markers can univariately and multivariately classify with moderate accuracy the samples with seropositivity and seronegativity for αAQP4 and αMOG.

Pathobiology, Diagnosis, and Current Biomarkers in Neuromyelitis Optica Spectrum Disorders.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is characterized by chronic inflammation of the central nervous system (CNS), particularly the optic nerves and spinal cord. Although it displays some clinical features similar to multiple sclerosis (MS), the etiology and treatment are distinct, and therefore accurate diagnosis is essential. Autoantibodies targeting the water channel protein aquaporin-4 (AQP4) and the myelin sheath protein myelin oligodendrocyte glycoprotein are the major antigen-specific serological biomarkers known to date, with destruction of astrocytes as the primary mode of CNS damage in AQP4-positive disease. CONTENT: This mini-review summarizes the pathobiology, clinical features, and current methods of serological testing used to assess NMOSD and differentiate this disorder from MS. A brief summary of emerging therapies is also presented. SUMMARY: NMOSD can be distinguished from MS through a combination of clinical findings, imaging investigations, and serological analysis. Seronegative cases are particularly difficult to diagnose and can pose a challenge to clinicians. As knowledge deepens, new therapies and biomarkers are expected to improve treatment of this rare debilitating disease.

Evaluation of Three Anti-SARS-CoV-2 Serologic Immunoassays for Post-Vaccine Response.

BACKGROUND: In North America, both messenger RNA (mRNA) vaccines, Pfizer-BioNTech BNT162b2, and Moderna mRNA-1273, each utilizing a 2-dose regimen, have started to be administered to individuals. METHODS: We evaluated the quantitative serologic antibody response following administration of either a single dose or both doses of an mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in a cohort of 98 participants (88 healthcare workers [HCW] and 10 solid organ transplant [SOT] recipients). Antibody levels were compared across 3 immunoassays: Elecsys Anti-SARS-CoV-2 S (Roche Diagnostics), SARS-CoV-2 TrimericS IgG (DiaSorin), and SARS-CoV-2 IgG II Quant (Abbott). RESULTS: Among HCW, sensitivity ranged from 100% (Roche), 99% (Abbott) and 98% (DiaSorin). The SARS-CoV-2 IgG II Quant and SARS-CoV-2 TrimericS IgG assays showed good agreement with a Pearson correlation coefficient of R = 0.95. Pearson correlation coefficients of R = 0.82 and 0.83 were obtained for Elecsys Anti-SARS-CoV-2 S vs SARS-CoV-2 TrimericS IgG and SARS-CoV-2 IgG II Quant vs Elecsys Anti-SARS-CoV-2 S, respectively. Significant differences in antibody levels between HCW and SOT recipients were observed. A decrease in antibody levels from time of vaccine administration to blood draw was evident. Among those with a second dose, an increase in antibody levels with increased time between administration of the first and second dose was observed. CONCLUSIONS: The absolute values generated from each of the assay platforms are not interchangeable. Antibody levels differed with increased time between vaccine administration and with increased time between administration of the first and second dose. Further, significant differences in antibody levels between HCW and SOT recipients were observed.

Aspirin: Bitter pill or miracle drug?

Acetylsalicylic acid (ASA) or brand name Aspirin is a widely available medication used to relieve inflammation, fever and pain. It has also been frequently prescribed as prevention for cardiovascular disease due to its anti-thrombotic qualities. However, ASA is also connected to increased internal bleeding, leading to concerns that this harmful side effect may outweigh its cardioprotective properties in some populations. In this review, we summarize data from several recent, large-scale clinical trials that put into the question the long-standing recommendations about prescribing ASA for primary cardiovascular disease. We also provide a detailed overview of the role of ASA in cancer, surgery and female reproductive health. Finally, we discuss the ASA prescription guidelines of several major medical organizations and suggest that this new evidence may lead to updates to these influential and longstanding recommendations.

The challenges and mental health issues of academic trainees.

