Vera Tang

In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remains untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the mouse dentate gyrus and subventricular zone (SVZ), and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The majority of the cells resid...

Rapid induction of type 1 interferons (IFN) expression is a central event in establishing the innate antiviral response and requires pathogen-inducible, activation of transcription factors that function in a synergistic fashion to induce gene expression. Vesicular stomatitis virus (VSV) multiplication is highly sensitive to the antiviral effects of type 1 interferons (IFN), as well as other innate immune effectors that strongly inhibit VSV in non-malignant cells and tissues; malignant cells acquire diminished responsiveness to IFN action and are specifically infected and killed by oncolytic viruses (OVs) such as VSV. Although tumor growth is delayed or eliminated in numerous animal models following treatment with OVs, several cancer models remain partially or completely resistant to viral oncolysis. To overcome this resistance, experimental strategies are now combining OVs with different cytotoxic compounds to improve OV efficacy. In the current study, we investigated the capacity o...

Accurate characterization of extracellular vesicles (EVs) is critical to explore their diagnostic and therapeutic applications. As the EV research field has developed, so too have the techniques used to characterize them. The development of reference materials is required for the standardization of these techniques. This work, initiated from the ISEV 2017 Biomarker Workshop in Birmingham, UK, and with further discussion during the ISEV 2019 Standardization Workshop in Ghent, Belgium, sets out to elucidate which reference materials are required and which are currently available to standardize commonly used analysis platforms for characterizing EV size, concentration, refractive index, and epitope expression. Due to their predominant use, a particular focus is placed on the optical methods nanoparticle tracking analysis and flow cytometry.

Rapid induction of type 1 interferons (IFN) expression is a central event in establishing the innate antiviral response and requires pathogen-inducible, activation of transcription factors that function in a synergistic fashion to induce gene expression. Vesicular stomatitis virus (VSV) multiplication is highly sensitive to the antiviral effects of type 1 interferons (IFN), as well as other innate immune effectors that strongly inhibit VSV in non-malignant cells and tissues; malignant cells acquire diminished responsiveness to IFN action and are specifically infected and killed by oncolytic viruses (OVs) such as VSV. Although tumor growth is delayed or eliminated in numerous animal models following treatment with OVs, several cancer models remain partially or completely resistant to viral oncolysis. To overcome this resistance, experimental strategies are now combining OVs with different cytotoxic compounds to improve OV efficacy. In the current study, we investigated the capacity of triptolide (TPL) – a natural molecule derived from the medicinal herb, Tripterygiumwilfordii Hook F, with demonstrated anti-tumor and anti-inflammatory activities – to enhance vesicular stomatitis virus (VSV) oncolysis in OV-resistant cancer cells. TPL treatment increased VSV replication in the human prostate cancer cell line PC3 and in other VSV-resistant cells in a dose and time-dependent manner in vitro and in vivo. Furthermore, TPL enhanced VSV-induced apoptosis in a dose proportional fashion in VSV-resistant cells, as measured by annexin-V, cleaved caspase-3 and B-cell lymphoma 2 (Bcl-2) staining. Mechanistically, TPL inhibited the innate antiviral response by blocking the type I interferon (IFN) signaling pathway, downstream of IRF3 activation. In vivo, using TSA mouse xenograft model, combination treatment with VSV and TPL delayed tumor growth and prolonged survival of tumor-bearing animals by enhancing viral replication. Together, these results demonstrate for the first time the synergic effect of TPL and VSV, and indicate that TPL treatment increases VSV replication and the subsequent apoptosis of tumor cells by inhibiting IFN signaling.

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rap...

ABSTRACTWhile P-selectin glycoprotein ligand-1 (PSGL-1/CD162) has been studied extensively for its role in mediating leukocyte rolling through interactions with its receptor, P-selectin, recently, it was identified as a novel HIV-1 host restriction factor. One key mechanism of HIV-1 restriction is the ability of PSGL-1 to be physically incorporated into the external viral envelope, which effectively reduces infectivity by blocking virus attachment through the steric hindrance caused by its large ectodomain. Importantly, a large portion of the literature demonstrating the antiviral activity of PSGL-1 has utilized viruses produced in transfected cells which express high levels of PSGL-1. However, herein we show that virion-incorporated PSGL-1 is far less abundant on the surface of viruses produced via infection of physiologically relevant models (T cell lines and primary cells) compared to transfection (overexpression) models. Unique to this study, we show that PSGL-1 is incorporated ...

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as t...

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus infected cells are well known to release soluble viral proteins, defective viruses and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small particle flow cytometry, is an emerging analytical method enabling...