In the last decade, mental health issues have come to the foreground in academia. Literature surrounding student mental health continues to grow as universities try to implement wellness services and study the mental health of their students. Studies vary greatly in terms of measurement tools, timeframe, sample demographics, as well as the chosen threshold of symptom severity for diagnosis. This review attempts to summarize, contextualize and synthesize papers that pertain to the challenges faced by academic trainees at the undergraduate, graduate and post-graduate level. The evidence for, and against, the common claim of increasing prevalence of mental health issues among students in recent years is discussed. While some studies support this claim, it is difficult to reach a definitive conclusion due to numerous confounding factors such as increased help-seeking behaviour, greater awareness of mental health issues and weak methodology. The prevalence of depression, anxiety, suicidal and self-injurious behaviour, distress and general mental illness diagnoses are discussed. Other issues known to influence mental health, such as sexual assault and bullying, are briefly addressed. Finally, select studies on a few wellness strategies that may improve mental health of trainees, such as mindfulness, are summarised, along with diverse recommendations for individual students, universities, and academia as a whole.

The current status and future prospects of precision medicine.

Over the last 5 years I have been coordinating a graduate course on genomic technologies and their applications in medicine. The course is offered to graduate students in the Department of Laboratory Medicine and Pathobiology at the University of Toronto. In attending the diverse lectures, I came to better understand the burgeoning field of "personalized" or "precision" medicine (PM) and its current status and future prospects. Below, I provide my personal views on this topic.

A miRNA-based classification of renal cell carcinoma subtypes by PCR and in situ hybridization.

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors the most prevalent of which are clear cell, papillary, and chromophobe RCC. Accurate distinction between the typically benign-behaving renal oncocytoma and RCC subtypes is a frequent challenge for pathologists. This is critical for clinical decision making. Subtypes also have different survival outcomes and responses to therapy. We extracted RNA from ninety formalin-fixed paraffin-embedded (FFPE) tissues (27 clear cell, 29 papillary, 19 chromophobe, 4 unclassified RCC and 11 oncocytomas). We quantified the expression of six miRNAs (miR-221, miR-222, miR-126, miR-182, miR-200b and miR-200c) by qRT-PCR, and by in situ hybridization in an independent set of tumors. We developed a two-step classifier. In the first step, it uses expression of either miR-221 or miR-222 to distinguish the clear cell and papillary subtypes from chromophobe RCC and oncocytoma (miR-221 AUC: 0.96, 95% CI: 0.9132-1.014, p < 0.0001 and miR-222 AUC: 0.91, 95% CI: 0.8478-0.9772, p < 0.0001). In the second step, it uses miR-126 to discriminate clear cell from papillary RCC (AUC: 1, p < 0.0001) and miR-200b to discriminate chromophobe RCC from oncocytoma (AUC: 0.95, 95% CI: 0.8933-1.021, p < 0.0001). In situ hybridization showed a nuclear staining pattern. miR-126, miR-222 and miR-200b were significantly differentially expressed between the subtypes by in situ hybridization. miRNA expression could distinguish RCC subtypes and oncocytoma. miRNA expression assessed by either PCR or in situ hybridization can be a clinically useful diagnostic tool to complement morphologic renal tumor classification, improving diagnosis and patient management.

Candidate recommendations for protein electrophoresis reporting from the Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group.

Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.

Liquid biopsy: a step forward towards precision medicine in urologic malignancies.

There is a growing trend towards exploring the use of a minimally invasive "liquid biopsy" to identify biomarkers in a number of cancers, including urologic malignancies. Multiple aspects can be assessed in circulating cell-free DNA, including cell-free DNA levels, integrity, methylation and mutations. Other prospective liquid biopsy markers include circulating tumor cells, circulating RNAs (miRNA, lncRNAs and mRNAs), cell-free proteins, peptides and exosomes have also emerged as non-invasive cancer biomarkers. These circulating molecules can be detected in various biological fluids, including blood, urine, saliva and seminal plasma. Liquid biopsies hold great promise for personalized medicine due to their ability to provide multiple non-invasive global snapshots of the primary and metastatic tumors. Molecular profiling of circulating molecules has been a stepping-stone to the successful introduction of several non-invasive multi-marker tests into the clinic. In this review, we provide an overview of the current state of cell-free DNA-based kidney, prostate and bladder cancer biomarker research and discuss the potential utility other circulating molecules. We will also discuss the challenges and limitations facing non-invasive cancer biomarker discovery and the benefits of this growing area of translational research.

An integrated proteomic and peptidomic assessment of the normal human urinome.

BACKGROUND: Urine represents an ideal source of clinically relevant biomarkers as it contains a large number of proteins and low molecular weight peptides. The comprehensive characterization of the normal urinary proteome and peptidome can serve as a reference for future biomarker discovery. Proteomic and peptidomic analysis of urine can also provide insight into normal physiology and disease pathology, especially for urogenital diseases. METHODS: We developed an integrated proteomic and peptidomic analytical protocol in normal urine. We employed ultrafiltration to separate protein and peptide fractions, which were analyzed separately using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) on the Q-Exactive mass spectrometer. RESULTS: By analyzing six urines from healthy individuals with advanced age, we identified 1754 proteins by proteomic analysis and 4543 endogenous peptides, arising from 566 proteins by peptidomic analysis. Overall, we identified 2091 non-redundant proteins by this integrated approach. In silico protease activity analysis indicated that metalloproteases are predominantly involved in the generation of the endogenous peptide signature. In addition, a number of proteins that were detected in normal urine have previously been implicated in various urological malignancies, including bladder cancer and renal cell carcinoma (RCC). CONCLUSIONS: We utilized a highly sensitive proteomics approach that enabled us to identify one of the largest sets of protein identifications documented in normal human urine. The raw proteomics and peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003595.

Proteomics and peptidomics: moving toward precision medicine in urological malignancies.

Urological malignancies are a major cause of morbidity and mortality worldwide. Advances in early detection, diagnosis, prognosis and prediction of treatment response can significantly improve patient care. Proteomic and peptidomic profiling studies are at the center of kidney, prostate and bladder cancer biomarker discovery and have shown great promise for improved clinical assessment. Mass spectrometry (MS) is the most widely employed method for proteomic and peptidomic analyses. A number of MS platforms have been developed to facilitate accurate identification of clinically relevant markers in various complex biological samples including tissue, urine and blood. Furthermore, protein profiling studies have been instrumental in the successful introduction of several diagnostic multimarker tests into the clinic. In this review, we will provide a brief overview of high-throughput technologies for protein and peptide based biomarker discovery. We will also examine the current state of kidney, prostate and bladder cancer biomarker research as well as review the journey toward successful clinical implementation.

miR-221/222 Are Involved in Response to Sunitinib Treatment in Metastatic Renal Cell Carcinoma.

Sunitinib is a multitargeting tyrosine kinase inhibitor used for metastatic renal cancer. There are no biomarkers that can predict sunitinib response. Such markers are needed to avoid administration of costly medication with side effects to patients who would not benefit from it. We compared global miRNA expression between patients with a short (≤12 months) versus prolonged (>12 months) progression-free survival (PFS) under sunitinib as first-line therapy for metastatic renal cell carcinoma. We identified a number of differentially expressed miRNAs and developed miRNA statistical models that can accurately distinguish between the two groups. We validated our models in the discovery set and an independent set of 57 patients. Target prediction and pathway analysis showed that these miRNAs are involved in vascular endothelial growth factor (VEGF), TGFß, and mammalian target of rapamycin (mTOR)-mediated signaling and cell-cell communication. We tested the effect of these miRNAs on cellular proliferation and angiogenesis. We validated the negative correlation between miR-221 and its target, VEGFR2.miR-221 overexpression was associated with a poor PFS while its target, VEGFR2 was associated with longer survival. Gain of function experiments showed that miR-221 and miR-222 decreased angiogenesis and cellular proliferation in human umbilical vein endothelial cells (HUVEC) while increasing cellular proliferation in ACHN cells. miRNAs represent potential predictive markers for sunitinib response.