Nanoscale flow cytometry (NFC) is becoming a method of choice for the phenotypic analysis of viruses and extracellular vesicles (EVs). However, many of these particles are smaller than 200 nm in diameter, which places them at the limit of detection for many commercial flow cytometers. The use of reference particles of size, fluorescence, and light-scattering properties similar to that of the small particles of interest is therefore imperative for accurate and reproducible data acquisition and reporting across different instruments and analytical technologies. We show here that an engineered murine leukemia virus (MLV) can act as a fluorescence reference particle which robustly satisfies these criteria. MLV can be engineered to express proteins of interest at high, but biologically relevant levels, on the surface of its viral envelope, which is derived from the cell plasma membrane. These recombinant proteins display consitent expression in the released virus population, and are read...

To assess blood levels of cortisol and cytokines (inflammatory and non-inflammatory) in members of the regular Canadian Armed Forces (CAF), and examine the associations between sex, age and adiposity and circulating levels of cortisol as well as pro- and anti-inflammatory cytokines. As part of a larger ranging project, 331 blood samples were collected from a representative population of the total CAF, which included officers and non-commissioned women and men from the Air Force, Navy and Army. The blood samples were analyzed for levels of cortisol, C-Reactive Protein, adiponectin and 20 cytokines (that included interleukins, interferons and Tumor Necrosis Factors). Higher levels of adiponectin were found in women compared to men (median and IQR; 16.71 (7.68-25.32) vs 5.81 (3.52-13.19) µg/ml), and higher levels of IL-18 in men compared to women (89.25 (84.03-94.48) vs 75.91 (69.70-82.13) pg/ml). An association between age and levels of stress and inflammatory cytokines was observed, ...

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that i...

Although the genital tract is the main portal of entry for sexually transmitted infections in women, we still have limited understanding of the immune responses in the local tissue and the factors that impact their generation. Genital herpes is the most common sexually transmitted infection in the world; affecting close to one tenth of the global population. There is as

Although the female genital tract is the main portal of entry for sexually transmitted infections in women, we still have limited understanding of the generation, maintenance and characteristics of memory T cells in the local tissue. Here, we utilized a mouse model of intravaginal HSV-2 infection and tetramers against the immunodominant HSV glycoprotein B epitope recognized by CD8+ T cells to examine the generation, maintenance and characteristics of anti-HSV memory T cells in the genital tract following acute infection. Our results show that the highest percentage of HSVgB-specific CD8+ T cells was found in the genital tract compared to the spleen or iliac lymphnode. Indeed, although the actual number of CD8+ T cells contracted following viral clearance, approximately one quarter of the CD8+ population that remained in the genital tissue was HSVgB-specific. Memory gB-tetramer+CD8 T cells in the genital tract were positive for CD127 and KLRG1 and negative for CD62L and CCR7, thus confirming that HSV-specific CD8 cells were effector memory T cells that lack the capacity for homing to lymphoid tissues. Functionally, both memory CD8+ and CD4+ HSV-specific populations in the genital tract produced IFNγ when stimulated in vitro and CD4+ cells also produced TNFα. Genital HSVgB-specific memory T cells expressed tissue-homing integrins CD103 (αE integrin) and CD49a (VLA-1 or α1 integrin). Our findings suggest that HSV-specific memory T cells are retained in the genital tract, poised to act as an early line of defense against future virus encounter.

Alterations in lipid oxidation during exercise have been well studied, but limited data exists on the effects of passive heat exposure and exercise in the heat on changes in lipid oxidation. This study was designed to examine: (1) the effects of heat exposure on lipid metabolism during passive heating and subsequent exercise in the heat by focusing on changes in whole-body lipid oxidation and plasma lipid concentrations, and (2) the effects of extended passive pre-heating on exercise performance in the heat. Male participants (n=8) were passively heated for 120min at 42°C, then exercised on a treadmill in the same temperature at 50% V̇O2max for 30min (HEAT). This same procedure was followed on a separate occasion at 23°C (CON). Results showed that lipid oxidation rates were not different between HEAT and CON during passive heating or exercise. However, non-esterified fatty acid (NEFA) concentrations were significantly higher following passive heating (618µM, 95% CI: 479-757) compared to CON (391µM, 95% CI: 270-511). The same trend was observed following exercise (2036µM, 95% CI: 1604-2469 for HEAT and 1351µM, 95% CI: 1002-1699). Triacylglycerol, phospholipid and cholesterol levels were not different between HEAT and CON at any point. Four of 8 participants could not complete 30min of exercise in HEAT, resulting in a 14% decline in total external work. Rate of perceived exertion over the final 5min of exercise was higher in HEAT (9.5) than CON (5). We conclude that: (1) heat exposure results in increased circulating NEFA at rest and during exercise without changes in whole-body lipid utilization, and (2) passive pre-heating reduces work capacity during exercise in the heat and increases the perceived intensity of a given workload.

Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under ...